A HELD SWQY CGNCEQNENG THE
EFFEQT {3F ZENC UPC-N Y3Y?’?QP‘HAN AWE
PROTEiN IN TWO VARIETIES
OF NAVY BEANg,

IS.‘

Thesis for {PM Dogma o? M. S.
MICHIGAN STATE UNIVERSETY

Mamuel J. Woods
1968

 

ichigan State
University

lug mu; “MEN“ w lfl‘ m mm m; \\ w w E: ,8 R A p 7:,

 

 

 

 

ABSTRACT

A FIELD STUDY CONCERNING THE EFFECT OF
ZINC UPON TRYPTOPHAN AND PROTEIN IN
TWO VARIETIES OF NAVY BEANS,
PHASEOLUS VULGARIS (L. )

 

by Samuel J. Woods

Two varieties of navy beans were exposed to zinc stress
for different lengths of time in the field. Zinc was applied to the
soil in a water solution of zinc sulfate (ZnSO4).

In Sanilac, zinc deficiency symptoms appeared in untreated
plants two weeks after planting. No Visible zinc deficiency symp-
toms appeared in Saginaw during the course of the experiment.

The total quantity of protein produced in whole-plant tissue
was greater for Saginaw when zinc was not applied. Zinc application
to Sanilac resulted in an increased amount of foliage protein.

Protein percentage decreased in both varieties with
advanced maturity. However, longer periods of zinc stress were
associated with higher whole-plant protein percentages in Sanilac,
whereas the period of zinc stress had little effect upon the protein

percentage of Saginaw.

Samuel J. Woods

The tryptophan percentage of Kjeldahl protein was measured
in whole-plant tissue and in the seed. No significant differences in
tryptOphan percentage due to zinc treatment were obtained in the
whole-plant tissue of either variety. The seed proved to be a
stronger indicator of tryptophan variability. As zinc was applied at
successively later dates during the season, the tryptOphan compo-
sition of the seed in both varieties increased.

No statistically meaningful relationships were obtained
between zinc concentration and tryptophan percentage in the foliage
or seed of either variety. A strong positive correlation existed
between zinc concentration and protein percentage in the seed of

Saginaw.

A FIELD STUDY CONCERNING THE EFFECT OF
ZINC UPON TRYPTOPHAN AND PROTEIN IN
TWO VARIETIES OF NAVY BEANS,

PHASEOLUS VU LGARIS (L. )

 

By

Samuel J. Woods

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Crop Science

1968

quafig
5" ab'ég

ACKNOWLEDGMENTS

The author wishes to express his appreciation to Dr. M. W.
Adams, not only for his help in preparing this manuscript, but also
for his sincere interest in the author' 8 intellectual development.
Drs. Selma Bandemer and E. J. Benne were very instrumental in
providing the technical assistance needed for performing the chemi-
cal analyses.

Special gratitude is offered to the author' s wife for her

unselfish contributions to this work.

ii

TABLE OF CONTENTS

ACKNOWLEDGMENTS
LIST OF TABLES

LIST OF FIGURES
INTRODUCTION

REVIEW OF LITERATURE

Zinc in relation to tryptophan synthesis
Zinc in relation to protein synthesis .

MATERIALS AND ME THODS

Field management . .

Procedure for sampling of plant material
Tryptophan analysis

Zinc determination

Protein determination

RESULTS

Varietal growth and development

Whole- -plant analysis for protein and tryptophan .

Whole- -plant zinc analysis .

Zinc application vs. protein content of Sanilac
Zinc application vs. protein content of Saginaw
Seed tryptophan analysis

Seed protein analysis

111

Page

ii

vii

10
11
12
12

14

14
19
24
26
26
29
31

Page

DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 34
Plant growth and deveIOpment . . . . . . . . . . . . . 34
Protein analysis . . . . . . . . . . . . . . . . . . . 35
Tryptophan analysis . . . . . . 36
Relationships between zinc, tryptophan, and protein . . . 36

SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . 38

LITERATURE CITED . . . . . . . . . . . . . . . . . 41

iv

Table

LIST OF TABLES

A guide to codes representing the various
zinc application dates .

Varietal protein percentage based on whole-
plant samples for three harvesting periods
representing five different zinc application
dates

Varietal tryptOphan composition expressed as
a percentage of the protein found in whole-
plant samples

Summary of the analysis of variance performed
at three sampling periods for tryptophan
percentage of protein and protein percentage

in whole-plant tissue

Zinc concentration in micrograms per gram
of dry matter for Saginaw and Sanilac, based
upon whole-plant analysis .

Correlation coefficients relating zinc,
tryptophan, and protein in whole-plant tissue

Varietal mean tryptophan percentages for
four zinc application dates

Functional analysis of variance for tryptophan
composition of seed protein .

Seed zinc content of Saginaw and Sanilac as
affected by four different dates of zinc
application

Page

13

19

20

21

25

25

29

30

31

Table Page
10. Correlations between zinc concentration,

tryptophan content, and protein percentage
in the seed of Saginaw and Sanilac 33

vi

Figure

LIST OF FIGU RES

Varietal dry matter production patterns
for five zinc application dates

Zinc deficiency in Sanilac three weeks
after planting .

