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thesis entitled

Enzyme Activity, and Ethanol Accumulation as
Related to Zinc Uptake in Four Varieties of
Beans (Phaseolus Vulgaris L.) Under an

 

Aeration Stress.

presented by

Normah Mohamad Noor

has been accepted towards fulfillment
of the requirements for

M.S.

. Cr & S 11
degreem op o

 

 

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ENZYME ACTIVITY AND ETHANOL ACCUMULATION
AS REIATED TO ZINC UPTAKE IN FOUR VARIEI‘IES
OF BEANS (figSEOLUS moms L.) UNDER
AN AERATION STRESS

By

Normah Mohamad Noor

A THESIS

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

MASTER OF SCIENCE
Department of Crop and Soil Sciences
1979

ABSTRACT

ENZYME ACTIVITY AND ETHANOL ACCUMULATION
AS RELATED TO ZINC UPTAKE IN FOUR VARIETIES
OF BEANS (PHASEOLUS VULCARIS L.) UNDER
AN AERATION STRESS

By

Normah Mohamed Noor

Relationships between alcohol dehydrogenase and pyruvate decarboxy-
lase activities. ethanol accumulation, and zinc concentration in the roots
grown under aerated and anaerobic conditions were studied. A green-
house experiment was conducted using four varieties of Phaseolus
vulgaris L.; Seafarer, MSU 31908, NEP-Z, and San Fernando grown at O and
0.2 ppm levels of zinc. An anaerobic condition was established by
flooding at flowering for thirty-six hours.

Variety Seafarer had the highest ethanol accumulation, the highest
alcohol dehydrogenase activity and the highest zinc concentration in the
roots. MSU 31908 had the least response to treatments except for
greater pyruvate decarboxylase activity. NEP-Z and San Fernando showed
intermediate responses. Alcohol dehydrogenase activity and ethanol
accumulation were hypothesized to be correlated with zinc concentration
in roots. The varietal differences were then explained according to

the developed hypothesis.

To my father.

ii

ACKNOWLEDGMENTS

The author wishes to express her gratitude to:
Dr. M. W. Adams for his guidance and encouragement during the
course of her study.
Dr. A. J. M. Smucker for his constructive criticism.
Dr. J. Kofmann and Dr. G. Safir'as members of the guidance committee.
The Training Division, Majlis Amanah Rakyat (M.A.R.A.) of Kuala
Lumpur, Malaysia, for their financial support during her stay in the

United States.

iii

LIST OF TABLES . . . .
INTRODUCTION . . . . .
LITERATURE REVIEW . .
MATERIALS AND METHOD .
Enzyme Extraction
Protein Analysis
Ethanol Analysis
Zinc Analysis . .
RESULTS AND DISCUSSION
SUMMARY AND CONCLUSION
APPENDIX . . . . . . .

LITERATURE CITED . . .

TABLE

OF CONTENTS

iv

Page

occur)

10
1o
11
13
23
25
29

Table

LIST OF TABLES

Alcohol dehydrogenase activity of Seafarer, MSU 31908,
NEP-Z, and San Fernando grown at two levels of zinc

and subjected to two levels of flooding (Greenhouse
experiment, February - May 1979) . . . . . . . . . . . .

Zinc concentrations in roots of Seafarer, MSU 31908,
NEP-Z, and San Fernando grown at two levels of zinc
and subjected to two levels of flooding (Greenhouse
experiment, February - May 1979) - . . . . . . . . . . .

Analysis of variance of zinc concentrations in the
roots (Greenhouse experiment, February - May 1979) . . .

Ethanol concentrations in xylem exudate of Seafarer,
MSU 31908, NEP—Z, and San Fernando grown at two levels
of zinc and subjected to two levels of flooding
(Greenhouse experiment, February - May 1979) . . . . . .

Pyruvate decarboxylase activity of Seafarer, MSU 31908,
NEP-Z, and San Fernando grown at two levels of zinc and
subjected to two levels of flooding (Greenhouse experi-
ment,February-May1979)-..............

