AN ABSTRACT OF THE THESIS OF
Sudarshan Lai Sabharwal
(Name)
in
Title:
Horticulture
(Major)
for the
Doctor of Philosophy
(Degr&e)
presented on ^^/^i^/^^ /^//
//^
/jj&te)
RESPONSE OF STRAWBERRY PLANTS TO MANGANESE
NUTRITION
Abstract approved:
Ralph Garren,
Jr.
The effects of manganese nutrition on growth, mineral composition,
carbohydrate and nitrogen metabolism, IAA oxidase and phenols
of 'Northwest' strawberry plants are reported.
Growth and mineral
composition response were investigated in sand culture and Hoagland's
solution 2 modified to contain 0. 0, 0. 5,
125. 0 ppm substrate manganese.
2. 5, 12. 5,
25. 0, 62. 5 and
The remaining aspects of the study
were conducted using the Hoaglands solution in a hydroponics system
with manganese levels of 0. 0, 0. 5,
2. 5,
1 2. 5 and 25. 0 ppm in the
nutrient solution.
In sand culture,
symptoms of manganese toxicity occurred first
in old leaves after three weeks of 125. 0 ppm Mn treatment; after five
weeks at 62. 5 ppm; and after 13 weeks at 25. 0 ppm substrate Mn.
Tox-
icity symptoms appeared as necrotic spots along the margins of leaves;
these spots enlarged greatly within a few days and ultimately killed all
the leaves.
All the plants treated with 62. 5 and 125. 0 ppm Mn died
within 7 and 11 weeks, respectively.
Young leaves of runners from the plants treated with 0. 0 ppm Mn
developed interveinal chlorosis and cupping after 12 weeks.
Later,
these leaves developed chlorotic spots which expanded to chlorotic
bands.
Plant weight and total leaf area of the plants harvested after 20
weeks was greatest with 2. 5 ppm Mn treatment, and decreas-e.d both at
lower or higher substrate manganese.
The most leaves and runners
were produced at 25. 0 ppm treatment; whereas area per leaf was highest at 0. 5 ppm Mn.
Manganese present in tissue was directly correlated to substrate
manganese levels and was also related to the plant part.
Accumulation
of manganese occurred in all tissues at high substrate Mn levels,
indicating that the tolerance of strawberry plants to high Mn is not
because of low uptake but due to their ability to endure large amounts
of accumulated Mn.
In Mn deficient plants, the differences in the con-
centration of Mn in the various tissues were small, suggesting the
possibility of redistribution of the element under conditions of deficiency.
Highest levels of tissue iron were found in 0. 0 ppm Mn treatment followed in order by 2. 5, 0. 5, 1 2. 5 and 25. 0 ppm substrate Mn.
A strong inverse relationship was noted between the tissue iron/
manganese ratio and the substrate Mn.
The deficiency and excess of
Mn resulted in a decrease in the concentration of Mg and P in the
leaves .
Highest levels of these elements were observed with 2. 5 ppm
Mn treatment.
The most vigorous plants were produced at 2. 5 ppm Mn treatment when old and young leaves contained 955. 7 and 617.4 ppm Mn,
respectively.
Typical toxicity symptoms were shown by plants when
Mn in young leaves reached about 4, 000 and in old leaves 9, 000 ppm.
In the hydroponic system, manganese deficiency and toxicity
resulted in a decrease in sugar as well as starch content in the leaves,
except in old leaves at 25. 0 ppm Mn treatment which contained the
highest levels of starch.
Total and insoluble N increased whereas nitrate N decreased with
the increase in substrate Mn.
Lowest levels of soluble and soluble
reduced N were found at 2. 5 and 0. 5 ppm Mn treatment, respectively.
Both the excess and deficiency of Mn resulted in the accumulation of
amino acids.
The highest concentrations of nitrate and lowest concen-
trations of insoluble N observed in Mn deficient plants indicate that the
Mn is not involved in nitrate N absorption but may be concerned in
protein synthesis.
High IAA oxidase activity was found in enzyme extracts of young
and old leaves of deficient and toxic plants.
Total and bound phenols,
in general, were directly related to substrate manganese.
Low levels
of free phenols were found in conditions of excess and deficiency of Mn.
The increase in the level of LAA oxidase activity at high level of
tissue Mn was accompanied by simultaneous decrease in the concentration of o-dihydroxyphenols.
These results indicate the possible
involvement of phenols in the inhibition of IAA oxidase in the leaves of
strawberry plants and manganese seems to affect the levels of the
inhibitor(s).
Response of Strawberry Plants
to Manganese Nutrition
by
Sudarshan Lai Sabharwal
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
June 1972
APPROVED:
" v^-c-T -y^--—
^T faO^. tf fc-M-JC" ■■■^r
Professor of Horticulture
in charge of major
•y^ »• •^
Head of Department of Horticulture ^^
Dean of Graduate School
Date thesis is presented
^/yy^V g/(>\. /<?"7/
Typed by Barbara Eby/fpg Sudarshan Lai Sabharwal
ACKNOWLEDGEMENT
I would like to express my sincere appreciation to Dr. Ralph
Garren, Jr. for his interest, guidance, encouragement and support
during the entire graduate program and to Dr. O. C. Compton for the
suggestions and inspiration throughout the period of this study.
I would like to express my sincere thanks to committee members
Dr. N. I. Bishop, Dr. R. H. Converse, and Dr. T. L. Jackson and to
Dr. E. Hansen for thoughtful criticism of the manuscript.
I would also like to express my thanks and appreciation to the
staff members of the Department of Horticulture for their guidance,
cooperation, and help during my period of study.
TABLE OF CONTENTS
INTRODUCTION
GROWTH AND MINERAL COMPOSITION RESPONSE
OF STRAWBERRY PLANTS TO MANGANESE
NUTRITION
Materials and Methods
Plant material
Sand culture studies
Purification of nutrient solution
Sampling of plants
Chemical analysis
Results
Manganese toxicity and deficiency symptoms
Percentage of leaves shewing deficiency or toxicity
symptoms
Effect of substrate manganese on plant growth
Concentration and distribution of tissue manganese
Concentration and distribution of tissue iron
Iron manganese ratio and distribution
Concentration and distribution of tissue magnesium
Concentration and distribution of tissue phosphorous
Discussion
Literature Cited
1
PART I.
INFLUENCE OF MANGANESE NUTRITION ON
CARBOHYDRATE AND NITROGEN METABOLISM
OF STRAWBERRY LEAVES
Materials and Methods
Water culture studies
Analytical methods
Results and Discussion
Carbohydrate content
Total nitrogen
Insoluble and soluble nitrogen
Soluble reduced and nitrate nitrogen
Amino acids
Literature Cited
2
3
3
4
5
5
6
6
6
8
8
11
13
13
16
16
18
23
PART II.
26
29
29
29
30
30
33
33
35
36
41
PART III.
INFLUENCE OF MANGANESE NUTRITION ON IAA
OXIDASE AND PHENOLS OF STRAWBERRY LEAVES
Materials and Methods
Solution culture studies
Extraction and purification for assay of IAA oxidase
LAA oxidase assay
Assay for co-factor
Extraction for determination of phenols
Estimation of phenols
Determination of manganese
Results and Discussion
Tissue manganese
Characterization of IAA oxidase enzyme assay
Effect of Mn on IAA oxidase activity of dialyzed extract
Effect on phenols
Interaction of tissue Mn, o-dihydroxyphenols and IAA
oxidase
Literature Cited
45
47
47
47
48
49
49
49
50
50
50
50
55
58
61
63
LIST OF FIGURES
Figure
1. 1
Page
Effect of manganese on the growth of strawberry plants.
10
Effect of manganese on the magnesium content
in strawberry plants.
17
Effect of manganese on the phosphorous content in strawberry plants.
17
Carbohydrate content in relation to manganese
nutrition.
32
Nitrogen content in relation to manganese
nutrition.
32
Soluble and insoluble nitrogen in relation to
manganese nutrition.
34
Soluble reduced and nitrate nitrogen in relation
to manganese nutrition.
34
IAA oxidase activity of dialyzed and nondialyzed extracts of strawberry leaves.
52
3. 2
Effect of time of dialysis on enzyme activity.
52
3. 3
Effect of enzyme concentration on oxygen
uptake.
54
3. 4
Effect of co-factors on IAA oxidase activity.
54
3. 5
pH optimum of IAA oxidase of strawberry
leaves.
56
3. 6
Effect of substrate IAA on IAA oxidase activity.
56
3. 7
Effect of substrate manganese on IAA oxidase
activity of strawberry leaves.
57
1. 2
1. 3
Z. 1
2. 2
2. 3
2. 4
3. 1
Figure
3. 8
3. 9
3. 10
Page
Effect of substrate manganese on total and
bound phenols of strawberry leaves.
59
Effect of substrate manganese on free phenols
of strawberry leaves.
59
Interaction of tissue manganese, o-dihydroxyphenol
and IAA' oxidase.
62
LIST OF TABLES
Table
1. 1
1. 2
1. 3
1. 4
2. 1
2. 2
3. 1
Page
Percentage of leaves showing deficiency or
toxicity symptoms.
9
Effect of manganese on the tissue manganese
of strawberry plants.
12
Effect of manganese on the tissue iron of
strawberry plants.
14
Effect of substrate manganese on the iron/
manganese ratio of strawberry plants.
15
Influence of substrate manganese on the
amino acids in young strawberry leaves.
37
Influence of substrate manganese on the
amino acids in old strawberry leaves.
39
Manganese content of young and old leaves.
51
RESPONSE OF STRAWBERRY PLANTS TO
MANGANESE NUTRITION
INTRODUCTION
A wide variation in the concentration of manganese in strawberry
leaves has been observed in Oregon from samples taken from various
locations.
The effect of this on the growth and yield of the strawberry
is not known.
The purpose of this study was to investigate the relation-
ship between growth and biochemical status of strawberry plants grown
in media varying from deficient to toxic levels of manganese.
Since
manganese has been observed to act as an antagonist or synergist to
other elements, the mineral composition status of other elements was
also examined.
The dissertation is divided into three parts.
In Part I, the
effects of substrate manganese on 'Northwest' strawberry plants are
described.
Since the reports on manganese deficiency and/or toxicity
for the crop are limited, the symptoms of deficiency and toxicity are
verified, and the optimum requirements of Mn indicated.
In Part II, the changes in carbohydrate and nitrogenous compounds
as affected by substrate manganese are examined.
The concentration
of these compounds is correlated with levels of the nutrient manganese.
In Part III, the interrelationship of tissue manganese, IAA oxidase and phenols is investigated in an attempt to explain growth differences in plants grown at manganese levels ranging from deficient to toxic.
GROWTH AND MINERAL COMPOSITION RESPONSE OF
STRAWBERRY PLANTS TO MANGANESE NUTRITION
Strawberries are grown successfully on soils that vary widely in
texture and reaction, yet little information is available on the relationships between growth and tissue manganese.
Poor plant growth has
been reported due both to low and excess tissue manganese (24, 25).
A number of workers have shown that the availability of manganese in soil increases or decreases as soil acidity increases or
decreases.
Mulder and Gerretsen (26) stated that soils with pH less
than 5. 5 contain most of their manganese in a water-soluble or
exchangeable form.
However, as the pH of the soil is increased, the
manganese is converted to the less available manganic oxide.
Most manganese toxicity occurs on highly acid soils.
Hewitt
(10) stated that the deleterious effects of high acidity were the results
of a complex of factors of which manganese toxicity was most importa'nt.
Fried and Peech (7) indicated that poor plant growth on acid
soils was primarily because of the toxic effects of soluble manganese
and not necessarily a lack of calcium.
It has been shown that the
plants grown in a solution or sand culture containing toxic quantities of
Mn developed abnormalities similar to those observed in plants grown
on acid soils (1, 2, 16, 25).