Sanilac treated one week after planting
(right) and untreated (left) six weeks
after planting .

Varietal protein percentage relationships
for five different zinc treatments at
three sampling dates

Varietal protein quantity relationships for
several different zinc treatments at three
sampling dates

Seed protein percentages for Saginaw and

Sanilac when treated with zinc on four
different application dates

vii

Page

15

18

18

22

27

32

INTRODUCTION

In Michigan' 5 Thumb area, the use of large quantities of
phosphorus has seriously reduced the zinc supplying capability of
the soil. Of all the different crops grown in the area, the navy bean
has proved most susceptible to zinc deficiency.

Differential response to zinc-deficient conditions has been
observed among navy bean varieties. The most dramatic contrast
exists between the variety Saginaw, which appears to grow and
develop normally under zinc—deficient conditions, and Sanilac, a
very susceptible variety, responding to zinc—deficiency by displaying
short internodes, small, deformed leaves, interveinal necrotic
lesions, few flowers and delayed maturity.

Major differences between the two varieties in response to
low levels of zinc appeared to be in the growth processes. There-
fore, it was postulated that varietal differences existed with respect
to certain physiological manifestations of growth.

For several years plant physiologists have known that zinc
plays a role in auxin synthesis. Strong evidence now exists that

zinc is required for the synthesis of the amino acid, tryptophan,

which serves as a natural precursor for auxin. Zinc also has been

shown to affect protein synthesis by being essential for the produc-

tion of RNA.

(1.)

(2)

(3)

(4)

This study was conducted with four main objectives in mind:
To compare the total protein contents of Saginaw and
Sanilac after subjection to zinc—deficient conditions for dif-
ferent periods of time in a field situation.

To compare the two varieties with respect to protein quality
by analyzing for tryptophan content.

To provide evidence for a relationship between zinc concen-
tration, protein percentage, and tryptophan content in
whole-plant tissue.

To analyze the seed of both varieties to provide evidence

for a possible zinc, protein, tryptophan relationship.

REVIEW OF LITERATURE

Zinc in relation to
tryptophan synthesis

 

 

Information concerning the relationship of zinc to modern
plant nutrition was practically nonexistent before 1940. One of the
first to explore the physiologic role of zinc in modern plant nutrition

was Skoog (13). Working with tomato, Lycopersicum esculentum,

 

and sunflower, Helianthus annuus, Skoog discovered that plants

 

deprived of zinc in a Hoagland nutrient solution showed depressed
auxin content. Both species were placed in a zinc-deficient nutrient
solution until the older leaves became necrotic. At this point zinc

at a concentration of . 01 m/l was added to the nutrient solution.
After only a twenty-four hour period the auxin content of both species
began to increase. The disappearance of auxin in zinc deficient
plants was accompanied by an increase in peroxidases. Skoog con-
cluded that zinc had an indirect effect by preventing auxin oxida-
tion.

Tsui (16) explored the relationship between zinc and auxin

more thoroughly. Working with tomato (variety, John Baer), he

found that in addition to the auxin content gradually decreasing on
exposure to a zinc-deficient medium, the tryptophan level fluctuated
as well. The tryptophan level of zinc—deficient plants was found to
be lower when compared to the level found in normal plants. When
zinc was supplemented in the deficient nutrient medium, the trypto-
phan level increased.

In a subsequent experiment Tsui (16) subjected tomato leaf
disks to tryptophan infiltration. An increase in auxin measurement
after tryptophan infiltration indicated that there must have been an
enzyme present in the leaf tissue capable of transforming the added
tryptophan to a growth substance. The capacity for tryptophan con-
version was essentially the same in both zinc-deficient and sufficient
tissue. Tsui concluded that zinc is not required principally for the
synthesis of auxin. This left the possibility that zinc affected trypto-
phan biosynthesis, which could serve as a precursor for auxin.

Extended work on the relationship between zinc and trypto-
phan was to no avail until a pathway for tryptophan biosynthesis in
higher plants had been established. Available enzyme separation
techniques were inadequate to define a pathway in plants. Therefore,
emphasis was placed on establishing a pathway in microorganisms
with later application to higher plants. Yanofsky (14), with the use

of Escherichia coli mutants for tryptophan synthesis, discovered a

 

functional pathway in bacteria. It was discovered that an enzyme
(tryptophan synthetase) was responsible for the condensation of indole
and serine during the last stage of tryptophan synthesis.

Basic information gained from Yanofsky' s microbial work
made it possible to continue the search for a similar mechanism in
higher plants. Greenberg and Galston (3) found the first evidence
for the indole—serine condensation in Alaska Pea extracts, Pisum
sativum. By the use of Ehrlich' s reagent, indole was measured
before and after subjection to the extract. Results showed that indole
disappearance was greater with the addition of D—L-serine. Black
Valentine bean seedlings were also investigated but no clear evidence
for the indole-serine reaction could be found. However, Mudd and
Zalik (10) demonstrated the dependency of indole disappearance on
serine concentration using tissue slices from various parts of the
tomato plant.