ADH and PDC activities, ethanol concentration and zinc
concentration in Seafarer (Greenhouse experiment,
February " May 1979) e e e e o e e e e e e e e e e e e o

ADH and PDC activities, ethanol concentration and zinc
concentration in MSU 31908 (Greenhouse experiment,
February - May 1979) e e e e e e e e e I e e I I e e e e

ADH and PDC activities, ethanol concentration and zinc
concentration in NEP-Z (Greenhouse experiment,
Februazy - May 1979) e e e e e e e e e e e e e e e e e e

ADH and PDC activities, ethanol concentration and zinc
concentration in San Fernando (Greenhouse experiment,
Febmry-May1979)eeeeeeeeeeeeeeeeee

1n

15

17

18

20

25

26

27

28

INTRODUCTION

Anaerobic conditions in soil can be caused by a period of heavy
rainfall. When a soil is flooded, oxygen concentration in the soil
becomes limiting for plant growth. The anaerobic condition is en-
hanced especially in a compacted soil where poor drainage exists.

Oxygen deficiency for a one day period can have a great influence
on growth and on yield of bean plants (Smucker, 1975). Navy beans
were found to be susceptible to poor soil drainage and to aeration
stress, especially at the pre—blossom stage. Twenty-four’and forty-
eight hours of flooding significantly reduced beanyield (Smucker
ei al- , 1978).

Navy beans (Phaseolus vulgaris var. Seafarer) also produce a
large quantity of ethanol during Short periods of oxygen stress to
plant roots and with greater accumulation when the stress is imposed
at the flowering stage. The production of ethanol is due to the limited
oxidation in the mitochondria, thus inducing this anaerobic respira-

tion reaction:

 

 

C02 NADH NAD+
Pyruvate / ; Acetaldehyde \ f > Ethanol .
Pyruvate Alcohol
decarboxylase dehydrogenase

An increase in alcohol dehydrogenase (ADH) and pyruvate decarboxy-

lase (PDC) activity is associated with the production of ethanol.

Alcohol dehydrogenase in turn requires zinc as a component of its
active structure, while pyruvate decarboxylase requires Mg** as its
cofactor. Since zinc deficiency is one of the major'production problems
for growing beans in Michigan, and the bean crop is always fertilized
with a zinc-containing fertilizer, a correlation of ethanol production
and alcohol dehydrogenase with zinc uptake can be postulated.

Thus, the objectives of this research were: (1) To determine the
activity of enzymes ADH and PDC in four varieties of field beans
(Phaseolus vulgris L.); i. Seafarer, ii. MSU 31908, iii. NET—2,

iv. San Fernando under normal and aeration stress conditions. (Sea—
farer and NEP—Z have been found earlier to be susceptible to aeration
stress while the other two were more tolerant to the condition (Adams
gt al., 1977)); (2) To determine the amount of ethanol produced in the
xylem exudate; (3) To compare ethanol accumulation and ADH activity
with the amount of zinc in the roots; (A) To fit the individual
observation into a working hypothesis or a model whiCh is consistent
with field observations of other workers and the result of other

experiments.

LITERATURE REVIEW

Oxygen is a limiting factor for root and.plant growth under water-
saturated soil conditions. It has been shown that diffusion of oxygen
through liquid is in the order of 104 times slower than in air
(Steward, 1960). Gas exchange is also reduced as water films around
the root becomes larger (Grable, 1966). In a saturated soil the
oxygen concentration generally approaches zero twenty-four hours after
flooding (Van Doren, 1958; Purvis and Williamson, 1972).

Unger and Danielson (1965) suggested that reduced oxygen supply
rather than a build-up of carbon dioxide might be responsible for
reduced growth of young corn plants under poorly aerated soil.
Williamson (1970) demonstrated that tobacco was permanently injured by
flooding and this injury was primarily due to the lack of oxygen in the
root zone and not due to excess carbon dioxide. Later, Purvis and
Williamson (1972) also showed that the primary cause of injury and
reduced growth of flooded corn was due to the lack of oxygen.

Much work has been done on the effects of flooding on plant roots
and plant growth. Kramer (1951) showed that twenty-four hours of
flooding was sufficient to produce serious injury to the roots of
tomatoes, sunflower, and tobacco. Kramer also concluded that flooding
causes a rapid decrease in the capacity of roots to absorb and conduct
water. Flooding of more than twenty-four hours duration, can result in

permanent injury (Kramer and Jackson, 1954). Reduced growth of soybeans,

corn, and sorghum was noted when such cr0ps were grown at a high water-
table (Williamson, 1964). williamson (1970) also showed that pure
nitrogen treatment imposed on tobacco for twenty-four hours essentially
prevented further growth and.after forty-eight hours the roots and
Shoot were essentially dead.