The manganese content of strawberry leaves growing under
acidic conditions is often very high, and yet the plants sho-w no signs of
abnormalities.
Mn levels up to 1. 8% have been observed without
visible symptoms of toxicity (29).
Lohnis (21) stated that the tolerance
to high available Mn may be caused by either weak absorption, as observed
in mustard and marigold, or to strong tolerance within the plant tissue
as appears to be true for strawberry, potato and probably for broadbeans.
Gerloff et al. (8) also concluded that species with high concen-
trations of Mn in their tissues showed tolerance of the element.
Lohnis (20) found that the strawberry is not only resistant to Mn
toxicity but also to Mn deficiency.
Johanson and Walker (18) observed
the first deficiency symptoms in solution cultures after 88 days;
whereas, Dennis and O'Brien (5) noticed growth retardation after two
months in sand culture and symptoms of deficiency after four months.
The present work was undertaken to study the effects of substrate
and tissue manganese on growth and foliage-nutrient composition of
'Northwest' strawberry plants.
Materials and Methods
Plant material.
All the strawberry plants for this study were of
the cultivar Northwest and "certified" free of diseases and pests by
the Oregon State Department of Agriculture.
Before separating the
plants into different weight categories, they were washed with tap water;
the leaves were removed; and the roots were trimmed to four inches.
Plants handled as above and -weighing six to eight grams were held in
storage at 32 F for four months prior to use.
At the initiation of the
experiment, plants were removed from storage and grown in the greenhouse at 75 F day and 60 F night temperature with a 16-hour day
length.
Plants were grown in 11 liter plastic containers filled with
distilled water and aerated at a constant pressure.
The water in the
containers was changed twice daily.
Sand culture studies.
Plants grown for 15 days under the above
described conditions, having 2-3 leaves, were selected for uniformity
and transferred to two-gallon, glazed crocks having a single drainage
hole.
Crocks were filled with acid-washed quartz sand after the
manner of Hewitt (11).
Five plants were planted in each container,
which was individually connected to an 18 liter, gray-painted glass
bottle containing Hoagland Solution #2, minus manganese.
The plants
were irrigated automatically, by a time-clock mechanism, four times
a day by pumping air into the 18 liter bottles for 14 seconds at a time.
Mn treatments were started ten days after transplanting into the sand.
The levels of substrate manganese as MnSO
were 0. 0, 0. 5,
2. 5, 12. 5, 25. 0, 62. 5, and 125. 0 mg per liter of solution.
treatments were replicated four times.
The seven
Nutrient solutions were
adjusted to pH 5. 5 and were changed fortnightly to assure a constant
supply of nutrients.
Iron was supplied as iron tartrate, at the rate of
1 ml per liter (1 ppm), twice a week, and the level of solutions was
1 •
Supplied as amendments to the Hoagland Solution #2.
brought to the 18 liter mark.
Iron was not supplied as chelated iron
because it has been reported that chelated iron interferes with Mn
uptake (35).
Purification of nutrient solution.
The zero level of manganese
used in the study required that all nutrient salts be purified.
The
macro-nutrient stock solutions were purified by the calcium carbonate
phosphate coprecipitation procedure (11).
Further removal of man-
ganese was carried out by extraction with 8-hydroxyquinoline in chloroform at pH 7 (12).
Iron was purified by a modified isopropyl ether
extraction technique described by Piper (27).
Ferric chloride was
dissolved in concentrated hydrochloric acid and extracted three times
with isopropyl ether.
The iron was recovered from the ether fraction
by adding deionized water and heating to drive off the ether.
The
solution was then analyzed for iron and brought to volume by the addition of five grams of tartaric acid and five milliliters of concentrated
H SO per five grams of pure ferric chloride.
Other micro-nutrients
■were purified by recrystallization.
Sampling of plants.
Blossoms were removed as they occured,
but all runners were permitted to develop.
The plants were observed
twice a week for deficiency and toxicity symptoms in relation to substrate levels.
The time of appearance and the distribution of the symp-
toms was recorded.
One plant from each pot was harvested after every
four weeks for observations on roots.
The last plants 'were harvested
after ZO weeks of treatment.
Samples were washed in EDTA (ethylene
diamine tetra acetic acid) detergent solution; rinsed with demineralized water; separated into roots, petioles and leaves; and dried in an
oven at 65 C.
total weight.
The dry -weight of each fraction was combined to give the
The roots, petioles and leaves were ground separately.
The area of the leaves was determined by the method described by
Darrow and Waldo (4) using the length and width measurements of each
leaflet.
Chemical analysis.
The oven-dried samples were ground in a
Wiley mill to pass through a 40 mesh screen.
An aliquot of 0. 25 to
1. 0 gram of each sample was weighed and ashed in an electric muffle
o
o
furnace at 300 C for one hour and then at 55 0 C for three hours.
Ashed samples were cooled, taken up in 0. IN HCl, digested with 10 ml
of 2. 5 HCl and brought to volume in 0. IN HCl.
The determination of
Mg, Mn and Fe was made with an atomic absorption spectrophotometer.
Phosphorous was determined co.lori.metrica.Uy by the molybdate
method (6).
Results
Manganese toxicity and deficiency symptoms.
Plants grown at
the 125. 0 ppm Mn level died after 7 weeks, while the plants at the
62. 5 ppm Mn level survived up to 11 weeks.
Thus, the analytical
results are largely confined to the 0. 0, 0. 5, 2. 5, 12. 5, and 25. 0 ppm
Mn treatments.
Symptoms of manganese toxicity developed three weeks after
treatment with 125. 0 ppm Mn.
Symptoms of toxicity were also observed
in plants with 62. 5 and 25. 0 ppm manganese treatment after 5 and 13
weeks, respectively.
The first deficiency symptoms were noticed in
some plants after 12 weeks
in 0. 0 ppm (minus) manganese treatment.
No symptoms, either of toxicity or deficiency, were observed in plants
at substrate manganese levels of 0. 5 (control), 2. 5 and 1 2. 5 ppm.
Symptoms of toxicity appeared first in old leaves as small necrotic
spots around the margin of leaves and enlarged greatly within a few
days.
Necrotic spots appeared also around the mid rib.
The marginal
necrosis extended inward, and ultimately killed the whole leaf.
The deficiency symptoms which developed first in the young
leaves of the runner plants were interveinal chlorosis and cupping of
leaves.
The leaves developed chlorotic spots which expanded to form
the chlorotic band.
No deficiency symptoms were observed in leaves
arising from the parent plant throughput the duration of the experiment.
The substrate Mn also affected the development of roots, and
there were practically no new roots after 4 and 8 weeks in plants
treated with 125. 0 and 62. 5 ppm Mn, respectively.
In these two treat-
ments, roots taken at various intervals showed a remarkable correlation between their browning and the number of leaves showing toxicity
8
symptoms.
Slight browning of roots was also observed in 16 and 20-
week old plants treated with 25. 0 ppm manganese.
Percentage of leaves showing deficiency or toxicity symptoms.
The percentage of leaves showing toxicity symptoms was directly
related to the concentration of substrate manganese, and the rate of
appearance of toxicity symptoms was highest at high substrate manganese concentrations (Table 1. 1).
Within four weeks of the appear-
ance of the first toxicity symptoms, 100 percent of the leaves were
affected in plants treated with 125. 0 ppm Mn, while in other plants
treated with 62. 5 ppm Mn showed toxicity symptoms after six weeks.
On the other hand, only 11. 0% of leaves showed toxicity even after six
weeks in plants treated with 25. 0 ppm Mn.
However, there was not
much change in the percentage of leaves showing deficiency symptoms
even after six weeks of the appearance of the first deficient leaf.
Effect of substrate manganese on plant growth.
Plant vigor
implies a well balanced growth which can be quantatized by various
indices.
In this investigation leaf and runner number, area of leaves,
dry plant weight and tissue analysis were used to measure quantitative
growth.
Increase in substrate Mn resulted in an increase in the number
of leaves and runners as shown in Figure 1. la.
Leaf numbers
increased subs'tantiallly with each increment of added Mn but only the
highest Mn levels produced significantly more runners.
The increase
Table 1.1.
Substrate
Manganese
(ppm)
Percentage of leaves showing deficiency or toxicity
symptoms.
0
Percentage Leaves Affected
Weeks After the Appearance of First Symptom
1
2
3
4
5
6
Toxicity Symptoms
125.0
5.4
18.8
47.5
81.4
100.0
62. 5
2. 5
5.6
21.0
37.8
69.0
92.5
100. 0
25. 0
0. 5
1.9
3. 7
9.5
13.0
12.4
11. 0
4.2
5.2
5.5
Deficiency Symptoms
0. 0
0.5
1.2
2.0
3.1
Figure 1.1a.
Effect of manganese on the number of runners and
leaves in strawberry plants. Vertical lines
indicate L. S. D. at . 05 level.
Figure 1.1b.
Effect of manganese on leaf area and dry weight of
strawberry plants. Vertical lines indicate L. S. D.
at . 05 level.
oo
<►
o>
TOTAL LEAF AREA Cm2xlOO
ro
LEAF AREA (Cm2)
PLANT WEIGHT (gm)
ro
OJ
01
ro
^
oo
o
NUMBER OF LEAVES
NUMBER OF RUNNERS
01
11
in the number of leaves and runners seems to be a direct response to
the increase in the level of substrate Mn.
The data on size of leaf, leaf area per plant and total dry weight
per plant are shown in Figure 1.1b.
Aside from a slight but non-
significant increase at 0. 5 ppm Mn, leaf size decreased progressively
with increase in substrate Mn, signficance being attained only at the
two highest levels.
Total leaf area per plant was greatest at 2. 5 ppm
Mn and decreased with either lower or higher Mn.
Changeis in total dry
weight per plant were similar to those for total leaf area.
The most
vigorous plants were produced at 2. 5 ppm Mn.
Concentration and distribution of tissue manganese.
The man-
ganese content of all the fractions increased with each increase in substrate Mn (Table 1. 2).
The old leaves treated with 1 2. 5 and 25. 0 ppm
Mn accumulated 2 to Zj times more manganese than the young leaves,
while in the remaining treatments they accumulated only 1. 5 times
more Mn.
The concentration of Mn in the petioles was lowest of all the
fractions sampled.
The 1 2. 5 and 25. 0 ppm treatments produced signif-
icantly higher petiole Mn than did 0. 0, 0. 5 and 2. 5 ppm Mn.
However,
the Mn content of petioles at the highest substrate level was only 3
times the control; whereas, in leaves the range was 10 (young leaves)
to 1 6 (old leaves) times the control.
12
Table 1. 2.
Substrate
Manganese
(ppm)
Effect of manganese on tissue manganese of strawberry
plants.
Manganese Content of Tissue
(ppm)
Old Leaves
Young Leaves
Petiole
Roots
0. 0
283. 0
e
203. 4
e
206. 5 c
368. 0
d
0.5
641.5
d
450.3
d
220.4 c
418.9
d
2.5
955.7
c
617.4
c
284.0 c
590.7
c
12.5
2645.6
b
1252.8
b
401.9 b
775.4
b
25.0
9691.1
a
4308.0
a
628.1 a
1825.7
a
Each figure is the mean of four replications.
2
Means followed by the same letter in each column are not significantly
different at the 0. 05 level of probability.
13
In 2. 5, 1 2. 5 and 25. 0 ppm substrate manganese, the roots contained significantly more Mn than the 0. 0 and 0. 5 ppm Mn treatments.
The roots of the minus Mn treatment contained 368. 0 ppm Mn as compared to 418. 9 ppm for the control.
Roots of plants treated with 2. 5,
12. 5 and 25. 0 ppm Mn contained 1.4, 1.9 and 4. 4 times the Mn as in
the control.
Concentration and distribution of tissue iron.
Decreasing the
substrate Mn from 0. 5 to 0. 0 resulted in an increase in the iron content of leaves and roots and a decrease in the iron content of petioles
(Table 1. 3).