Crown gall tissues of Boston Ivy, Parthenocissus tricuspidatus,

 

were cultured by Klein (8) on a zinc-deficient nutrient medium. Two
types of cultures were used: cultures receiving no exogenous auxin
and those receiving exogenous auxin. The cultures receiving auxin
grew normally while growth of the cultures receiving no auxin was
decreased by 50 percent. Results showed that exogenous tryptophan

permitted normal growth also.

The effect of zinc on the biosynthesis of tryptophan has been
studied in barley (9). Plants were cultured in several different con-
centrations of zinc. Maximum growth, as measured by plant height,
occurred at a zinc concentration of 8 X 10-4M. Maximum tryptophan
concentration in the plant occurred with optimum zinc concentration
in the nutrient medium. Tryptophan content fell below that of the
control when an 8 X 10—2M concentration of zinc was used in the
nutrient medium.

Zinc in relation to
protein synthesis

 

 

Growth is manifested by cell division and (or) enlargement.
Steward (15) reported that some form of protein synthesis always
occurs with the growth process.

A major prerequisite for protein synthesis is the presence
of ribonucleic acid (RNA). Working with cultures of Rhizopus
nigricans, Wegener and Romano (17) discovered that the addition of
zinc immediately stimulated RNA synthesis. After a brief period,
protein synthesis increased at the same rate as RNA synthesis. The
. pattern observed for growth was similar to that found for protein
synthesis. Key and Barnett (7) suggested that the RNA synthesis
required for growth is restricted to a certain type called messenger

RNA. The use of specific metabolic inhibitors revealed a probable

relationship between auxin-induced growth and protein synthesis.
Actinomycin D, a strong inhibitor of RNA and protein synthesis, was

applied to mature soybean (Glycine max) hypocotyls. When auxin and

 

inhibitor were added simultaneously to the plant tissue, inhibition of
auxin-induced growth paralleled the inhibition of RNA and protein
synthesis.

Both messenger and soluble ribonucleic acids are needed
for protein synthesis in the living cell. Hall and Cocking (4) dis-
covered that an enzyme, RNA-ase, was effective in degrading both
m RNA and 8 RNA, which in turn impaired the incorporation of
amino acids into protein. Further investigation resulted in the dis-
covery that both copper sulphate and zinc acetate were effective
inhibitors of crystalline RNA-ase.

Kessler (6) established RNA-ase as a guide for the deter-
mination of zinc deficiency in citrus. By assuming that zinc was
either directly or indirectly reSponsible for protein synthesis,
Kessler assayed zinc-deficient leaves for zinc and RNA-ase activity.
One half of the leaf was used for zinc determination and the other
half for measuring the percentage of substrate (RNA) hydrolysis per
hour. In healthy leaves, regardless of variety, species, or location,
RNA-ase activity was below 40 percent of total substrate hydrolysis

per hour. In zinc-deficient leaves the percentage of total substrate

hydrolysis was higher than 50. High RNA-ase activity was asso-
ciated with 16 ppm zinc in orange trees. Only 10 ppm in Delicious
apples produced the same effect. Kessler concluded that optimum
levels of zinc in the leaf keeps RNA—ase activity at a minimum, thus
promoting RNA and protein synthesis.

Nason (11) investigated total protein levels in Neurospora

 

as affected by zinc. A dramatic decrease in total protein was expe-
rienced in zinc—deficient mycelia as compared to mycelia cultured

on zinc -deficient media.

MATERIALS AND METHODS

Field management

 

This investigation was conducted on the Clifford Schiann
farm, five miles east of Saginaw, Michigan. The experimental site
consisted of a calcareous Wisner clay loam soil. To insure the lack
of available zinc before treatment, superphosphate, at the rate of
91 kilograms/hectare was broadcast and worked in. One hundred
and seventy kilograms/hectare of 5-20-20 fertilizer was applied in
a band one inch to the side and two inches below the seed. Plots
were planted with a one row International Harvester planter modi-
fied for experimental use. Seeds were Spaced approximately five
centimeters apart in the row with 1. 8 meters between rows. Weed
control consisted of a pre—plant application of Eptam at the rate of
3. 10 kilograms of active ingredient per hectare. During the growing
season each plot was cultivated twice with a standard two row culti-
vator.

The experimental design consisted of a Split-plot arrange-
ment with varieties as main plots. Each main plot was divided into

8 individual one-row subplots representing eight successive weeks

10

of zinc application. Zinc application dates were replicated five
times.

Zinc sulfate (ZnSO4) dissolved in well water was applied by
bucket to individual plots at an approximate rate of 12. 7 kilograms]
hectare. For this soil 6. 75 kilograms/hectare is barely adequate for
growth of Sanilac. Trenches were made on both sides of the row to
prevent run-off.

Procedure for sampling of
plant material

 

 

Four zinc application dates, in addition to an untreated
check, were sampled at three separate times during the growing
season. The first sampling was July 13 with two subsequent sam-
plings at two week intervals.