A reduction in growth can and usually does lead to a reduction in
yield. Tomatoes can be severely stunted and the yield reduced if
oxygen deficiency occurs early in the life of the plant (Erickson and
Van Doren, 1960). They also showed that yield of peas can be reduced
by one-third by a one-day oxygen deficient period at the early bloom
stage. Oxygen deficiency also decreased corn, sorghum and sugarbeet
yields (Williamson, 1969; Erickson and Van Doren, 1960). The magnitude
of the reduction in yield, however, depended on the species and variety
of the plant and its stage of development as well as on light, tempera-
ture, fertility, etc. (Erickson and Van Doren, 1960).

Kramer (1951) sUggested that injury of the roots and death of the
leaves may be caused at least in part by toxic substances moving up
from the dead roots or even from the surrounding soil. Kenefick (1962)
found that ethanol accumulated in sugar beet under oxygen stress.
Later, Fulton (1963) showed that ethanol appeared in xylem exudates of
tomatoes when the soil supplied less than 38 x 10—2 ug oxygen cm-Z

min-1

and the ethanol concentration increased as supply of oxygen was

further reduced. Ethanol concentration of xylem exudate samples taken
from plants flooded in the light was greater than from plants flooded

in the dark (Fulton and Erickson, 1969).

In Crawford's (1967) study, there was an increase in ethanol

production in flood-sensitive plants. However, in rice, a flood-tolerant

plant , ethanol was also produced under anaerobic conditions (App and
Meiss, 1958; John and Greenway, 1976, Avadhani pp a_I_., 1978). Natural
anaerobiosis during germination also induces ethanol production
(Leblova e_t g._l_. , 1969; Leblova _e_t pl. , 1971+; Avadhani gt al- , 1978).

Ethanol was shown to be toxic to tomato plants when added to
Hoagland water culture media with concentrations corresponding to those
observed in the xylem exudates (Fulton, 1963). Kiyosawa (1975) showed
in Nitella that alcohol molecules interact with the cell membranes to
make equivalent pore radii of the membranes narrower without changing
the nature of the water flow, causing a decrease in water permeability.
This might be the cause of the reduction of the absorption and trans-
location of water by bean plants in an anaerobic condition, as shown by
Smucker (1975).

App and Meiss (1958) showed that ADH (alcohol dehydrogenase)
activity increased in proportion to ethanol production. ADH activity
has also been reported to increase during flooding in flood-sensitive
plants (Crawford, 1967), in corn seedlings (Hageman and Flesher, 1960),
in flood-sensitive subspecies of Trifolium subterraneum (Francis _e_t_ pi. ,
1974), and in rice (John and Greenway, 1976; Wignarajah _e_Ԥ_ al- , 1976).
ADH activities were also found in natural anaerobiosis of germinating
seeds (Cossins and Turner, 1962; Kolloffel, 1968; Leblova _e_i_:_ _a_l_. ,

1969; Leblova pp pl. , 1974).

John and Greenway (1976) also showed that an increase in PDC
(pyruvate decarboxylase) activity in rice is maximum after twenty-four
hours of anaerobiosis. They also showed that absolute activities of
PDC were about fifteen times lower than for ADH and the degree of

increase in activity in response to anaerobiosis was smaller for PDC

than for ADH.

An increase in ADH activity was found to be due to dg‘ggyg
synthesis of the enzyme (McManmon and Crawford, 1971; John and
Greenway, 1976). App and Meiss (1958) suggested that ethanol concen-
tration in rice shoots might control ADH activity, however, acetaldehyde
was found to be a natural inducer of ADH in corn seedlings by Hageman
and Flesher (1960), in Senecio intolerant species by Crawford and
McManmon (1968), and in germinating seeds by Leblova 93 gl_. (1974).

John and Greenway (1976) suggested that PDC activity may be en-
hanced by an increase in NADH levels and a decrease in NAD+ levels,
which occur during anaerobiosis.

Vallee and Hock (1955) showed that the ADH of yeast is a zinc
metalloenzyme containing four moles of zinc firmly bound to one mole
of protein. They also showed that the activity of the enzyme is
directly dependent on zinc. Zinc atoms are thought to stabilize the
quarternary structure of the enzymes through the formation of bridges
between monomers to form the enzymatically active tetramer (Kagi and
Valle, 1960).