Also, an increase in substrate Mn to 2. 5 ppm resulted in
an increase in the iron content of leaves (young and old) and petioles
but a small decrease in the iron content of the roots.
Any further
increase in substrate levels of Mn resulted in an appreciable decrease
in iron content of all the fractions sampled.
The highest concentration of iron was found in roots, followed by
old leaves, young leaves and petioles, except for the 25. 0 ppm Mn
treatment where young leaves contained more iron than the old leaves.
Iron manganese ratio and distribution.
A strong inverse relation-
ship was noted between the tissue iron/manganese ratio and the substrate Mn level (Table 1.4).
The highest iron/.manganese ratio always
occurred in the root fraction regardless of the substrate Mn level.
Next in order was young leaves, followed by old leaves and the petioles.
14
Table 1. 3.
Substrate
Manganese
(ppm)
Effect of manganese on tissue iron of strawberry plants.
Old Leaves
0.0
1301.2
0.5
651.9
2.5
I
a
2
Iron Content of Tissue
(ppm)
Young Lea/ves
Petiole
Roots
821.0
a
137. 4 ab
3958.7 a
b
582.7
b
163.7 a
3894.6 a
702.5
b
602.4
b
177. 2 a
3863.2 a
12.5
481.0
c
375.1
c
144. 2 ab
3654.9 ab
25.0
201.4
d
243.7
d
109.0 b
3486.0 b
Each figure is the mean of four replications.
2
Means followed by the same letter in each column are not significantly
different at the 0. 05 level of probability.
15
Table 1.4.
Substrate
Manganese
(ppm)
Effect of manganese on the iron/manganese ratio of
strawberry plants.
Fe/Mn Ratio
Nutrient
Solution
Old
Leaves
Plant Fractions
Young
Petiole
Leaves
Roots
0.0
200.00
4.60
4.04
0.67
10.76
0. 5
2.00
1. 02
1. 29
0. 74
9. 30
2.5
0.40
0.73
0.98
0.62
6.54
12. 5
0. 08
0. 18
0. 30
0. 36
4. 71
25. 0
0. 04
0. 02
0. 06
0. 17
1. 91
Each figure is the mean of four replications.
Figure 1. 2.
Effect of manganese on the magnesium content in strawberry plants. Vertical lines indicate L. S. D. at . 05 level.
Figure 1. 3.
Effect of manganese on the phosphorous content in strawberry plants. Vertical lines indicate L, S. D. at . 05 level.
17
oOLD LEAVES ^YOUNG LEAVES AROOTS»PETIOLES
.2 5^^
0" 0.5
2.5
LOG Mn
12.5
25.0
12.5
25.0
(PPM)
>8
0" 0.5
2.5
LOG
Mn (PPM)
18
and Mg were similar in that they did not show wide variations among
the different fractions.
Discussion
The work described above was designed to find the critical substrate and tissue manganese levels associated with the deficiency and
toxicity symptoms in strawberry plants.
Further studies were made
to determine the effect of tissue manganese on growth and mineral
composition of various fractions.
The results showed that the minus
and high substrate Mn levels restricted growth and altered the mineral
composition.
The visual symptoms of deficiency in strawberry plants are
difficult to establish.
The first symptom of deficiency was noticed
after 12 weeks of minus manganese treatment while, after 18 weeks,
only 5. 5% leaves showed deficiency symptoms, which developed only
on the runner leaflets.
Iwakiri and Scott (14) noticed no Mn deficiency
symptoms in 'Temple' strawberry. . However, Johanson and Walker
(18) reported the appearance of the first Mn deficiency symptom after
88 days on runner leaflets and within another two weeks in the intermediate leaves of parent plants.
Dennis and O'Brien (5) observed
small reddened and crinkled leaves after four months in sand culture;
whereas, Lott (22) observed, similarly, yellowish-green coloration of
leaves -with veins remaining darker green but with no purple stippling.
19
This effect, however, was more pronounced in old leaves (3).
Lohnis (20) found that strawberry is resistant not only to Mn
deficiency but also to Mn toxicity.
The resistance to Mn toxicity
seemed to be correlated with lower Mn uptake (3).
However, when the
strawberry plants were grown -with a high substrate Mn, the plants
developed the toxicity symptoms which closely paralleled the characteristic chlorosis, necrotic spots and cupping of leaves observed in
other crops.
Plants showed the typical toxicity symptoms when Mn in
young and old leaves reached about 4000 and 9000 ppm, respectively.
Thus, the results indicate that the 'Northwest' strawberry is rather
tolerant of high concentrations of substrate Mn not because of low
uptake but because of its ability to endure large amounts of accumulated
Mn(17, 20).
Increase in substrate Mn from 0. 0 to 2. 5 ppm resulted in greatly
increased growth as measured by the dry weight of plants, whereas
decreases in growth were experienced with more than 2. 5 ppm.
dry weight of the minus Mn plants was 71. 1% of the control.
The
The
decrease in growth capacity was in part due to leaf chlorosis and to
reduction in photosynthetic area (9).
The reduction in growth of plants
grown with 1 2. 5 and 25. 0 ppm Mn was correlated to excess Mn.
Medappa and Dana (23) stated that dry weight of cranberry shoots was
not affected by Mn concentration in the external solution; whereas,
20
Hiatt and Ragiand (13) and others (1, 32) observed that the dry weight
was inversely related to Mn concentration in substrate or tissue.
Numerous reports show that a direct relationship exists between
substrate and tissue manganese.
Results of this study were consistent
with this relation (2, 16, 24, 26, 34).
Manganese concentrations in
different tissues were found to vary over a wide range.
contained more Mn than roots and petioles.
The leaves
From the comparison of
the tissue manganese between the 0. 0 and 0. 5 ppm Mn plants, this is
evident that the difference in the amount of manganese required for
good growth and that which results in deficient plants is small.
addition,
In
these results indicate that as the substrate manganese be-
came limiting,
there was an increase in translocation of available
manganese to the plant parts which were most active physiologically.
This is supported by the observation that in the 0. 0 ppm Mn plants,
there was not much difference in the levels of Mn in young and old
leaves as well as in petioles and roots .
Application of Mn to foliage
has been shown to result in redistribution, mainly to the developing
regions (30, 34).
Jackson (5) concluded that the transfer of Mn from
old leaves could occur but the rate of redistribution was very sluggish.
The excess of Mn has been reported to hinder the translocation
of iron by causing the iron in the plants to be converted to an insoluble
form (31).
It was suggested that, with a low level of Mn in the plant,
iron is in the ferrous form and may cause iron toxicity, -while at a high
21
Mn level, iron may be in the ferric form and Fe deficiency may result
(26).
Hiatt and Ragland (13) found that substrate Mn did not change the
concentration of iron in tobacco tissue.
In this study, the inverse
relationship between tissue Mn and iron, as reported by Somers and
Shive (31), was found in all plant fractions except in leaves which contained little more iron at 2. 5 ppm Mn than in the control (0. Oppm).
Rieckels and Lingle (28) found that increasing the Mn in the nutrient
solution increased Fe uptake in tomato plants at low levels of Mn but
decreased Fe uptake at high levels.
Kirsch et al. (19) suggested that
Mn does not interfere with iron utilization in the leaf but produces
chlorosis by depressing the absorption of iron by the roots.
An inverse relationship was found between the substrate Mn level
and the iron/manganese ratio in the plant fractions studied.
The ratio
varied in the leaves from a value of more than 4. 6 with 0. 0 ppm substrate manganese to less than 0. 02 with 25. 0 ppm substrate manganese.
The ratio in the roots was of a smaller order than that reported by
Vlamis and Williams (3 3) for rice roots.
However, the ratio only
varied by a factor of 230 and 67 in the young and old leaves of the
strawberry compared to as much as 1000 times in rice leaves.
Somers and Shive (31) found that for good growth (without any
deficiency symptoms) in soybean, the Fe/Mn ratio could only fluctuate
slightly from 2. 0 in the nutrient solution.
Iron/Mn ratios as high as
200 (minus manganese) and as low as 0. 04 (25. 0 ppm Mn) were imposed
22
in this experiment, but the detrimental effects (symptoms of deficiency
and toxicity) were not observed in leaves when the Fe/Mn ratio ranged
from 0. 08 to 2. 0 in the nutrient solution.
This indicates that in
'Northwest' strawberry the substrate balance between Mn and Fe in the
nutrient solution is not as critical as for the soybean.
Similar to these results, Johanson (17) reported that Mn deficiency
reduced the phosphorous level in the leaf blade of strawberry by 50
percent and magnesium by 35 percent.
According to Lohnis (20, 21),
there is a clear uptake antagonism between Mn and Mg, so that the
addition of Mg prevents the appearance of injury symptoms in a .medium
with an excess of manganese.
In the results presented above, highest
levels of P and Mg were observed when the Mn in leaves ranged from
617. 4 (young leaves) to 995. 7 (old leaves) ppm.
Also, the maximum
plant growth, as measured by the plant dry weight, was observed at
these levels of Mn in the leaves.
23
LITERATURE CITED
1.
Berger, K. C. , and G. C. Gerloff. 1947. Manganese toxicity of
potato in relation to strong soil acidity. Soil Sci. Soc. Amer.
Proc. 12:310-314.
2.
Bolle-Jones, E. W. 1955. The effect of varied nutrient levels
on the concentration and distribution of manganese with potato
plant. Plant Soil 6;45-60.
3.
Boyce, B. R. , and D. L. Matlock. 1967. Strawberry nutrition,
p. 518-598. In: N. F. Childers [ed. ] Nutrition of Fruit Crops.
Rutgers-the State University, New Brunswick, N. J.
4.
Darrow, G. M. , and G. F. Waldo. 1932. Effect of fertilizers
on plant growth, yield and decay of strawberries in North
Carolina. Proc. Amer. Soc. Hort. Sci. 29:318-324.
5.
Dennis, R. W. G. , and D. G. O'Brien. 1937. Boron in agriculture. West Scot. Agr. College, Plant Husbandry Dept. Res.
Bui #5, p. 98.
6.
Fiske, C. H. , and Y. Subbarow. 1925. The co.lorimetric
determination of phosphorous. Jour. Biol. Chem. 66:375-400.
7.
Fried, M. , and M. Peech. 1946. The comparative effects of
lime and gypsum upon plants grown in acid soils. J. Amer. Soc.
Agron. 38:614-623.
8.
Gerloff, G. C. , D. G. Moore, and J. T. Curtis. 1966. Selective absorption of mineral elements by native plants of Wisconsin. Plant and Soil 25:39 3-405.
9.
Gerretsen, F. C. 1949. Manganese in relation to photosynthesis.
Plant and Soil 1:346-35 8.
10.
Hewitt, E. J. 195 2. A biological approach to the problems of
soil acidity. Trans. Congr. Intern. Soc. Soil Sci. (Dublin)
1:107-117.
11.
Hewitt, E. J. 1966. Sand and water culture methods used in the
study of plant nutrition. Technical Communication No. 22.
Commonwealth Bureau of Horticulture and Plantation Crops, East
Mailing, Maidstone, Kent. p. 547.
24
12.
Hewitt, E. J. , and E. W. Jones. 1947. The production of
molybdenum deficiency in plants in sand culture with special
reference to tomato and brassica crops. J. Pomol. 23:254-262.
13.
Hiatt, A. J. , and J. L. Ragland. 1963.
burley tobacco. Agron. J. 55;47-49.
14.
Iwakiri, B. , and L. E. Scott. 1951. Mineral deficiency symptoms of the Temple strawberry grown in sand culture. Proc.
Amer. Soc. Hort. Sci. 57:45-52.
15.
Jackson, W. A. 1967. Physiological effects of soil acidity.
p. 43-124. In: R. W. Pearson and F. Adams [ed. ] Soil acidity
and liming. Monograph 12. Amer. Soc. Agro. Madison,
Wisconsin.
16.