Samples consisted of three plants taken in succession
within the one-row plot. To minimize contamination, each plant
was carefully put through a series of washings. Plants were first
submerged in tap water, followed by rinsing in a dilute HCl solution
to render soluble all free zinc clinging to roots and foliage. Finally,
each sample was rinsed in deionized water and placed in clean paper
bags. The bags were then placed in a forced air dryer set at 80 C.
for a 36 hour period. Upon drying, each whole plant (including roots)

was ground in a Wiley mill using a 60 mesh screen. After grinding,

11

each sample was revolved in a large piece of wrapping paper to insure
proper mixing. Samples were then placed in a sealed glass wash
bottle to await chemical analysis. During the grinding procedure, the
samples undoubtedly took up moisture. Therefore, before chemical
analysis, samples were redried at 90 C. for six hours and placed in

desiccators until weighing.

Tryptophan analysis

 

The method used for tryptophan analysis was a modification
from Roth (12) by Dr. Selma Bandemer of the M. S.U. Biochemistry
Department. One-half gram of plant material was weighed out, using
a triple beam analytical balance, and placed in a 125 ml. Erlenmeyer
flask for digestion. The acid solution used to digest the plant mate-
rial consisted of 98 ml. concentrated sulfuric acid (H2S04), 63 ml.
water, and 84 ml. of concentrated nitric acid (HNOB). Forty m1. of
the acid solution was poured into each flask. The flasks were then
placed on an 85 C. steam bath for 18 hours. During the first two
hours of digestion a heavy brown vapor of nitrous oxide evolved.
After several hours all that remained of the plant material was a
gelatinous precipitate of silica. After digestion, contents of each
125 ml. flask were washed into a 50 ml. volumetric flask and brought

up to volume using deionized water. Upon cooling, the solution was

 

 

12

filtered through Whatman no. 50 hardened filter paper into a 50 ml.
glass stoppered Erlenmeyer flask. The filtered acid solution was
then read at 440 millimicrons in a Bauch and Lomb spectrophotome-

ter.

Zinc determination

 

Zinc determination was accomplished with a Perkin-Elmer
303 atomic absorption Spectrophotometer. In order to obtain zinc
in large enough quantities for analysis, a 0. 3-1. 0 gram sample was
placed in a crucible for dry-ashing at 500 C. for 18 hours. The
ash was then dissolved in 5 ml. of 2 N HCl and filtered through
Whatman no. 2 filter paper into a 125 ml. Erlenmeyer flask. Con-
tents of the flask were brought up to a 50 m1. volume by the use of
an automatic pipet. Samples were then read at a wavelength of

2 14 millimicrons.

Protein determination

 

The official Kjeldahl procedure, as outlined in the AOAC
manual (1), was used to determine percent amino nitrogen. The
nitrogen percentage was then multiplied by a factor of 6. 25 to obtain

total protein.

13

Table 1.—-A guide to codes representing the various
zinc application dates.

 

 

Code Application date

TO . . . . . . . . June 15
T1 . . . . . . . . June 22
T2 . . . . . . . . June 29
T3 . . . . . . . . July 6

T4 . . . . . . . . July 13
T5 . . . . . . . . July 20
T . . . . . . . . July 27

RESULTS

Varietal growth and development

 

Varietal dry matter accumulation patterns for three
sampling dates are presented in Figure 1. Saginaw displayed a
significantly higher dry matter accumulation for two of the three
sampling dates than Sanilac. Means representing dry matter per
plant were lower for Saginaw when zinc was added than when no
zinc was applied. However, the differences were not statistically
significant, suggesting that zinc application had little effect upon
the growth of Saginaw.

Plots of Sanilac treated at T1 (one week after planting) had
a more rapid rate of dry matter accumulation in July as compared
to the other application. dates. However, the same treatment date
showed a large decrease in the rate of dry matter accumulation
during the July 27 to August 10 interval. All other treatments,
including the check, displayed their most rapid rate of dry matter
accumulation during the July 27 to August 10 period. The effect of
the early post-emergence applications of zinc on Sanilac is illustrated

in Figure 3. Application of zinc to Sanilac at planting time resulted

14

15

Figure 1. -- Varietal dry matter production patterns for five zinc
application dates.

 

Grams dry matter per plant

12—

11—

10—

 

16

 

SAG INAW

   

00000000000

 

 

   

No zinc

Aug 10

Grams dry matter per plant

11—

 

 

17

No zinc

l l
le 27 Aug 10

Figure 1. -- Continued.

OOOOOOO

 

 

Grams dry matter per plant

11—

 

 

17

No zinc

1 1
le 27 Aug 10

Figure 1. -- Continued.

0000000

 

 

18

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19

in a 30 percent increase in dry matter over the check on August 10.

Treatment at T

2 resulted in 20 percent more dry matter per plant

on August 10 than those untreated.

Whole-plant analysis for
protein and tryptophan

 

 

Tables 2 and 3 present treatment means for protein per-
centage and tryptophan composition of protein, respectively. A
Table 2. -- Varietal protein percentage based on whole-plant samples

for three harvesting periods representing five different
zinc application dates.