Zinc deficiency occurs mostly on calcareous soils (Thorne, 1957).
High pH causes the solubility of an+ in soils to decrease and thereby
reduces the uptake and availability of zinc to plants (Linsay, 1972).
High soil phosphorus levels also induce zinc deficiency by restricting
zinc movement within the plant, resulting in accumulation in the roots
and deficiency in the tops (Vitosh pp 11,, 1973).

In Michigan, zinc deficiencies have been identified in navy beans,
especially in the Sanilac variety (Robertson and Lucas, 1976). The

Saginaw variety, however, is less affected by low zinc availability in

the soil (Ellis, 1965; Polson, 1968).

Smucker (1977) later found that greater accumulation of ethanol
occurred in the xylem exudates of flooded navy bean plants having
greater quantities of tissue zinc than plants with lower concentration
of zinc. Thus, the production of ethanol in xylem exudates might be

further induced by higher application of zinc.

MATERIALS AND METHOD

Four varieties of field beans (Phaseolus vulgris L.); Seafarer,
MSU 31908, NEP-Z and San Fernando were grown in one gallon plastic milk
bottles filled with acid-washed pea-sized gravel. The gravel was
washed with distilled water twice to remove the acid.

The plants were grown in a modified Hoagland's solution
(Shellenberger, 1970) containing 300 ppm P and Zn concentrations of O
and 0.2 ppm by formulation. In actuality, the concentrations of Zn at
the low level was 0.03 ppm, and at the high level was 0.23 ppm, as
determined by analysis of leachate. Phosphorus concentration was in-
creased above Hoagland's to insure zinc deficiency. The micronutrients
were according to Hoagland with the exception of the varying Zn concen-
trations.

Each pot was watered with 300 ml of nutrient solution three to
four times a day. The nutrient solution was collected in an acid bottle
and reused for two or three days after which it was collected and
tested for zinc using a Perkin-Elmer model 303 atomic absorption
spectrophotometer.

The plants were grown in a greenhouse under natural light supple-
mented by artificial light from gro-lux tubes, providing a total
intensity of 214 uE m"2 sec~1. The photoperiod was 14 hours.

The design of the experiment was a split-plot with plots being

completely randomized. The plots were the flooding and zinc levels

while the sub-plots were the varieties.

At flowering plants were flooded with nutrient solution for 0 and
36 hours. The control plants were never under water stress as they
were watered normally. There were also no visible symptoms of water
stress appeared for both non-flooded and flooded plants.

Flooding was accomplished by stopping drainage from the pots and
completely submerging the gravel and roots. After 36 hours xylem
exudates were collected from all plants by severing the stem at the
first internode from the root and attaching a piece of surgical rubber
tubing to the excised stem. When a sufficient sample of exudate
accumulated in the tubing, the stem was cut and the open end of the
tubing was closed by folding and tying it with a piece of capper wire.
The entire sample was held in a labelled test-tube and frozen until
ready for ethanol analysis.

Shoots were weighed and dried. Roots were cleaned of gravel and

kept in a 5°C room until ready for extraction.

m Extraction 2&2 A_§_s_a_y

Root extraction procedure used for alcohol dehydrogenase (ADH) and
pyruvate decarboxylase (PDC) was essentially the same as that reported
by John and Greenway (1976). Root tissue was homogenized in a high-
speed blender. The extraction buffer (5 ml per gram fresh weight) con-
tained 50 mM HEPES (N-2-hydroxyethyl piperazine-N'-2 ethanosulphonic
acid), 5 mM MgCL2 (magnesium chloride), 2 mM cysteine hydrochloride and
2% (w/v) PVP-40 (polyvinyl pyrrolidone). TPP (thiamine pyrophosphate)
at 0.5 mM was included for extracting PDC. Extraction buffer was made
up to pH 8.0 for ADH and pH 6.0 for PDC using KOH (potassium hydroxide).

After filtration through Miracloth (Chic0pee Mills, Inc.),

lO

extracts were centrifuged fer 20 minutes at 25,000 g. Crude extracts
(supernatants) were desalted on Sephadex 025 (coarse) columns, length
2 cm and column width 1 cm. The first 2 ml was collected for assay.

Residues were dried for zinc determination.