Jackobson, H. G. M. , and T. R. Swanback. 1932. Manganese
content of certain Connecticut soils and its relation to growth of
tobacco. J. Amer. Soc. Agron. 24:237-245.
17.
Johanson, F. D. 1963. Nutrient deficiency symptoms, p. 7580. In: C. R. Smith and N. F. Childers [ed. ] The Strawberry.
Rutgers-the State University, New Brunswick, N. J.
18.
Johanson, F. D. , and R. B. Walker. 1963. Nutrient deficiency
and foliar composition of strawberries. Proc. Amer. Soc. Hort.
Sci. 83:431-439.
19.
Kirsch, R. K. , M. E, Harward.and R. G. Peterson. I960.
Interrelationships among iron, manganese and molybdenum in the
growth and nutrition of tomato grown in culture solution. Plant
Soil 12:259-275.
20.
Lohnis, M. P.
1950. Symptoms of Mn toxicity in cultivated
crops. T. N. O. Nieuws 5:150-155.
21.
Manganese toxicity of
' Lohnis, M. P. I960. Effect of magnesium and calcium supply
on the uptake of manganese by various crop plants. Plant Soil
12:339-376.
22.
Lott, W. L. 1946. Strawberries need small amounts of Mn and
Zn. N. Car. Agr. Exp. Sta. Ann. Rept. 69, p. 66.
23.
Medappa, K. C. , and M. N. Dana. 1970. Tolerance of cranberry plants to manganese, iron and aluminum. J. Amer. Soc.
Hort. Sci. 95:107-110.
25
?4.
Morris,, H. D. 1969. The soluble manganese content of acid
soils and the relation to growth and manganese content of sweet
clover and lespedeza. Soil Sci. Soc. Amer. Proc. 13:362-371.
25.
Morris, H. D. , and W. H. Pierre. 1949. Minimum concentrations of manganese necessary for injury to various legumes in
culture solutions. Agron. J. 41:107-113.
26.
Mulder, E. G. , and F. C. Gerretsen. 195 2. Soil manganese in
relation to plant growth, p. 221-227. In: A. G. Norman [ed. ]
Advances in Agronomy Vol. IV. Academic Press Inc. N. Y.
27.
Piper, C. S. 1941. Marsh spot of peas.
disease. J. Agric. Sci. 31:448-452.
28.
Riekels, J. W. , and J. C. Lingle. 1966. Iron uptake and translocation by tomato plants as influenced by root temperature and
manganese nutrition. Plant Physiol. 41:1095-1101.
29.
Roberts, A. N. , and A. L. Kenworthy. 1956. Growth and
composition of the strawberry plant in relation to root temperature and intensity of nutrition. Proc. Amer. Soc. Hort. Sci.
68:157-168.
30.
Romney, E. M. , and S. J. Toth. 1954. Plant and soil studies
with radioactive manganese. Soil Sci. 77:107-117.
31.
Somers, L I., and J. W. Shive. 1942. The iron-manganese
relation in plant metabolism. Plant Physiol. 17:5 82-602.
32.
Sutton, C. G. , and E. G. Hallsworth. 1958. Studies on the
nutrition of forage legumes. I. Toxicity of low pH and high
manganese supply to lucerne as affected by climatic factors and
calcium supply. Plant Soil 9:305-317.
33.
Vlamis, J. , and D. E. Williams. 1964. Iron and manganese
relations in rice and barley. Plant and Soils 20:221-231.
34.
Vose, P. B. 1963. The translocation and redistribution of
manganese in Avena. J. Exp. Bot. 14:448-45 7.
35.
"Wallace, A. 1958. Effect of chelated iron and manganese on
the manganese content of soybeans grown in solutions culture.
Agro. J. 50:285-286.
A manganese deficiency
26
INFLUENCE OF MANGANESE NUTRITION ON CARBOHYDRATE
AND NITROGEN METABOLISM OF STRAWBERRY LEAVES
The effects of manganese on the metabolism of plants are numerous.
The metal serves as an activator or a co-factor for a wide
variety of enzyme systems involved in carbohydrate and nitrogen
metabolism (13, 25).
Although the effects of Mn deficiency have been
studied in a number of crops, information is lacking on the effects of
Mn, ranging from near deficient to near toxic levels on the nitrogen
and carbohydrate metabolism of strawberry plants.
Manganese plays a primary role in photosynthesis and is involved
mainly with oxygen evolution in photosynthesis (9, 18, 37).
The inhibi-
tion of photosynthesis in Mn deficient plants has been observed to be
independent of any influence' oh chlorophyll level, and occurs in the
early stage of manganese deficiency prior to any real chlorosis (27, 28).
Manganese deficient plants are characterized by low levels of
soluble carbohydrates (37).
Reduction in levels of starch and sugar
were found in Mn deficient leaves of oats and tomatoes (10, 32, 37);
also Hewitt (12) observed that total carbohydrate in normal chlorella
cells was higher than in Mn deficient cells.
In the leaves, Mn pro-
motes the development of an effective carbohydrate apparatus only
during the early formative period; and a deficiency at that time cannot
be made good by the subsequent addition of more Mn (27, 37).
27
Mn is also believed to be involved in the chain of reactions beginning with the reduction of NO
plants.
and ending in protein synthesis in higher
Burstrom (4) concluded that Mn deficiency resulted in nitrate
accumulation for lack of a carbon skeleton due to a decrease in carbohydrate supplies following impaired photosynthesis.
Nance (23) sug-
gested that Mn was involved in reduction of nitrite rather than nitrate.
Later it was shown that Mn is involved as a constituent of the flavoprotein of hydroxy.la.mine reductase (17, 24).
Steward and Margolis
(34) concluded that Mn is not implicated in reduction of NO
immediate conversion to organic compounds.
or its
Mn does promote a rapid
build-up of soluble N compounds through its effects on the supply of N
acceptor compounds via the citric acid cycle.
In Mn deficient plants,,the assimilation of both NO
disturbed.
Because of Mn deficiency,NO
and NH
is
assimilation in algae was
inhibited (30); whereas it accumulated to abnormally high levels in
cauliflower leaves (14).
Accumulation of soluble N compounds was
also observed in plants deficient in Mn (16, 27, 33).
Cain and Holly
(5) found, in lime-induced chlorotic leaves of blueberries, a higher
accumulation of soluble nitrogenous compounds, as compared to green
leaves.
A number of workers have reported the accumulation of amino
acids in Mn deficient plants.
Bar-Akiva (2) found that Mn deficient
citrus leaves accumulated more free arginine, asparagine, alanine
28
and serine.
Accumulations of aspartic acid and proline and a reduction
in "y-amino butyric acid were also observed in citrus leaves (35).
Gilfillan _et al. (11) found that Mn deficient chlorotic leaves of macadamia
contained significantly higher amounts of homoserine; whereas
Labanauskas e_t al. (19) reported a significantly higher accumulation of
free aspartic acid, threonine, giutamic acid, proline, alanine, valine,
isoleucine, leucine, tyrosine and phenyla.lanine.
Scheffer et al. (31), working with oats and wheat, observed
phenyla.lanine and histidine only in normal plants; whereas alanine was
totally absent in Mn deficient plants, and proline was found only in
plants showing toxicity of Mn.
Lljin (16) studied N metabolism of
various fruit plants affected by lime-induced chlorosis and found that
3 to 7 times more amino acids accumulated in the chlorotic leaves than
in green leaves.
However, a decrease in total free amino acids was
observed by Lewis e_t al. (20) in pear leaves in conditions of Mn deficiency and excess.
The effects of Mn deficiency or excess on the carbohydrate and N
metabolism, of plant's, in general, and strawberry plants in particular,
are still obscure.
The purpose of this study was to evaluate the effects
of Mn deficiency and excess in strawberry leaves on the carbohydrate
and N fractions of plants grown in water culture.
29
Materials and Methods
Water culture studies.
Strawberry plants as described in a pre-
vious experiment were transplanted in ■water culture solution after
growing 15 days in distilled water (29).
Five young plants were trans-
planted to each 11 liter colored plastic container.
Each container was
fitted with a wooden lid having five holes drilled in a circular pattern
and plants were placed through the holes and supported by packed,
sterile glass wool.
through an air stone.
immediately.
The solutions were aerated with filtered air passed
The manganese treatments were imposed
The substrate levels of manganese were 0. 0, 0. 5, 2. 5,
12. 5 and 25. 0 pprn with three replications in each treatment.
The nutrient solution, made from purified salts (29), was completely changed after a week and iron was supplied as iron tartrate on
the 3rd and 5th day of change of solutions (15).
trusses were removed periodically.
Runners and flower
Plants were harvested after 84
days, immediately fractionated into young and old leaves, and
lyophilized.
Analytical methods.
with 100 ml of 80% ethanol.
Samples of 0. 5 to 1. 0 gram were extracted
The alcohol fraction was made to 25 0 ml
volume and stored under refrigeration for further analysis.
The con-
centrations of nitrate N, soluble reduced N, total soluble N, and soluble carbohydrates 'were determined in the alcohol fraction.
30
Total hot-water-soluble carbohydrate (starch) was determined
from the oven-dried residue of 80% ethanol extract.
The mixture of
25-50 mg of dried residue, plus 2 ml 80% ethanol and 25 ml of water,
was placed on a steam bath for 30 minutes.
This was transferred
quantitatively to a centrifuge tube and centrifuged.
was brought to 100 ml by addition of water.
The supernatant
Carbohydrates were deter-
mined colorimetrically by the phenol sulfuric acid method (7, 22).
Total N was determined by Kjeldahl, the reduced soluble N by
the micro Kjeldahl and NO
(6).
- N by the phenol disulfonic acid method
The total soluble N was obtained by adding the nitrate and soluble
reduced N.
The total insoluble N was the difference between total N and
soluble N.
For the determination of free amino acids, the leaf material was
extracted according to the method of Thompson et aU (38).
The individ-
ual amino acids in each sample were then separated and quantitatively
determined by the Technicon Amino Acid Autoanalyzer using two
buffers as outlined by Efron (8).
Results and Discussion
Carbohydrate content.
Both the substrate Mn concentration and
the type of leaf affected the 80% ethanol-soluble (sugar) and hot-watersoluble (starch) carbohydrate fractions of strawberry leaves harvested
after 84 days of Mn treatment (Figure 2. 1 ).
31
The sugar content of young as well as old leaves increased with
increases in substrate Mn but only up to 1 2. 5 ppm Mn treatment.
The
lowest sugar content was observed in Mn deficient leaves (0. 0 ppm).
However, at low (0. 0 and 0. 5 ppm) and high (25. 0 ppm) substrate Mn,
the sugar content was higher in old leaves than in young leaves.
In young leaves, the starch content increased in treatments up to
2. 5 ppm Mn, but no definite trend was observed in old leaves.
Young
and old leaves in all Mn treatments contained substantially more
starch than the minus Mn treatment.
The low levels of sugars and starch observed in the Mn deficient
leaves agree with the findings of other workers.
Gerretsen (10)
observed low levels of both sugar and starch in Mn deficient leaves of
oats, while Miller (21) observed only low levels of soluble carbohydrate under similar conditions.
The reduction in the content of carbo-
hydrates in the Mn deficient leaves may have been due to low photosynthesis.
A decrease in the amount of reducing sugar in potato and tomato
on fertilization with Mn was observed by Abutalybov et al. .(1). The
levels of Mn in these plants prior to treatment were not indicated.
It
may be that the treatment caused an excess of Mn and a decrease in
carbohydrates.
leaves, also.
This effect of excess Mn is apparent in the strawberry
Figure 2. 1.
Carbohydrate content in relation to manganese nutrition.
Figure 2. 2.
Nitrogen content in relation to manganese nutrition.
32
IZ
UJ
-1
I
28
o
UJ
UJ
24
V)
o
o
dzo
16
- -^
^ YOUNG LEAVES
o OLD LEAVES
SUGAR
STARCH
r
0.5
2.5
12.5
LOG.