 

 

 

 

 

Variety
Zinc
application Saginaw - Sanilac
date a b
le 13 le 27 Aug 10 le 13 le 27 Aug 10
T0 24.73 22.34 18.86 24.46 22.28 18. 96
T1 23.75 22.34 18.52 22.19 20.98 17.25
T2 23.31 22.40 17.54 23.25 21.71 17.05
T3 _ 24.87 22.93 17.81 24.44 22.37 17.98
T4 ----- 21.75 17.44 ----- 23.23 17.72
No zinc 24.69 23.19 19.12 25.10 23.80 20.28

 

 

 

aJuly 13 data represents the mean of three replications.

bJuly 27 and August 10 data represents mean of five replica-

tions .

20

Table 3. -- Varietal tryptophan composition expressed as a percent-
age of the protein found in whole-plant samples.

 

 

 

 

 

Variety
Zinc
application Saginaw Sanilac
date a b
le 13 le 27 Aug 10 le 13 le 27 Aug 10

TO 2.79 2.65 3.14 2.54 2.58 3.04
T1 3.22 2.77 3.07 2.52 2.72 2.91
T2 2.84 2.89 2.95 2.63 2.72 3.40
T3 . 2.74 2.98 2.93 2.52 2.78 3.08
T4 ---- 2.74 3.02 ---- 2.73 3.09

No zinc 2.69 2.75 3.53 2.49 2.82 2.95

 

 

 

aJuly 13 data represents the mean of three replications.

bJuly 27 and August 10 data represents the mean of five rep-
lications.

separate analysis of variance for the data appearing in Tables 2 and 3
was performed for each sampling date. A summary of these analyses
is found in Table 4.

Protein percentage was greatly affected by zinc application
date for each sampling period. A significant varietal difference
occurred in total protein percentage for the July 13 sampling date.
Interactions between zinc application date and variety were not

significant for either tryptOphan or total protein percentage.

21

Table 4. -- Summary of the analysis of variance performed at three
sampling periods for tryptophan percentage of protein and
protein percentage in whole—plant tissue.

 

 

Source of Tr y PtOPhan Total protein percentage

variation

 

le 13 le 27 Aug 10 le 13 le 27 Aug 10

 

Variety * *

Z inc
application
date >:<>:< x: >:< a: *

Variety
X zinc
application

 

 

 

*--Significant at . 05 level.

*>:<—-Significant at .01 level.

Figure 4 illustrates the whole—plant protein percentage rela-
tionship between Saginaw and Sanilac for three sampling dates. Both
varieties tended to decrease in protein percentage with maturity.
When zinc was not applied to either variety, Sanilac possessed a
higher protein percentage. Zinc application lowered the protein per—
centage of both varieties. The decrease in protein as a result of
zinc application was more pronounced in the Sanilac variety. With
the exception of the preemergence treatment, a decrease in protein

of Sanilac occurred with earlier dates of zinc application. Although

22

 

 

 

 

 

 

 

 

 

 

 

26 OT 26 0—
24.5— 24.5—
.5 23.0 a 23.0
.9 '8
821 5 g 21.5
A a.
E$20.0 is 20.0
18.5 18.5
17.0 . 17.0
le 13 le 27 Aug 10 le 13 le 27 Aug 10
T1-- June 22 zinc application date T2" June 29 zinc application date
26.0—
24.
c: .5 23
'8 3
3 ° 21
L.
E 0.
is E>320.
18.
17.0 ' ‘ 17.
le 13 le 27 Aug 10 le 13 le 27 Aug 10

T3" July 6 zinc application date T4" July 13 zinc application date

Saginaw ——————

 

Sanilac

Figure 4. -- Varietal protein percentage relationships for five different
zinc treatments at three sampling dates.

% Protein
H N N [\3
CD 0 H 00

H
4

 

 

23

[\D
00

% Protein
N
H

 

 

 

 

20.
18.
0 I I 17. 0 I I
le 13 le 27 Aug 10 le 13 le 27 Aug 10
No Zinc To" June 15 zinc application date
Saginaw — — —- — —
Sanilac

 

Figure 4. -- Continued.

24

somewhat variable, the pattern of response for the protein percent-
age of Saginaw remained relatively constant among treatments. Pre-
emergence application of zinc had the effect of reducing the magni-
tude of difference in protein percentage between varieties.

On July 13 Saginaw possessed a higher tryptophan content
than Sanilac for all zinc application dates (Table 4). The varietal
difference in tryptophan percentage disappeared as the growing sea-
son progressed. An increase in tryptophan content occurred in both
varieties as a result of plant growth and development. This increase
was observed for each application date, and was more pronounced in
Sanilac. Differences in tryptophan percentage resulting from zinc

application dates were not significant for either variety.

Whole -plant zinc analysis

 

Zinc content based upon whole-plant analysis proved to be
extremely variable (Table 5). Saginaw possessed a significantly
higher zinc content for the first two sampling dates, but not on
August 10.

Correlation coefficients were small, positive, and insig-
nificant for both varieties with regard to zinc concentration and
tryptophan content (Table 6). This suggests only a small degree of

relationship between the two variables. The correlation between

25

Table 5. -- Zinc concentration in micrograms per gram of dry matter

for Saginaw and Sanilac, based upon whole-plant analysis.