Enzymes were assayed at 28°C by spectrophotometric determination of
oxidation or reduction of pyridine nucleotides at 340 nm. ADH assay con-
tained 0.48 mM pyr0phosphate buffer, pH 8.8, 1 mM ethanol, 0.025 mM NAD
(Nicotinamide adenine dinucleotide), and 0.1 ml of enzyme (Worthington,
1978). PDC assay contained 0.186 M citrate buffer,;pR 6.0, 30 mM
pyruvate, 0.32 mM NADH (reduced nicotinamide adenine dinucleotide), 33
ug/ml of ADH and 0.02 ml of enzyme solution (Bergmeyer, 1970).

A minute was calculated from a linear portion of the curve.

Enzyme activity was determined by the following calculation:

A min
Units/mg PIOtein 3 6.2 x.mg proteih7ml reaction mixture

 

Protein Analysis
The method of Lowry gt gl. (1951) was used for*protein analysis.
Protein assay consisted of 1 ml of Reagent C (alkaline capper solution

which is a mixture of 50 ml of Reagent A (2% NaZCO in 0.10 NaOH) and

3

1 ml of Reagent B (0.5% Cuso4 - 5H20 in 1% solution tartrate)), 0.10 ml

of Reagent E (diluted Folin reagent), and 0.2 ml of protein sample.
Readings were made at 660 nm on a Beckman DU spectrophotometer.

Protein values were calculated from a standard curve.

Ethanol Analysis
1 microliter of the xylem exudate was injected in the column of

gas chromotograph:

11

Model: Beckman GC 72-5

Column: 6' x %"

Packing: PPQ 100/200 mesh

Temperature: 150°C

Flow rate: 60 ml/minute

Detector: Hydrogen flame

The recorder was equipped with an integrator. Ethanol concentra-
tion was calculated by counting the number recorded by the integrator

for standards and for samples and using the equation:

_ unknown
Ethanol ppm — SIEREEEE x ppm of the standard

Zinc Analysis

Root residues were ground in a Wiley mill to pass a 20-mesh screen.
Samples of one half to one gram were used. They were dry-ashed in
porcelain crucibles at 500°C for four hours. The ash was moistened
with deionized water and was taken up in 5 ml of'ZN HCl and filtered
through a Whatman No. 2 filter*paperu Four rinsings of 10 ml each were
made with deionized water: the crucible twice, the filter'paper once
and the funnel once. The final solution volume was made Up to 50 ml
with deionized water. The resulting solution was then analyzed for
zinc with a Perkin-Elmer model 303 atomic absorption spectrOphotometer.

Absorption readings were converted to absorbence by a formula,

2- log A. This was done by drawing a standard curve on a one-cycle
semi-log paper with the numbering reversed. Concentrations of zinc
were then calculated from this standard curve.

Concentration for the amount of tissue used in microgram/gram

was calculated by the equation:

12

Vol
ume of solution

g of sam
ple used x co
nc
entration of soluti
on -
— micro
gram/gram

RESULTS AND DISCUSSION

Due to experimental variability not attributable to replications,
no statistical analysis was performed on enzyme and ethanol responses.

Results on alcohol dehydrogenase (ADH) activity (Table 1) show
that for varieties Seafarer, NET-2, and San Fernando, there were
increases in the activity in the roots of flooded plants grown at
high levels of zinc. The degree of increase was highest with Seafarer,
followed by NET-2 and San Fernando, respectively. MSU 31908 did not
show any ADH activity at either level of treatment.

ADH activity was also found in flooded plants grown at the low zinc
level but the activity was not as much as for those that grew at the
high zinc level.

The presence of ADH activity at 0 level of flooding was presumably
due to some anaerobic microsites in the pots, thus causing anaerobic
respiration in some parts of the roots.

As can be noted in Table 1 and Table 2, there was a discernible
relationship between ADH activity and zinc concentration in the roots.
Seafarer again contained the most zinc while MSU 31908 contained the
least. This agrees with Vallee and Hoch (1955) who reported that
activity of ADH in yeast was directly dependent on zinc. The difference
in the amount of zinc in the roots for flooded and non-flooded plants
was probably due to greater utilization of zinc by plants growing in an

aerobic condition than when growing in an anaerobic condition. Also,

13

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16

oxygen stress might have reduced translocation of zinc to the top,
resulting in greater accumulation in the roots under anaerobiosis.

Analysis of variance of zinc concentrations (Table 3) shows that
there are significant varietal differences in the amount of zinc in the
roots. There are significant interactions between flooding and variety
and also between zinc and variety.