25.0
Mn (PPM)
3.3 r-
2.5
LOG.
Mn
12.5
(PPM)
25.0
33
Total nitrogen.
The N content was also affected by the type of
leaf and substrate Mn levels (Figure 2. 2).
Increase in the substrate
concentration of Mn was associated with increase in the total N content
of young as well as old leaves.
However, the young leaves contained
more total N than the old leaves at all levels of substrate Mn.
The
increases in total N were considerably greater when Mn was increased
from 0. 0 to 2. 5 ppm than from 2. 5 to 25. 0 ppm.
Voth ^t al. (39) also found a positive correlation between the total
N and Mn in leaf tissue of a number of strawberry varieties.
They
attributed increases in Mn at high N application either to its release"
from the exchange complex by the NH
ions produced by nitrification.
ions of the fertilizer or by H
Later, they found the relationship
true irrespective of soil pH (40).
In the majority of treatments, the
highest carbohydrate contents were at intermediate levels of leaf Mn
and N, similar to the observations of Blatt (3).
Insoluble and soluble nitrogen.
Insoluble N increased with
increases in the amounts of Mn in the substrate in young as well as old
leaves (Figure 2. 3).
In both types of leaves, the insoluble N was
substantially lower in Mn deficient plants (0. 0 ppm).
On the other
hand, high levels of soluble N were observed both at low and high levels
of substrate Mn.
The lowest level of soluble nitrogen was at 2. 5 ppm.
In all treatments, young leaves contained higher soluble and insoluble
N than old leaves; the differences in the amount of insoluble N bet-ween
Figure 2. 3.
Soluble and insoluble nitrogen in relation to manganese
nutrition.
Figure 2. 4.
Soluble reduced and nitrate nitrogen in relation to manganese nutrition.
34
i56
2.5
LOG Mn(PPM)
O
-
\
x
[\\
ro
O
a.
/
v
££
^^^^
x
tf
"
0.5
s
o
s
x
s
'
\
37
cr
en
or
-- '
S
-'-$■''
1
y \
'
"—^~——^
-* "
' ^~~~~~~~~~-*-^z-'
^ "
^ "*
^^^^*v\
^
"
^^^*^^*»^
-"'
^-^
" X6^^
-*4S
'
V
\
\\ \\^
\V\
\\ \
13
S.R.N
\
A
\
I
A 40O
"
N
\\
i
- NO3-N
-
O
O
25.0
YOUNG LEAVES
OLD LEAVES
^
17 K
12.5
1
H34
^^^
1
2.5
12.5
LOG Mn (PPM)
31
25.0
a.
35
the young and old leaves were smaller at 0. 0 ppm Mn.
These results indicate that Mn deficiency resulted in a decrease
in protein (insoluble N fraction) synthesis.
Steward ej: aX.
Nason et al.(25) and
(32, 33) proposed that protein synthesis was affected
adversely in the Mn deficient leaves of cauliflower and tomato.
Higher
concentrations of insoluble N in young leaves of cotton plants grown in
minus Mn nutrient solution were found by Taylor (36) who proposed
that the Mn •was not involved in protein synthesis.
Accumulations of
soluble N were also observed in tomato plants by Steward and Margolis
(34) under conditions of Mn deficiency.
Soluble reduced and nitrate nitrogen.
High levels of soluble
reduced N 'were found in leaf samples taken from plants treated -with low
as well as high levels of Mn (Figure 2. 4).
In young leaves, the lowest
levels of soluble reduced N were found at 0. 5 ppm and in old leaves at
2. 5 ppm.
The accumulation of soluble reduced N in leaves with Mn
deficiency has been reported in cauliflower by Hewitt et aA.
(14) and
in tomato plants by Steward and Margolis (34).
The levels of nitrate in the leaves were negatively correlated
with substrate Mn and were higher in young than old leaves.
et al. (14), working with caulifower,
required for NO
this conclusion.
Hewitt
concluded that Mn was not
reduction, and Steward and Durzan (33) supported
Jones et al. (17) showed that Mn was involved as a
co-factor with hydroxy.lamine reductase.
The high level of NO
36
observed at low Mn indicates that Mn is not involved in NO
but in its utilization.
A high turnover rate at high Mn levels resulted
in the low level of NO
Amino acids.
absorption
observed.
The concentrations of free amino acids in young
strawberry leaves were greatly increased by Mn deficiency or excess
as shown in Table 2. 1.
Mn deficient leaves contained more glycine,
valine, aspartic acid, lysine, arginine, serine, phenylalanine, asparagine and glutamine than leaves with more Mn.
The concentrations of
alanine, threonine, glutamic acid, proline and Y-amino butyric acid
increased slightly; whereas leucine, isoleucine, histidine, methionine
and tyrosine decreased a little.
Mn treatments above 0. 5 ppm produced variable influence on the
content of free amino acids in strawberry leaves.
There was a decrease
in the concentration of glycine, leucine and arginine when the substrate
Mn was increased to Z. 5 ppm. All other amino acids increased a
slight amount.
Further increase in substrate Mn resulted in an increase
of all amino acids and the highest amounts were found in young leaves
taken from the 25. 0 ppm Mn treatment.
Table 2. 2 gives the results of the analysis of free amino acids in
old leaves of strawberry.
As in young leaves, the concentrations of
amino acids in old leaves also were greatly increased by Mn deficiency
and excess.
Mn deficiency in old leaves resulted in an increase of all
37
Table 2. 1.
Influence of substrate manganese on amino acids in young
strav/berry leaves.
Amino Acid
0.0
Substrate Manganese (ppm)
0.5
2.5
12.5
25.0
jiM/g
glycine
2. 62
0. 79
0. 07
0. 69
0.97
alanine
10. 08
8. 28
6. 10
6. 01
13.41
valine
2. 15
0. 62
0. 94
2. 16
1. 84
leucine
1. 04
1. 27
1. 71
1. 11
1.92
isoleucine
0. 91
0. 96
0. 97
1. 60
1.88
threonine
3. 65
3. 31
3. 85
4. 07
5. 78
aspartic acid
7. 96
0. 51
0. 63
1. 05
7.90
glutamic acid
8. 79
5. 53
6. 13
6. 05
8. 19
lysine
0. 65
0. 06
0. 12
0. 45
0. 76
arginine
4. 69
1. 77
1. 60
4. 78
9.51
histidine
0. 09
0. 65
0. 83
1. 41
1.53
methonine
0. 10
0. 14
0. 14
0. 15
0. 21
proline
2. 04
1. 83
2. 23
2. 36
2.57
tyros ine
0. 14
0. 25
0. 25
0. 38
0. 39
20. 81
14. 28
16. 23
16. 84
18. 04
11. 45
3. 86
6. 32
10. 22
8.52
phenylalanine
0. 39
0. 05
0. 27
0. 32
0. 39
asparagine
8. 67
5. 23
4. 12
4. 92
5. 78
glutamine
5. 38
3. 17
3. 58
4. 69
7. 84
91. 66
52. 56
55. 46
69. 24
97.43
Y- amino
butyric acid
serine
TOTAL
Data given are average of three samples.
38
amino acids except leucine, arginine, histidine and tyrosine, which
decreased slightly.
The level of alanine,
lysine, histidine, and tyrosine decreased
when the substrate Mn concentration was increased from 0. 5 to Z. 5 ppm.
Further increase in substrate Mn resulted in an increase of all free
amino acids with very high concentrations at 25. 0 ppm Mn.
Mn deficiency in strawberry leaves produced changes in the concentrations of free amino acids, which are partially in agreement with
the results reported by Possingham (26), who found more aspartic acid,
glutamic acid, asparagine, arginine, -y-amino butyric acid, valine and
proline and less glutamine, histidine, lysine, serine, glycine, threonine,
alanine, phenylalanine and leucine in Mn deficient tomato leaves.
Hewitt et al. (14), Steward et al. (32, 33), and Labanauskas et al. (19)
also reported similar results in experiments with other crops.
Excess Mn in the plant tissue also produced an increase in all
the amino acids.
In contrast, Lewis et al. (20) reported increases
only in glutamic acid, serine, and alanine and a decrease in arginine
and asparagine in pear leaves grown with excess Mn.
Scheffer et al.
(31) also observed an accumulation of methionine, threonine, glutamic
acid and proline and reduction of the remaining amino acids in leaves
of wheat plants grown with 100 ppm Mn in the substrate.
In strawberry leaves (Table 2. 1 and 2. 2) and also in other crops
(16, 19),the total amino acid content increased tremendously under
39
Table 2. 2.
Influence of substrate manganese on amino acids in old
strawberry leaves.
Amino Acid
0.0
Substrate Manganese (ppm)
0.5
2.5
12.5
25.0
M-M/g
glycine
3.40.
0. 97
0. 69
1. 25
0. 97
alanine
19. 14
12. 32
10. 24
13. 01
19. 21
valine
3.98
1.61
1. 81
1. 84
3. 16
leucine
0.49
1. 28
1. 35
1.46
1.61
isoleucine
2. 16
1. 08
1.86
1. 48
1.97
threonine
7.94
4. 04
4. 38
4. 16
5. 01
aspartic acid
8. 14
3. 55
4. 05
4. 81
4.96
glutamic acid
3.64
2. 07
3. 15
2. 05
4. 35
lysine
1. 75
0. 86
0. 5 3
0. 89
0.94
arginine
0. 22
0. 73
0. 80
0.96
1. 26
histidine
0.0 3
0. 05
0. 01
0.04
0.07
methonine
0.46
0. 12
0. 13
0. 15
0. 17
proline
2. 68
1. 27
2.42
2.97
5.98
tyrosine
0. 27
0.41
0. 23
0.45
0. 71
21. 19
20. 76
20.99
22. 62
24. 04
11.03
9. 72
11. 16
11. 50
12. 05
phenylalanine
0. 51
0, 37
0.48
0. 56
0.69
asparagine
7.91
6.48
7. 64
9. 70
10. 77
g.lutamine
4. 83
3. 81
4. 57
5. 15
5. 89
82.19
85. 05
103. 81
•y amino
butyric acid
serine
TOTAL
99. 77
71. 5
1 Data given are average of three samples.
40
conditions of deficiency and toxicity.
The work of Lewis ej: al. (20)
with pear leaves, on the other hand, found a decrease in the total
amino acid content with an increase and a decrease in the Mn nutrition.
Hewitt (12), reviewing the effects of micronutrients on the metabolism of free amino acids in plants, observed that a deficiency or
toxicity of a plant nutrient could increase or decrease the contents of
free amino acids in plants.
Thus, the concentration of an individual
amino acid or all amino acids cannot be used to distinguish between
the deficiency or toxicity of Mn in leaves.
41
LITERATURE CITED
1.
Abutalybov, M., I. Buniatov, and A. Mardanov. 1956. Importance of manganese in oxidation reduction processes in plant
organism. Uchen. Zap. Azerbaidzh. Univ. No. 9, 47-57 [inter.
Abst. Biol. Sci., 11:(2348), 1958].
2.
Bar-Akiva, A. 1964. Visible symptoms and chemical analysis
vs. biochemical indicators as a means of diagnosing iron and
manganese deficiencies in citrus plants, p. 9-24. In: C.
Bould, P. Prevot, and J. R. Magness [ ed. ] Plant Analysis and
Fertilizer Problems, IV.
3.
B.latt, C. R. 1967. Response of 'Acadia' strawberry to two
forms and three rates of nitrogen at two pH levels. Proc. Amer.
Soc. Hort. Sci. 92;346-35 3.
4.
Burstrom, H. 19 39. Uber die Schwermetallkatalyze der
Nitratassimil'ation.
Planta. 29;292-305.
5.
Cain, J. C. , and R. W. Holly. 1955. A comparison of chlorotic
and green blueberry leaf tissue with respect to free amino acid
and basic cation contents. Proc. Amer. Soc. Hort. Sci.