 

 

 

 

 

 

 

 

 

 

 

 

Variety
Zinc
application Saginaw Sanilac
date
le 13 le 27 Aug 10 le 13 le 27 Aug 10
To 69.00a 87.80 70.80 58.42 69.60 87.60
T1 106.33 72.10 76.90 87.41 65.50 54.80
T2 104.00 72.70 93.70 63.16 63.20 68.90
T3 99.23 81.60 70.90 72.83 65.87 61.00
T4 ------ 110.00 80.80 ----- 85.00 79.70
No zinc 57.25 55.40 105.60 63.80 75.60 75.30
aData represents mean of five replications.
Table 6. -- Correlation coefficients relating zinc, tryptophan, and
protein in whole-plant tissue.
Variety
Correlation
Sanilac Saginaw
Zinc and tryptophan . 127 . 385
Zinc and protein -. 046 -. 129
Protein and tryptophan -. 877** -. 537**

 

 

 

>'.<>'.<—- Significant at the . 01 percent level.

26

zinc and tryptophan was greater for Saginaw than Sanilac. Only a
small negative relationship existed between zinc concentration and
protein percentage in both varieties.

A very strong negative correlation existed between pro-
tein percentage and tryptophan content. Although both varieties de-
creased in protein percentage and increased in tryptophan content over
the time-course of the experiment, the tendency proved greater for
Sanilac (Tables 2 and 3).

Zinc application vs. protein
content of Sanilac

 

 

A larger quantity of protein occurred in plants treated
with zinc at T0 (Figure 5). When zinc was applied at T1 the rate of
protein accumulation during July was high when compared to the other
treatments, but decreased markedly during early August. Only a

small variation in protein content was observed when zinc was

applied subsequent to T1.

Zinc application vs. protein
content of Saginaw

 

 

Protein content remained low at all three sampling dates

when zinc was applied at T0 and T1. A moderate increase occurred

with the later treatments. Plants remaining untreated contained a

27

   

 

 

 

 

 

 

 

 

 

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‘a
CU -- H p-
32. O Q 2. O
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8.1. 9‘ 1.
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8 1. 8 1.
a. o.
"’ E
E
8 8
O (D
0 I l 0 l 1
le 13 le 27 Aug 10 le 13 le 27 Aug 10
To" June 15 zinc application date T1" June 22 zinc application date
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45 +9
3 2. E 2.
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8 8
O (D
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T2“ June 29 zinc application date T3“- July 6 zinc application date
Saginaw —————
Sanilac

Figure 5. -- Varietal protein quantity relationships for several dif-
ferent zinc treatments at three sampling dates.

28

 

 

 

 

 

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L. L.
8, 8.
c: 1. .5 1.
.0...) 3
*5 0
El. ‘5. 1.
"’ ‘8
8
8 8
(5 CD
0 1 1 o ’ I i
le 13 le 27 Aug 10 le 13 le 27 Aug 10
T4" July 13 zinc application date No Zinc

Saginaw P—— — —

Sanila c

Figure 5. -- Continued.

29

larger quantity of protein on August 10 than any particular zinc treat-

ment.

Seed tryptophan analysis

 

The percentage of tryptophan found in total seed protein for

four zinc application dates is presented in Table 7. A functional

Table 7. -- Varietal mean tryptophan percentages for four zinc
application dates.

 

 

 

 

 

Zinc Variety
application Treatment mean
date Saginaw Sanilac
June 15 1.94 1.82 1.88
June 29 1.97 2.25 2.11
July 13 2.21 2.26 2.24
July 27 2.36 2.48 2.42
Varietal mean 2. 12 2. 21

 

 

 

 

analysis of variance summarizing the data in Table 7 appears in
Table 8.

Significant differences between varieties regarding trypto-
phan percentage could not be discerned. The variety—treatment
interaction was not significant, which would indicate that both vari-

eties displayed the same pattern throughout the analysis.

30

Table 8. -- Functional analysis of variance for tryptophan composi—
tion of seed protein.

 

 

 

 

Source 8.8. D. F. M.S. F.
Variety 0. 0368 1 0. 0368 0. 6095
Replication 0. 1982 2 0. 0991 1.6404
Error A 0. 1208 2 0.0604
Zinc application date 0. 9393 3 0.3101 14. 6004***
(A) 1 .5370 26. 85 **
(B) 1 .3450 17. 25 *4
(C) 1 .0460 2. 30
Variety X zinc . 1178 . 3 1.85
Error B 12
Total 1. 6588 23

 

 

 

 

 

(A)--July 27 vs. the average of June 15, June 29, and July 13.
(B)—-June 15 vs.‘ the average of June 29 and July 13.
(C)--June 29 vs. July 13.

**—-Significant at . 01 level.

***-—Less than . 01% level of significance.

A very significant difference in tryptophan composition due
to zinc application was detected in the seed of both varieties. There-
fore, a set of orthogonal comparisons was used to render the dif-
ferences meaningful. Seed from the July 27 application date was
significantly higher in tryptophan than the average of all other dates.

When zinc was applied immediately after planting, the tryptophan

31

percentage proved to be significantly lower than the average of the
June 29 and July 13 dates. No significant difference in seed trypto—
phan percentage could be found between the June 29 and July 13
application dates.