For MSU 31908, which did not show any ADH activity, the differences
in the concentration of zinc at different levels of treatment were not
as much as for the other varieties which showed.an increase in ADH
activity.

Table 4 shows ethanol concentrations in xylem exudate of the four
varieties. Seafarer had the highest ethanol production under anaerobic
conditions. However, the difference in ethanol production at low zinc
and at high zinc was not as great as the difference in ADH activities
at the same levels. San Fernando at high zinc level produced higher
ethanol than NEP-Z at the same level when flooded.

Loss of ethanol through root exudates into the rhizosphere has been
reported by Bolton (1966) and Smucker and Erickson (1976). This then
might be the cause of the low concentration in the exudate and also
might be the cause of the uncorrelated values of ethanol to ADH
activities for*NEP-2 and.San Fernando.

MSU 31908 accumulated some ethanol which was the least among all
varieties.

The difference in the ethanol concentrations also might be due to
the varietal difference in the flow rate of the exudate. Seafarer’had
the highest flow rate while MSU 31908 had the least. Ethanol concentra-

tion in Seafarer then would be diluted more than that of NEP-Z,

Table 3. Analysis of variance* of zinc concentrations in the roots

(Greenhouse experiment, February - May 1979).

 

 

 

Source df Mean Square F
Flooding 1 1 . 756 319. 27***
Zinc 1 0.813 1&7.81***
Flooding x Zinc 1 0.018 3.35 +
Error (a) 20 0.006
Variety 3 0.719 153.04***
Flooding X Variety 3 0.06).; 13.6249”
Zinc x Variety 3 0.018 3.72 +
Flooding X Zinc

X Variety 3 0.021 4.u7**
Error 60 0.005

 

* Using transformed data (log x)
** Significant at at: 0.01

*** Significant at at = 0.005

+ Significant at an: 0.025

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19

San Fernando and MSU 31908, respectively.

Pyruvate decarboxylase (PDC) activities for each variety (Table 5)
were found to be higher with flooding. However, there was no general
pattern between activities for those plants grown at low zinc and for
those grown at high zinc level.

Roots of the variety MSU 31908 had the greatest PDC when flooded.
These differences, however, were small. During flooding, the reaction
for these varieties might have st0pped at acetaldehyde. Acetaldehyde
had been shown to induce ADH synthesis (Crawford and McManmon, 1968;
John and Greenway, 1976), but McManmon and Crawford (1971) suggested
that for tolerant plants acetaldehyde might cause negative feedback and
thus stop the reaction from pyruvate to acetaldehyde.

Seafarer, which was reported earlier to be sensitive to flooding,
had the highest increase in ADH activity and also had the highest con-
centration of zinc in the roots, while MSU 31908, which was reported to
be a non-ethanol producer, had the least ADH activity and had the least
concentration of zinc in the roots. Between NEP-Z and San Fernando,
there were small differences in the responses though NEP-Z Showed a
slightly higher ADH activity and also had a higher zinc concentration
than San Fernando. The similar responses of these two varieties are
presumably attributable to the fact that they are genetically closely
related (NEP-Z is a mutant of San Fernando).

These results show a general pattern or'a framework that suggest
a relationship of zinc uptake to ADH activity and ethanol production.
Consequently, it may be postulated that a plant sensitive to an
anaerobic condition could have a glycolytic pathway to pyruvate, and

acetaldehyde, with ethanol being the final electron acceptor. ADH

20

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21

activity and ethanol production would correlate with the amount of zinc
taken up by the roots. A tolerant plant to an aeration stress, however,
would accumulate less zinc in the roots, resulting in a limited ADH
activity and ethanol production. The metabolic pathway of a tolerant
plant also could possibly step at acetaldehyde which then acts as an
end product inhibitor, and diverting pyruvate into organic acids
production (McManmon and Crawford, 1971).

Variety Seafarer showed a fully consistent result to the suggested
hypothesis. There was a large increase of ADH activity, there was a
large amount of ethanol produced during flooding and also there was a
high amount of zinc uptake by the roots. For this variety, NADH may
have increased due to an increase in glycolysis in order to meet the
demand for ATP (adenosine triphosphate). The increase could have
enhanced the PDC activity causing the formation of acetaldehyde which in
turn might induce ADH activity. ADH which required zinc for its
activity then presumably catalyzed the reaction from acetaldehyde to
ethanol.