65:49-53.
6.
Chapman, H. D. , and P. F. Pratt. 1961. Methods of analysis
for soils, plants and waters. University of California, Riverside, California. p. 309.
7.
Dubois, M. , K. A. Gilles, J. K. Hamilton, P. A. Rebers, and
F. Smith. 1956. Colorimetric method for determination of
sugars and related substances. Anal. Chem. 28:350-356.
8.
Efron, M. L. 1965. Quantitative estimation of amino acids in
physiological fluids using a Technicon Amino Acid Analyzer.
' p. 637-642. In: L. T. Skegg [ ed. ] Automation in analytical
chemistry. Technicon symposium. Mediad Incorporated, New
York.
9.
Eyster, C. , T. E. Brown, H. A. Tanner, and S. L. Hood. 1958.
Manganese requirement with respect to growth, Hill reaction,
and photosynthesis. Plant Physiol. 33:235-241.
42
10.
Gerretsen, F. C. 1949. Manganese in relation to photosynthesis.
I. Carbon dioxide assimilation and the typical symptoms of manganese deficiency in oats. Plant and Soil ls346-358.
11.
Gilfillan, I. M. , and W. W. Jones. 1968. Effect of iron and
manganese deficiency on the chlorophyll, amino acid and organic
acid status of leaves of macadamia. Proc. Amer. Soc. Hort.
Sci. 93:210-214.
12.
Hewitt, B. R. 1959. Glucose assimilation in normal and manganese deficient chlorella cells. Physiol. Piantarum 12:45 2-455.
13.
Hewitt, E. J. 1963. The essential nutrient elements: the
requirements and interaction, p. 218-222. In: F. C. Steward
[ ed. ] Plant Physiology: a treatise. III. Inorganic Nutrition of
Plants. Academic Press, New York.
14.
Hewitt, E. J. , E. W. Jone, and A. H. Williams. 1949. Relation of molybdenum and manganese to the free amino acid content
of the cauliflower. Nature 163:681-682.
15.
Hoagland, D. R. , and D. J. Arnon. 195 0. The water culture
method for growing plants without soil. University of California
Circ. 347:29-31.
16.
Iljin, W. S. 1951. Metabolism of plants affected with limeinduced chlorosis. I. Nitrogen Metabolism. Plant and Soil
3:239-255.
17.
Jones, L. H. , W. B. Shepardson, and C. A. Peters. 1949.
The function of manganese in assimilation of nitrates. Plant
Physiol. 24:300-306.
18.
Kessler, E. 1955. On the role of manganese in the oxygen
evolving system of photosynthesis. Arch. Biochem. Biophys.
59:527-529.
19.
Labanauskas, C. K. , and M. F. Handy. 1970. The effects of
iron and manganese deficiencies on accumulation of nonprotein
and protein amino acids in macadamia leaves. Amer. Soc. Hort.
Sci. 95:218-223.
20.
Lewis, L. N. , N. E. Tolbert, and A. L. Kenworthy. 1963.
Influence of mineral nutrition on the amino acid composition of
Bartlett pear trees.
Proc. Amer. Soc. Hort. Sci.
83:185-192.
43
21.
Miller, L. P. 1933. Effect of manganese deficiency on the
growth and sugar content of plants. Amer. J. Bot. 20:621-631.
22.
Nalewaja, J. D, , and L. H. Smith. 1963. Standard procedure
for the quantitative determination of individual sugars and total
soluble carbohydrate materials in plant extracts. Argon. J.
55:523-525.
23.
Nance, J. F. 1948. Role of oxygen in nitrate assimilation by
wheat roots. Amer. J. Bot. 35:602-606.
24.
Nason, A. 195 6. Enzymatic steps in the assimilation of nitrate
and nitrite; in fungi and green plants, p. 109-136. In: W. D.
McElroy and H. B. Glass [ ed. ] Inorganic nitrogen metabolism.
Johns Hopkins Press, Baltimore, Maryland.
25.
Nason, A., and W. D. McElroy. 1963. Modes of action of the
essential mineral elements, p. 451-5 36. In: F. C. Steward
[ ed. ] Plant physiology: a treatise. III. Inorganic Nutrition of
Plants. Academic Press, New York.
26.
Possingham, J. V. 1956. The effect of mineral nutrition on the
content of free amino acids and amides in tomato plants. Austral.
J. Biol. Sci. 9:539-551.
27.
Prison, A. 1958. Manganese and its role in photosynthesis, p.
81-98. In: C. A. Lamb et al. [ ed. ] Trace elements. Academic
Press, New York.
28.
Ruck, H. C. , and B. D. Bolas. 1954. The effect of manganese
on the assimilation and respiration rate of isolated rooted leaves.
Ann. Bot. 18:267-297.
29.
Sabharwal, S. L. , R. Garren, and O. C. Compton. 1971.
Growth and mineral composition response of strawberry plants
to manganese nutrition. (In Preparation).
30.
Sadana, J. C. , and W. D. McElroy. 1957. Nitrate reductase
from Achromobacter fischeri. Purification and properties:
function of flavines and cytochrome. Arch. Biochem. Biophys.
67:16-33.
44
31.
Scheffer, F., E. Przemeck, and V. K. Saolapurkar. 1970.
Mineral nutrition and metabolic processes in young plants. II.
Effects of manganese nutrition on the levels of free amino acids
in oats and wheat. J. Ind. Soc. Soil Sci. 16; 135-141.
32.
Steward, F. C. , F. Crane, K. Millar, R. M. Zacharius, R.
Rabson, and D. Margolis. 1959. Nutritional and environmental
effects on the nitrogen metabolism of plants. In; Utilization of
Nitrogen and its Compounds of Plants, Symposia Soc. Exptl.
Biol. No. 13:148-176.
33.
Steward, F. C. , and D. J. Durzan. 1965. Metabolism of nitrogenous compounds, p. 397-602.
In; F. C. Steward [ ed. ]
Plant Physiology; a treatise, IV. A. Metabolism; Organic
nutrition and nitrogen metabolism. Academic Press, New York.
34.
Steward, F. C. , and D. Margolis. 1962. The effects of manganese upon free amino acids and amide of the tomato plants.
Contrib. Boyce Thompson Inst.
21s393-409.
35.
Stewart, L
1962. The effects of minor element deficiencies on
free amino acids in citrus leaves. Proc. Amer. Soc. Hort. Sci.
81:244-249.
36.
Taylor, D.
1965. The manganese nutrition of cotton.
Thesis. Texas A and M University, p. 138.
37.
Thomas, M. D0 1965. Photosynthesis (carbon assimilation):
Environmental and metabolic relationship, p. 125-128. In:
F. C. Steward [ ed. ] Plant Physiology: a treatise, IV. A.
Metabolism: Organic nutrition and nitrogen metabolism. Academic Press, New York.
38.
Thompson, J. F. , C. J. Morris, and R. K. Gering.
1959.
Purification of plant amino acids for paper chromatography.
Anal. Chem. 31:1028-1031.
39.
Voth, V., E. L. Proebsting, and R. S. Bringhurst. 1961.
Response of strawberries to nitrogen in Southern California.
Proc. Amer. Soc. Hort. Sci. 78:270-274.
40.
Voth, V., K. Uriu, and R. S. Bringhurst. 1967. Effect of
high nitrogen application on yield, earliness, fruit quality and
leaf composition of California strawberries. Proc. Amer. Soc.
Hort. Sci. 91:249-256.
Ph. D.
45
INFLUENCE OF MANGANESE NUTRITION OF IAA OXIDASE
AND PHENOLS OF STRAWBERRY LEAVES
Strawberry plants were observed to exhibit unusual growth
response to deficient and toxic levels of manganese during an investigation of absorption and distribution of the metal (28).
The quantita-
tive plant growth as measured by the plant dry weight showed a decrease
in plant vigor, both from deficiency and excess of manganese.
Since
the elongation of plant cells is known to be hormonally regulated, the
amount of growth accomplished by the cell is quantitatively dependent
on the concentration of such substances.
Any factor influencing the
concentration of one of these hormones,.such as IAA, would affect growth
within cell, tissue or organ.
Manganese and a number of phenolics are known to affect growth
by their regulatory action on IAA oxidase both in vivo and in vitro (1,
10, 18, 20, 34, 37, 40).
The role of Mn as a co-factor in the oxidation
of IAA was first reported by Wagenknecht and Burris (39) and has since
been observed by numerous workers (13, 14, 17, 29).
Increases of two
to three fold in the rate of IAA oxidation by horseradish peroxidase
with additions of Mn
have been reported (15).
Goldacre (7) reported
that Mn was an inhibitor of IAA oxidase; whereas Furuya and Galston
(4) observed that the preincubation of pea tissue homogenates with Mn
overcame the effects of IAA oxidase inhibitor (a g.lucoside of quercetin).
46
Goldacre, Galston and Weintraub (8) pointed out that IAA oxidation is usually activated by monophenols and inhibited by o-dipheno.ls,
an observation since supported by numerous workers (1, 9, 21, 23, 30,
38, 40).
Presumably, phenols which inhibit IAA oxidase activity have
a sparing effect on the IAA and thereby promote growth and vice versa.
Stutz (35) reported an obligate requirement for a phenolic co-factor
and Mn in the IAA oxidase system of lupine and found Mn to be inactive
in the absence of the phenolic co-factor.
Kenten and Mann (15) stated
that the manganous ions were oxidized to the manganic ions via a
phenol by the peroxidase-peroxide system.
In the system, Mn
seems
to function as a reducing agent for the oxidized peroxidase substrate,
the Mn
ion being subsequently reduced, possibly by IAA.
Recently, Stonier et al„ (32) suggested that a high molecular
weight auxin protector, found by them in Japanese morning glory and
sunflower, and the phenolic compounds described by others are concerned with regulation of peroxidase catalyzed redox reactions.
They
further stated that the auxin protector (which is inactivated by Mn)
acts as an antioxidant of IAA.
Morgan et a.l. (22) reported an inverse correlation between high
manganese and IAA oxidase inhibitor (gossypol andkaempferol) activity.
The degree of IAA oxidase stimulation was associated with the severity
of Mn toxicity.
Taylor et al.
(37) indicated that the biosynthesis or,
at least, the activity of the native co- factors and inhibitors of IAA
47
oxidase system was influenced by the concentration of manganese in
the leaf.
Both high and low levels of Mn modified IAA oxidase inhibitor
activity (22, 37).
This suggests that there is a relationship among
phenols, IAA oxidase and Mn nutrition of plants and would indicate an
in vivo role for the enzyme; such a role is compatible with other
evidence.
This paper deals with the effects of manganese nutrition on IAA
oxidase and phenols of strawberry leaves.
Tissue manganese was
varied by growing strawberry plants at substrate manganese concentrations ranging from deficient to toxic levels.
The effects of the
tissue Mn on IAA oxidase and phenols were tested with assay of enzyme,
co-factors and total, bound, free and o-diphenols.
Materials and Methods
Solution culture studies.
'Northwest1 strawberry transplants
were grown in solution culture in the greenhouse during 1969 and 1970,
as described previously (28).
Five treatments with five plants in each
11 liter plastic container were treated with 0. 0, 0. 5, 2. 5, 12. 5, and
25. 0 ppm Mn.
The leaves were harvested after 84 days and divided
into young and old leaves.
The leaves for enzyme assay were kept in
an ice bath or lyophilized for phenol and manganese determination.
Extraction and purification for assay of IAA oxidase.
Immedi-
o
ately on harvest, the leaves were homogenized in a cold room (40 F)
48
in four volumes (4 ml;
1 g fresh wt. ) of 0. 1 M KH PO -Na HPO
Li
Q
Li
Q
buffer (pH 6.0), containing 0. 3M sucrose and 200 |j,g/m.l each of penicillin G and streptomycin sulfate, and then filtered through a double
layer of cheesecloth.