Zinc content was less variable between treatments and
varieties when based upon seed analysis (Table 9). The difference

Table 9. -- Seed zinc content of Saginaw and Sanilac as affected by
four different dates of zinc application.

 

 

 

 

Variety
Zinc application date
Saginaw Sanilac
TO 37. 23 39. 66
T2 34. 91 53. 83
T4 45.16 40. 50
T6 39.25 40. 50

 

 

 

between varietal means was not statistically significant. No dif-

ference between treatment means were observed.

Seed protein analysis

 

Seed samples from plants selected from zinc application
dates were analyzed for protein'percentage. Results from the pro-

tein analysis are summarized in Figure 6.

32

 

 

 

25 r
24 —
.S
8
8 23 _
a.
83
/
/
\ /
\\\ /
\\\\\ //
\\ /
\
22 — \ \ \ //
\ /
\V
21 l L I
June 15 June 29 July 13 July 27
Saginaw ——————
Sanilac
Figure 6. -- Seed protein percentages for Saginaw and Sanilac when

treated with zinc on four different application dates.

33

The Sanilac variety was higher in seed protein percentage
for all treatment dates selected. Both varieties followed the same
general pattern over all treatment dates. Seed protein percentages
decreased with the delay of treatment date until July 13, and increased
markedly for the July 27 treatment date.
Table 10. —— Correlations between zinc concentration, tryptophan

content, and protein percentage in the seed of Saginaw
and Sanilac.

 

 

 

 

Variety
Correlation
Saginaw Sanilac
Zinc and tryptophan +. 180 -. 116
Zinc and protein +. 661’!< -. 048
Protein and tryptophan +. 422 -. 335

 

 

 

*--Significant at .05 percent level.

With one exception, no meaningful relationships were found
between zinc content, tryptophan percentage, and protein percentage
in the seed of either variety. A rather substantial and significant
correlation was recorded between zinc content and protein percentage
for Saginaw. It is interesting to note that all coefficients were posi-

tive for Saginaw and negative for Sanilac.

DISCUSSION

Plant growth and development

 

Sanilac demonstrated a need for supplemental zinc soon
after emergence. Plants showing deficiency sumptoms took approxi-
mately two weeks to recover after zinc was applied. Conversely,
Saginaw lacked deficiency symptoms in the early stages of growth
and showed little response to added zinc. This would suggest that
the requirement for zinc in the early developmental stages is more
critical for Sanilac than for Saginaw.

Soils with a large clay content tend to lose their structure
when wetted. Lack of macro—porosity upon redrying reduces aera-
tion of plant roots, resulting in poor nutrient uptake. It is believed
that reduced growth of Saginaw experienced when zinc in solution
was applied early was caused by altered soil structure. Develop-
ment of a more extensive root system before zinc application enabled
the plants to deal more effectively with the aeration problem.

Several possibilities exist to explain why Sanilac reacted
favorably to early zinc treatment: (1) A difference in tolerance to

low levels of soil aeration exists between varieties, especially

34

35

during the early growth period. (2) The need for zinc was the most
limiting factor for successful plant growth of Sanilac. (3) A combi-

nation of statements (1) and (2).

Protein analysis

 

Early postemergence zinc treatment permitted normal plant
development of Sanilac (Figure 3). Plants receiving no supplemental
zinc flowered two weeks later than those treated at T1 and T2.

The effect of zinc application upon plant maturity of Sanilac
was also reflected in foliage protein percentage. With the exception
of T0’ increased protein percentage occurred with extended zinc
stress. One of the most pronounced symptoms of zinc deficiency is
a tendency for the plants to remain at a juvenile stage of growth.

The protein percentage of young whole-plant tissue tends to be higher
than in older tissue, which possesses a greater amount of carbo-
hydrate in the form of secondarily thickened cell walls. A clear
effect of zinc application upon the protein percentage of Saginaw was
not discerned.

Several workers have demonstrated that protein synthesis
continues throughout the main course of cell division and enlarge-
ment (15). This would explain why patterns observed for the

total quantity of protein per plant strongly reflected the dry matter

response.

36

Tryptophan analysis

 

Both varieties increased in seed tryptophan content with
successively later dates of zinc application. Tentatively, this can
be explained by principles of plant development. Early in the season,
growth is taking place at a rapid rate. TryptOphan is being utilized
in large quantities in regions of meristematic activity. As the
growth rate decreases with increased plant maturity the developing
reproductive structures become the major recipients of plant
metabolites, such as tryptOphan. Any increase in tryptophan synthe-
sis would be detected in the developing seed.

Results obtained from the seed tryptophan analysis suggest
the possibility of altering the tryptophan content of bean seeds by
manipulating the available zinc supply in a field situation.

Results obtained from the foliage tryptophan analysis proved
to be inconclusive. A natural tendency for protein to increase in
tryptophan composition with plant development may have masked any
effect that zinc had upon tryptophan content. Analysis of a particular
organ taken from a specific position on the plant may have been more
effective.