Results with MSU 31908 agree with the suggested model for a
tolerant plant, in the sense that it had no or very little ADH activity,
it produced small amounts of ethanol during anaerobiosis and it also
accumulated small amounts of zinc in the roots. Due to the limited
amount of zinc present, the functional metalloenzyme molecule of ADH
[(ADH) Znn] might not be formed thus leading to no ethanol production.

Another possible reaction to have taken place in a tolerant plant
would have involved acetaldehyde end.product inhibition. Relatively
high PDC activity suggested that there was a reaction from pyruvate to

acetaldehyde. Acetaldehyde in this case, presumably acted as an

22

inhibitor and failed to induce ADH activity. Organic acids such as
malate, oxaloacetate, and shikimate might be the products of anaerobic
respiration such as reported by Crawford and Tyler (1969) and Tyler and
Crawford (1970). These organic acids, particularly malate and pyru—
vate, were also found to be inhibitors for.ADH (Leblova gt a;., 1977).
These organic acids produced are not toxic to plants and can be
fruther'metabolized when oxygen is restored.

For>NET-2 and San Fernando, the responses were intermediate
between those of Seafarer and MSU 31908. The reaction from pyruvate
to ethanol probably occurred but the ADH activity and ethanol
production were much less than those of Seafarer. This presumably was
due to a lesser amount of zinc in the roots. Acetaldehyde may still
induce ADH activity, especially in NEP-Z, however, with the amount of
zinc accumulated, ADH activity was assumed to be lower than those of
Seafarer. High PDC activity in San Fernando may also cause an acetalde-
hyde negative feedback and this could lead to the production of organic
acids such as suggested for MSU 31908. ERhanol produced in these
varieties also COUld have been excreted and also further metabolized
such as suggested by Cossins and Turner (1962).

The presence of isoenzymes of ADH such as found in maize (Marshall
gt a;., 1973). can also be suggested for the differenCes in the

response by the above varieties.

SUMMARY AND CONCLUSION

Short-term aeration stress has been implicated to be the cause of
yield reductions in beans. Ethanol is produced due to an induction of
or activation of alcohol dehydrogenase. This enzyme requires a zinc
cofactor for its activity.

In this greenhouse experiment, an attempt was made to develOp a
hypothesis for the relationship between ethanol production, alcohol
dehydrogenase and pyruvate decarboxylase activities, and zinc uptake
in four varieties of Phaseolus vulgaris L., namely, Seafarer, MSU 31908,
NET-2, and San Fernando under aerated and anaerobic conditions.

Seafarer had the highest alcohol dehydrogenase activity, the
highest ethanol accumulation, and the highest zinc concentration in the
roots. MSU 31908 had the lowest values of all the findings except for
pyruvate decarboxylase activity. NEP-Z and San Fernando were the inter-
mediates.

It was then hypothesized that zinc had an effect on the response
of the four bean varieties under an aeration stress. Under this
hypothesis the more zinc in Seafarer lead to a higher'alcohol dehydro-
genase activity while the lower uptake by NEP-Z, San Fernando, and
MSU 31908 presumably resulted in a lower activity of the enzyme. Also,
acetaldehyde was suggested to be an inhibitor to alcohol dehydrogenase
in a tolerant variety and.this might lead to another possible pathway,

that is, the production of organic acids.

23

20

Nevertheless, from the results obtained it can be postulated that
the increasing zinc application in fertilizer used in growing beans

might promote yield reduction under aeration stress.

APPEIN DI X

25

 

 

 

 

 

Table 6. ADH and PDC activities, ethanol concentration and zinc concen-
tration in Seafarer (Greenhouse experiment, February - my
1979)-
F'looding Zinc in Repli- ADH PDC Ethanol Zinc
(hours) nutrient cation (units/mg (units/mg (ppm/ g (micro-
solution protein) protein) dry wt.) grams/g)
(ppm)
1 0 0 0 59.0
2 O 0.073 0 59.10
0 3 0.42 0.212 0 59.50
4 0.51 0.197 0 63.85
5 1.01 0.034 0 64.0
6 0 0.139 0 63.65
0
1 0.256 0.117 1.136 79.45
2 0 0.303 0 72-73
0 2 3 0 0.149 0 70.60
' 4 2.62 0.50 0.667 79.55
5 0 0 0 70.45
6 0 0 0 70.0
1 0 1.40 3.46 90.4
2 0.034 0.169 0 87.5
0 3 0.466 0.269 6.183 95.0
4 1.965 0.811 0 102.28
5 0.209 0.126 14.71 93.18
6 0.131 0.109 11.23 90.9
36
1 1.468 0.126 0 190.9
2 1.680 0 2.60 200.0
2 0 3 9-77 0.691 3.99 220.4
' 4 0 0.737 7.79 231.0
5 16.67 1.10 14.84 225.3
6 45.62 0 17.91 233.0