Crude homogenate was centrifuged at 10, 000 x
g for 20 minutes at 40 F, and the resulting supernatant was used as
enzyme extract.
The homogenates were kept in an ice bath throughout
the preparative procedure.
One set of the prepared homogenates was dialyzed against a 200
fold volume of 0. 1M phosphate buffer (pH 6. 0) for 48 hours with three'
changes of buffer at 12-hour intervals, except as noted otherwise;
while the second set was not dialyzed.
at 40 F in a dark room.
The dialysis was carried out
After dialysis the enzyme extracts were
centrifuged as before and either assayed immediately or stored at a
o
temperature of 0 F.
IAA oxidase assay.
system (20, 29).
IAA oxidase was assayed by a manometric
The reaction mixtures as described by Taylor et a.l.
(37) were used with slight modification.
Each flask contained 0. 2 ml
of 20% KOH in the center well with a filter paper wick, 1 ml containing SOJJIM of IAA in the side arm and 0. 5 ml of 0. 1M sodium, phospate
(pH 6. 0), 0. 2|i.M of 2, 4-dichlorophenol, 0. 1 mM of MnCl , and 1 ml
of leaf extract and deionized water in the main compartment to give a
total volume of 3. 3 ml.
Unless otherwise indicated, the assay was
conducted at room temperature in light,
after 15 minutes of equilibration.
49
Duplicate flasks of each extract were assayed.
Assay for co-factor.
The assay for co-factor activity was identi-
cal to the IAA oxidase assay except that 2, 4-dichlorophenol was omitted
from the reaction mixture.
Appropriate blanks, containing boiled
extracts, were run to determine oxygen uptake due to the constituents
other than the co-factors.
Extraction for determination of phenols.
Freeze dried leaves
were ground to pass a 40 mesh screen and extracted thrice with boiling
95% ethanol.
The hot extract was filtered through a sintered glass and
reduced to semi-dryness under vacuum.
The residue was taken up in
a small quantity of hot water for subsequent extraction of free and
bound (acid and alkali hydrolysable) phenols (3, 12).
Acid hydrolysis was performed using 2N HC1 and refluxing on a
steam bath for an hour, and subsequently, extracted with ethyl ether in
a continuous liquid-liquid extractor for 24 hours.
Alkaline hydrolysis
was carried out using 2N NaOH and the preparation allowed to stand
at room temperature overnight in a nitrogen atmosphere and then
acidified to pH 2. 0 with HCl prior to ether extraction (16).
The ether
extract of alkaline hydrolysates was washed with 5% NaHC O . The
ether portion was discarded and the alkaline portion was acidified to
pH 2. 0 and re-extracted with ether.
Estimation of phenols.
The phenolic content of strawberry
leaves was estimated by the method of Rosenblatt and Peluso (27),
50
using the Folin-Denis reagent and calculated as chlorogenic acid.
The
Folin-Denis reagent used in this method lacks complete specificity for
polyphenols but was used because of its simplicity and convenience.
O-dihydroxyphenols were estimated by the method of Arnow (2).
The
Arnow reagent appears to be a modification of the Hoepfner reagent
(11) and is specific for the o-dihydroxy configuration.
Determination of manganese.
Levels of manganese in the leaves
were determined by atomic absorption spectrophotometer (28).
Results and Discussion
Tissue manganese.
The Mn content of leaf fractions after 84
days varied from 129. 0 ppm of leaf tissue from the young leaves of
plants grown with 0. 0 ppm Mn to 10,132. 5 ppm in old leaves in plants
grown with 25. 0 ppm Mn (Table 3. 1).
In all treatments, the concen-
tration of Mn was higher in old than in young leaves.
In a number of
other plants Millikan and Romney et al. (18, 26) also found more Mn in
mature than in younger leaves, and autoradiographic studies showed
prominent localized areas of Mn concentrations in old leaves.
Characterization of IAA oxidase enzyme assay.
Enzyme
extracts of young and old leaves of strawberry dialyzed for 48 hours
showed a decrease in lag period and a substantial increase in O uptake
when compared with similar non-dialyzed leaves (Figure 3. 1).
O
Low
uptake was observed in non-dialyzed extracts of old as compared to
51
Table 1.
Influence of substrate manganese on the manganese content
of young and old leaves of strawberry.
Substrate
Manganese
(ppm)
Old Leaves
(ppm)
Young Leaves
(ppm)
0.0
164. le
129. 0e
0.5
782. 4d
541. 2d
2. 5
1, ,137. 8 c
749. 1 c
12. 5
2, 9 36. 0 b
1, 448. 7 b
25. 0
10, 132. 5 a
4, 295. 4 a
Each figure is the mean of five replications.
2
Means followed by the same letter in each column are not significantly different at the 0. 05 level of probability.
Figure 3. 1„
IAA oxidase activity of dialyzed and nondia.lyzed extracts
of strawberry leaves.
Figure 3. 2.
Effect of time of dialysis on enzyme activity.
52
200
C?
o OLD DIALYZED
• OLD NON DIALYZED
a YOUNG DIALYZED
A YOUNG NON DIALYZED
150
UJ
CL
3
100
L±J
O
>X
o
60
90
120
150
180
60
72
MINUTES
200r
oOLD LEAVES
^ YOUNG LEAVES
_•
150
UJ
CL
Z>
100
UJ
o
>X
50
o
12
24
36
48
DIALYSIS TIME - HOURS
53
young leaves.
After dialysis the old leaves showed higher enzyme
activity than young leaves as measured by O uptake.
Decline in
enzyme activity was also observed when dialysis was extended beyond
48 hours (Figure 3. 2).
Taylor et al. (37) observed a similar lag in
O uptake in old leaves of cotton.
Dialysis for 72 hours removed heat-
stable, endogenous inhibitors which were causing the delay in O
uptake.
At relatively low concentrations of enzyme extract, its activity
was not proportional to concentration (Figure 3. 3).
The enzyme activ-
ity did increase from zero to 1. 0 ml of extract per reaction mixture.
The deviation from direct proportionality at low enzyme levels may be
due to product degradation (19, 29).
In all other enzyme assay experi-
ments, a concentration of 1. 0 ml (equivalent to 250 mg fresh weight
of leaves) was used as a standard.
Tang and Bonner (36) observed that
at low concentrations of pea seedling enzyme, rate of reaction was
proportional to enzyme concentration, while at high concentrations
substrate became limiting.
Morgan (20),
working with cotton leaves,
found 0. 2 ml of enzyme extract had a minimum lag period.
Quantities
above or below 0. 2 ml resulted in an increase in the lag period of O
uptake.
Both the dialyzed and undialyzed enzyme extract of strawberry
leaves required Mn (supplied as MnCl^) and 2, 4-dichlorophenol for its
maximum expression of activity (Figure 3. 4).
In its requirement for
Figure 3. 3.
Effect of enzyme concentration on oxygen uptake.
Figure 3„ 4.
Effect of co-factors on IAA oxidase activity.
54
200 r
o OLD LEAVES
A YOUNG LEAVES
^
150
LJ
?CL
Z
UJ
CD
>
X
100
50
o
.25
.50
.75
1.00
1.25
1.50
ENZYME EXTRACT (ml)
400
A
o
—
D
H300
A
•
UJ
■
CRUDE COMPLETE
MINUS Mn.
MINUS 2,4-D
DIALYZED COMPLETE
MINUS Mn.
MINUS 2,4-D
0- 200
UJ
o
X "00
-
o
120
180
MINUTES
360
55
Mn, enzyme extract in strawberry resembles the enzyme in bean and
pineapple (9, 14,
by even 10
-5
39) and differs from that in pea, which is inhibited
M Mn (4).
Phosphate borate buffer for determination of the pH optimum of
the enzyme system of strawberry leaves was prepared as described by
Moore (19).
(Figure 3. 5).
The optimum pH for the enzyme was found to be 6. 0
Strawberry leaf IAA oxidase differs strikingly from the
enzyme in pineapple, having a pH optimum of 3. 0 (9) but is similar in
range to IAA oxidase in peas and beans (36,
39).
In separate experiments in which the concentrations of IAA were
varied under otherwise optimum conditions (Figure 3. 6), the amount
of IAA decarboxylation (measured as O
uptake) was approximately a
linear function of initial IAA concentration at low levels but became nonlinear at concentrations above 50 |JLM.
This optimum concentration was
far more than observed for enzyme extracted from cotton (21) but low
as compared to enzyme obtained from omphalia and lupine (25,
35).
Effect of Mn on IAA oxidase activity of dialyzed extract.
The
effect of different levels of nutrient Mn on the IAA oxidase activity of
dialyzed extract of old and young leaves is shown in Figure 3. 7.
At
all manganese levels, IAA oxidase activity was found to be higher in
the old than in the young leaves.
Enzyme extracts of leaves from
25. 0 ppm Mn treated plants had the highest IAA oxidase activity.
The
order of activity of extracts from old leaves in the remaining treatments
Figure 3. 5„
pH optimum of IAA oxidase of strawberry leaves.
Figure 3„ 6.
Effect of substrate IAA on IAA oxidase activity.
56
200,
o OLD LEAVES
a YOUNG LEAVES
150
UJ
Q_
100
3
LU
O
>
X
o
50
3.o
4.5
5.0
5.5
pH
6.0
6.5
7.0
60
70
200.
o OLD LEAVES
a YOUNG LEAVES
"5. 150
UJ
a.
3
100
UJ
o
50
20
30
40
50
IAA CONCENTRATION ( pM)
Figure 3. 7.
Effect of substrate manganese on IAA oxidase activity of
strawberry leaves.
57
300
/25.0PPM
OLD LEAVES
(PPM of Mn.)
A 2.5 PPM
^^OOPPM
250
/
/
UJ
200
Q.
150
2.5PPM_^^
•
UJ
O
>X
100
o
50
//
^^^^/^OSPPM
l^^
■
/
/
15
i
30
45
200r
i
i
60
75
i
/25.0PPM
YOUNG LEAVES
(PPMofMn.)
/
-5. 150-\
/-
/
90
//
/I2.5PPM
^^^6!OPPM
UJ
<
3
0.5 PPM/'
100
/
/
UJ
o
>X
o
^^5PPM
50
eS*^
,
1
15
30
1
45
MINUTES
1
1
60
75
1
90
58
was 12. 5, 0. 0, 0. 5, and 2. 5 and in young leaves -was 12. 5, 0. 0, 2. 5
and 0. 5 ppm Mn.
The differences in the enzyme activity between the
extracts of old and young leaves from 0. 5 and 2. 5 ppm Mn treatments
were small as compared to the other treatments.
Results of this experiment are in agreement with the data of
Taylor ej: al. (37) that a toxic as well as a deficient level of Mn in
vivo results in higher activity of LAA oxidase enzyme system.
Kenten
et al. (15) and others (4, 10, 22, 29) in vitro tests observed an
increase in the activity of IAA oxidase with added Mn; whereas
Goldacre (7) reported that Mn was an inhibitor of IAA oxidase.
The
observed stimulation in IAA oxidase activity from toxic and deficient
leaves may be considered as an acceleration of the normal aging
process (6, 33).
Taylor et al. (37) found that the deficient leaves
needed no additional co-factor (2, 4-dichloropheno.l) for IAA oxidase
activity; whereas the toxic leaves needed this co-factor but no additional Mn.
Thus he concluded that the biosynthesis or, at least, the
activity of the native co-factors and inhibitors of the IAA oxidase
system was influenced by the concentration of manganese in the leaf,
which probably is true in strawberry leaves.
Effect on phenols.
The relative amounts of free, bound and
total phenols as affected by the substrate Mn concentration are shown
in Figure (3. 8 and 3. 9).
Both the total as well as bound phenol levels
increased with an increase in the concentration of the substrate or tissue
Figure 3. 8.
Effect of substrate manganese on total and bound phenols
of strawberry leaves.
Figure 3. 9.