Relationships between zinc,
tryptophan and protein

 

 

Negative correlations observed between protein percentage

and tryptophan content were expected, based on the data of Tables 2

37

and 3. The protein percentage decreased with advanced maturity,
while the quality of this protein was changed by increases in trypto-
phan composition. Steward and his co-workers found that plant
proteins are dynamic with respect to composition.

A positive correlation was observed between zinc content
and protein percentage in the seed of Saginaw, whereas practically
no relationship between the two variables was recorded for Sanilac.
These results, while statistically significant, are difficult to inter-
pret biologically. If zinc were closely related to protein synthesis
for different plant species, as supported by the literature, it would
seem logical to expect the same relationship to exist between varie—

ties of the same genus and Species.

SUMMARY AND CONCLUSIONS

Several plant responses were evaluated in two varieties of
navy beans (Saginaw and Sanilac) under differential conditions of
zinc stress in the field. Whole—plant and seed analyses for percent-
age and total protein, tryptophan composition, and zinc content were
performed at three different sampling dates during the growing
season.

(1) Saginaw and Sanilac responded differently in a field Situa-
tion of low zinc availability. Sanilac required greater
amounts of zinc in the early stages of growth than could be
supplied naturally by the soil, whereas Saginaw developed
normally during this time.

(2) The tryptophan content of both varieties, based upon whole—
plant analysis, increased with advancing plant maturity.
This phenomenon may have obscured any strong zinc-
tryptophan relationship.

(3) The seed of both varieties proved to be a more precise
indicator of tryptophan variability attributed to zinc treat-

ment.

38

 

(4)

(5)

(a)

(b)

39

An increase in seed tryptOphan content was observed in
both varieties when zinc was applied at successively
later dates.

Water applied with the zinc may have interacted to pro-

duce at least some of the effect attributed to treatment.

Relationship between zinc, tryptophan, and protein.

(a)

(b)

(C)

No meaningful relationships between zinc and trypto-
phan were observed in the seed or in whole-plant
tissues of either variety.

Inconsistent relationships with respect to variety were
recorded between zinc content and protein percentage in
the seed. Saginaw possessed a strong positive relation-
ship, while no relationship was observed in Sanilac.
Negative relationships between tryptophan and protein
percentage were found in whole-plant tissue of both

varieties. This was a consequence of plant maturity.

In Sanilac, higher whole—plant protein percentages were

associated with longer periods of zinc stress, with the

exception of treatment at planting time, while the protein

percentage of Saginaw remained stable among zinc treat-

ments.

40

(6) Total quantity of protein per plant was increased when zinc
was applied to Sanilac. A decreased amount of protein per

plant occurred with zinc application to Saginaw.

 

LITERATURE CITED

Association of Official Agricultural Chemists. Methods of
analysis. Washington, D.C. 7th edition. 1950.

Bonner, James, and J. E. Varner. Plant biochemistry.
New York. Academic Press. 1054 pp. 1965.

Greenberg, J. B., and A. W. Galston. Tryptophan synthetase
activity in pea seedling extracts. Plant Phys. 34: 489-494.
1959.

Hall, T. C., and E. C. Cocking. Studies on protein synthesis
in tomato cotyledons and leaves. II. Intermediate stages of
protein synthesis. Plant and Cell Physiology. 7: 343-356.
1966.

Hartman, Philip E. , and Sigmund R. SuSkind. Gene action.
Prentice-Hall, Inc. New Jersey. 158 pp. 1965.

Kessler, B. Ribonuclease as a guide for the determination of
zinc deficiency in orchard trees. 314-321. 1961. Ed. by
Walter Reuther. Plant analysis and fertilizer problems.
American Institute of Biological Science. Washington 6, D. C.

Key, Joe L., N. M. Barnett, and C. Y. Lin. RNA and protein
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Annuals of the New York Acad. of Sciences. 144: 49-62. 1967.

Klein, Richard M. , Emerita M. Caputo, and Barbara A.
Witterhold. The role of zinc in the growth of plant tissue
cultures. Amer. J. of Bot. 49: 323-327. 1962.

Mosev, Nikola, and Milan Kutacek. The effect of zinc on the

biosynthesis of tryptOphan, indole auxins, and gibberellins in
barley. Biologia Plantarum. 8(2): 142-151. 1966.

41

10.

11.

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l4.

15.

16.

17.

42

Mudd, J. B., and S. Zalik. The metabolism of indole by
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Nason, Alvin, Nathan 0. Kaplan, and Sidney P. Colowich.
Changes in enzymatic constitution in zinc-deficient Neurospora.
J. of Biological Chemistry. 188: 396-406. 1951.

 

Roth, H. , and Schuster P. Agew. Tryptophan analysis.
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Skoog, Folke. Relationships between zinc and auxin in the
growth of higher plants. Amer. J. of Bot. 27; 939-950. 1940. \

Smith, Oliver H. , and Charles Yanofsky. Enzymes involved in
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Steward, F. C., and D. J. Durzan. Metabolism of nitrogenous
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Tsui, Cheng. The role of zinc in auxin synthesis in the tomato
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Wegener, Warner, and Antonio Romano. Zinc stimulation of
RNA and protein synthesis in Rhizapus nigricans.

 

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