 

 

26

ADH and PDC activities, ethanol concentration and zinc concen-
tration in MSU 31908 (Greenhouse experiment, February - may
1979 -

 

 

 

 

 

 

Flooding Zinc in Repli- ADH PDC Ethanol Zinc
(hours) nutrient cation (units/mg (units/mg (ppm/g (micro-
solution protein) protein) dry wt.) grams/g)

(Ham)

1 0 0 0 26.0
2 0 0 0 22.73

0 3 0 0 0 25.5

4 0 0 0 30.0
5 0 o 0 27.25
6 0 0 0 23.73

0

1 0 0 0 38.05
2 0 0 0 37.50

0 2 3 0 0 0 34.1

' 4 0 0 0 47.5

5 0 0 0 52.3

6 0 0 0 49.0
1 0 0 1.997 62.50

2 0 0 0 40.0
0 3 0 0 0 40.90

4 0 0 0 30.0
5 0 o 0 29.55
6 0 0 0 34.10

36

1 0 0.205 0 38.0

2 0 0 0 38.4

0 2 3 0 0 0 75.0
' 4 0 1.059 0 72.75
5 0 5.0 1.44 38.50
6 o 0 2.97 84.55

 

Table 8 .

27

ADH and PDC activities, ethanol concentration and zinc concen-
tration in NEP-Z (Greenhouse experiment, February - May 1979).

 

 

 

 

 

 

Flooding Zinc in Repli- ADH PDC Ethanol Zinc
(hours) nutrient cation (units/mg (units/m (ppm/g (micro-
solution protein) protein dry wt.) grams/g)
(Ppm)
1 0 0 0 46.0
2 o 0 0 47.5
0 3 0.262 0 0 50.0
4 0 0 0 49.5
5 0 0 0 48.0
6 0.80 0 0 51.0
0
1 0 0 0 77.28
2 0 0 0 70.0
o 2 3 0 0 0 72.73
. 4 0 0 0 66.0
5 0 0 0 65.9
6 0 0 0 66.0
1 0.50 0.349 0 86.38
2 1.60 2.097 0 96.59
0 3 0.183 0 0 84.1
4 0.035 0.349 0 86.5
5 0.259 0.749 15.024 134.1
6 1.0 2.097 0 86.
36
1 5.242 0.411 0 170.45
2 0.142 4.194 0 129.55
0 2 3 2.725 0 0 145.45
° 4 1.922 0 26.29 140.9
5 0.839 0.456 0 130.0
6 2.097 0 0 140.0

 

Table 9.

28

ADH and PDC activities, ethanol concentration and zinc concen-

tration in San Fernando (Greenhouse experiment, February - May
1979 .

 

 

 

 

 

 

Flooding Zinc in Repli- ADH PDC Ethanol Zinc
(hours) nutrient cation (units/mg (units/mg (ppm/g (micro-
solution protein) protein) dry wt.) grams/g)

(mm)

1 0 0 0 41.0

2 0 0 0 41.5
0 3 o 0 0 38.65

4 0 0 0 38.5

5 o 0 0 38.4

6 0 0 0 38.0

0

1 0 0 0 56.83
2 0 o 0 56.83

0 2 3 0.419 0 0 59.2

' 4 0 0 0 52.3
5 0 0 0 52.25

6 0 o 0 52.25

1 0.139 0 1.52 80.0

2 0.419 0 1.92 86.5

0 3 0 6.29 0 76.2
4 0 0 0 76.15
5 0 0 10.51 77.28

6 2.446 0 0 84.1

36

1 0 0 0 101.0

2 8.387 0 7.76 135.0

0 2 3 0 0.362 11.87 125.0
' 4 0 0 15.84 122.73

5 2.097 0.599 0 120.0

6 0 0 0 109.0

 

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