Effect of substrate manganese on free phenols of strawberry leaves.
59
16
H 14
£
o
•
A
A
OLD LEAVES TOTAL PHENOLS
OLD LEAVES BOUND PHENOLS
YOUNG LEAVES TOTAL PHENOLS
YOUNG LEAVES BOUND PHENOLS
>Q |2
SX10
Q.
E
8
C -5S-
0.5
2.5
12.5
25.0
12.5
25.0
LOG. Mn (PPM)
12
O
o
10
>Q
8
ui
X
A YOUNG LEAVES
0.5
2.5
LOG. Mn (PPM)
60
manganese (Table 3. 1), except in 0. 5 ppm Mn treatments in which the
old leaves contained less phenols (bound and total) than the old leaves
from the 0. 0 ppm Mn treatment.
In all treatments, the old leaves con-
tained more phenols (total and bound) than young leaves.
There were slight increases in the concentration of phenols when
the concentration of substrate Mn was increased from 0. 0 to 2. 5 ppm.
When substrate concentration of Mn was increased from 2. 5 to 25. 0 ppm,
however, the increase in total and bound phenols was substantially more.
In contrast to total and bound phenols, the highest levels of free
phenols were observed at the intermediate substrate Mn in both young
and old leaves.
Also, the old leaves contained more free phenols at
all levels of Mn substrate than young leaves.
The presence of inhibitors of LAA oxidase in enzyme extracts
obtained from various plants has been demonstrated by a number of
workers (5, 9,
20, 21, 23, 29).
Addition of boiled extract resulted
in increased lag period (21, 22, 37), the duration of which was a function of the concentration of inhibitors.
Maclachlan et_ al. (17) and
Morgan (20) found that the activity of inhibitors followed total phenol
levels.
Morgan et al. (22) reported an inverse correlation between
high Mn and IAA oxidase inhibitor, which they thought to be kaempferol.
In this study, high IAA oxidase activity was observed both at deficient
and toxic levels of manganese in the tissue, but correlations of total
and bound phenols with IAA oxidase occurred only at concentrations
61
of Mn greater than 2. 5 ppm.
A negative correlation was found in the
levels of free phenols and IAA oxidase (Figure 3.7 and 3. 9).
This
indicates that free phenols are involved in the mechanism of IAA oxidation, and the observation is further supported by the fact that old leaves
with higher IAA oxidase activity have a higher concentration of free
phenols than do young leaves (Figure 3. 8).
Interaction of tissue Mn, o-dihydroxyphenols and IAA oxidase.
A short-term experiment was conducted to determine the relationship
of tissue Mn, o-diphenols and IAA oxidase activity.
Strawberry plants
were grown with 125 ppm substrate Mn and leaf samples were taken
at 5, 10, 15 and 20-day intervals.
The levels of IAA oxidase activity and o-dihydroxyphenols
increased slightly up to 15 days of Mn treatment,
but later o-dihy-
droxyphenols decreased and IAA oxidase, as indicated by oxygen uptake,
increased substantially (Figure 3. 10).
IAA oxidation is usually activated by monophenols and inhibited
by o-diphenols (8).
Recently, Stonier el al. (31, 32) stated that the
auxin protector acts as an antioxidant of IAA.
The auxin protector
found by them in Japanese morning glory leaves as well as in sunflower shoots was a phenolic compound of very high molecular weight.
Earlier, Furuya and Galston (4) considered a glucoside of quercetin
to be an inhibitor of IAA oxidase.
•with cotton,
Morgan et al. (21,
22), working
suggested that a low molecular •weight phenol, functioned
Figure 3. 10.
Interaction of tissue manganese, o-dihydroxyphenol and
LAA oxidase.
62
-225
UJ
Q_
UJ
O
>X
o
15
(23694)
(65670)
TISSUE Mn (PPM)
63
as an inhibitor of IAA oxidase.
The decrease in the level of o-dihy-
droxyphenol at a high tissue level of Mn, accompanied by a simultaneous increase in the level of IAA oxidase, suggests that a phenol may
be involved in the inhibition of IAA oxidase activity in leaves of strawberry.
In this experiment no attempts were made to isolate and
identify such a hypothetical compound.
The study also indicates that
the level of inhibition is functionally related to the tissue level of Mn.
A more elaborate experiment is necessary to elicit this relationship.
64
LITERATURE CITED
1.
Andreae, W. A., and G. Collet. 1968. The effect of phenolic
substances on the growth activity of indoleacetic acid applied to
pea root or stem sections. p. 553-561. In: F. Wightman and G.
Setterfield [ ed. ] Biochemistry and Physiology of Plant Growth
Substances. Proceedings of the 6th International Conference on
Plant Growth Substances. The Runge Press, Ottawa.
2.
Arnow, L. E. 19 37. Colorimetric determination of the components of 3, 4-dihydroxypheny.lalanine-tyrosine mixtures. Jour.
Biol. Chem. 118:5 31-5 37.
3.
Feldman, A. W. , and R. W. Hanks. 1966. Identification and
quantification of phenolics in the leaves and roots of healthy and
exocortis-infected citrus. Florida Agricultural Experiment
Station Journal Ser. No. 2423. p. 11.
4.
Furuya, M. , and A. W. Galston. 1961. Effect of in vitro preincubation with co-factors on the activity of the indoleacetic acid
oxidase of peas. Physiol. Plantarum 14:750-766.
5.
Furuya, M. , A. W. Galston, and B. B. Stowe. 1962. Isolation
from peas of co-factors and inhibitors of indolyl- 3- acetic acid
oxidase. Nature 193:456-457.
6.
Galston, A. W. 1967. Regulatory systems in higher plants.
Am. Scientist 55:144-160.
7.
Goldacre, P. L. 1963. The indole-3-acetic acid oxidase-peroxidase of peas. Plant Growth Regulation. Fourth International
Conference on plant growth regulation. The Iowa State University
Press, Ames, Iowa, U.S.A. p. 143-147.
8.
Goldacre, P. L. , A. W. Galston, and R. L. Weintraub. 195 3.
The effect of substituted phenols on the activity of IAA oxidase of
peas. Arch. Biochem. Biophys. 43:358-373.
9.
Gortner, W. A., and M. Kent. 1953. Indoleacetic acid and, an
inhibitor in pineapple tissue. J. Biology Chem. 204:59 3-603.
10.
Hillman, W. S. , and A. W. Galston. 1956. Interaction of Mn
and 2, 4-D in the enzymatic destruction of IAA. Plant Physiol.
9:230-325.
65
11.
Hoepfner, W. 1932. Two new reactions for caffeic acid and
chlorogenic acid. Chem-Zeit 56:991-993.
12.
Ibrahim, R. K. , and G. H. N. Towers. I960.
tion by chromatography of plant phenolic acids.
Biochem. Biophys. 87;125-128.
13.
Kenten, R. H. 1950. The oxidation of manganese by peroxidase
systems. Biochem. J. 46s67-73.
14.
Kenten, R. H. 1955. The oxidation of indolyl-3-acetic acid by
wax pod bean root sap and peroxidase systems. Biochem. J.
59:110-121.
15.
Kenten, R. H. , and P. J. G. Mann. 1952. Oxidation of manganese by enzyme systems. Biochem J. 52:125-130.
16.
Li, C. Y. , K. C. Lu, J. M. T.rappe, and W. B. Bo.llen. 1971.
Phenolic compounds in roots of Red Alder and Douglas fir. (In
Press).
17.
Maclachlan, G. A., and E. R. Waygood. 1956. Catalysis of
indoleacetic acid oxidation by manganic ions. Physiol. Plantarum
9:321-330.
18.
Mi.l.likan, C. R. 1950. Radio-autographs of manganese in plants.
Australian J. Sci. Res. B4:28-41.
19.
Moore, T. C.
1970. Comparative net biosynthesis of indoleacetic
acid from tryptophan in cell-free extracts of different parts of
Pi sum sativum plants. Phytochemistry 9:1109-1120.
20.
Morgan, P. W. 1964. Distribution of indoleacetic acid oxidase
and inhibitors in light grown cotton. Plant Physiol. 39:741-746.
21.
Morgan, P. W. , and W. C. Hall. 1963. Indoleacetic acid
oxidizing enzyme and inhibitors' from light grown cotton. Plant
Physiol. 38:365-370.
22.
Morgan, P. W. , H. E. Joham, and J. V. Amin. 1966. Effect
of manganese toxicity on the indoleacetic acid oxidase system of
cotton. Plant Physiol. 41:718-724.
23.
Rabin, R. S. , and R. M. Klein. 1957. Chlorogenic acid as a
competitive inhibitor of indoleacetic acid oxidase. Arch.
Biochem; Biophys. 70:11-15.
The identificaArchives
66
24.
Ray, P. M. I960. The destruction of indoleacetic acid^III.
Relationships between peroxidase action and indoleacetic acid
oxidation. Arch. Biochem. Biophys. 87jl9-30.
25.
Ray, P. M. , and K. V. Thimann. 1956. The distribution of
indoleacetic acid.
I. Action of an enzyme from Omphalia flavida.
Arch. Biochem. Biophys. 64:175-'192.
26.
Romney, E. M. , and S. J. Toth. 1954. Plant and soil studies
with radioactive manganese. Soil Sci, 77:107-117.
27.
Rosenblatt, M. , and J. V. Pleuso. 1941. Determination of
tannins by photocolorimeter. Jour. Assoc. Off. Agr. Chem,
24:170-180.
28.
Sabharwal, S. L. , R. Garren, Jr., and O. C. Compton. 1971.
Growth and mineral composition response of strawberry plants
to manganese nutrition. (In Prepaf-ation).
29.
Sacher, J. A.
1963. Effects of inhibitors on the kinetics of
indoleacetic acid oxidation. Amer. J. Bot. 50:116-122.
30.
Schaeffer, G. W. , J. G. Buta, and F. Sharpe. 1967. Scopoletin
and polyphenol-induced lag in peroxidase catalyzed oxidation of
indole-3-acetic acid. Physiol. Plants 20:342-347.
31.
Stonier, T. 1969. Studies on auxin protectors. VII. Association of auxin protectors with crown gall development in sunflower
stems.. Plant Physiol. 44:1169-1174.
32.
Stonier, T. , F. Rodriguez-Tormes, and Y. Yoneda. 1968.
Studies on auxin protectors. IV. The effect of manganese on
auxin protector of the Japanese morning glory. Plant Physiol.
43:69-72.
33.
Stonier, T. , and Y. Yoneda. 1967. Auxin destruction and
growth in Japanese morning glory stems. Ann. New York Acad.
Sci. 144:129-135.
34.
Straus, J. , and R. K. Gerding.
1963. Auxin oxidase and
growth control in tissue cultures of Ephedra. Plant Physiol.
38:621-27.
35.
Stutz, R. E. 195 7. The indoleacetic acid oxidase of Lupinus
a lb us L. Plant Physiol. 32:31-39.
67
36.
Tang, Y. W. , and J. Bonner. 1946. The enzymatic inactivation
of indoleacetic acid I. Arch. Biochem. Biophys. 13:11-25.
37.
Taylor, D. M. , P. W. Morgan, H. E. Joham, and J. V. Amin.
1968. Influence of substrate and tissue manganese on the IAAoxidase system in cotton. Plant Physiol. 43:243-247.
38.
Tomaszewski, M. , and K. V. Thimann. 1966. Interactions of
phenolic acids, metallic ions, and chelating agents on auxininduced growth. Plant Physiol. 41:1443-1454.
39.
Wagenknecht, A. C. , and R. H. Burris. 1950. Indoleacetic
acid inactivating enzyme from bean roots and pea seedlings.
Arch. Biochem. 25:30-5 3.
40.
Zenk, M. H, , and G. Muller. 1963. In vivo destruction of
exogenously applied indoly.l-3-acetic acid as influenced by
naturally occurring penolic acids. Nature 200:761-763.