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FIXATION, TRANSLOCATION AND ROOT EXUDATION OF

14C-LABELLED ASSIMILATES BY TWO GENOTYPES OF
PHASEOLUS VULGARIS L., SUBJECTED TO ROOT ANAEROBIOSIS

 

presented by

MARTHA MARY SHADAN

has been accepted towards fulfillment
of the requirements for

Master of Sciencedegree in Crop and Soil Sciences

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Dr. A. J. M. Smucker

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FIXATION, TRANSLOCATION AND
ROOT EXUDATION OF 14C-LABELLED
ASSIMILATES BY TWO GENOTYPES
OF PHASEOLUS VULGARIS L.,
SUBJECTED TO ROOT ANAEROBIOSIS

 

By

Martha Mary Shadan

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

1980

ABSTRACT

FIXATION, TRANSLOCATION AND ROOT

EXUDATION OF 14C- LABELLED ASSIMILATES

BY TWO GENOTYPES OF PHASEOLUS VULGARIS L.

 

SUBJECTED TO ROOT ANAEROBIOSIS

BY
MarthaMary Shadan

A study was conducted to investigate the effects of short term
anaerobic stresses on total root exudation, ethanol accumulation and
CO2 respiration of two dry bean varieties under axenic conditions.’
Roots were grown in glass mist chambers and shoots were continuously
fed 1l’COZ for 11 days. Stressed plants received two 48 hour anaerobic
treatments of 20% CO2 and N2. Roots of anaerobic Seafarer and San
Fernando respired 2.5 and 2.0 times more C02 per unit dry weight root
than the aerobic controls. Ethanol was not detected for aerobic
controls but accumulated in root assimilates in response to anaerobiosis
with a greater accumulation for Seafarer than San Fernando. 0f the
14C fixed, 35% and 25% more assimilates were detected for Seafarer and
San Fernando, respectively, under anaerobic than aerobic conditions.

The exudation potentials, which included 140 exuded and 14C0 respired,

2

for Seafarer and San Fernando, increased under anaerobiosis and were

approximately 1.8 and 1.4 times greater than their aerobic controls.

TO GERRY

ii

ACKNOWLEDGEMENTS

I wish to express my sincere appreciation to Kim Carlisle for
the ethanol analysis and to Shawn McBurney for his technical

assistance throughout my study.

Special appreciation is expressed to Dr. A. J. M. Smucker for

his guidance during my thesis.

Acknowledgement is also due to Dr. D. Penner, Dr. G. R. Hooper

and Dr. S. K. Ries.

iii

TABLE OF CONTENTS

LIST OF TABLES . .'. . . . . . . . . . . .
LIST OF FIGURES. . . . . . . . . . . . . . . .
INTRODUCTION . . . . . . . . . . . . . . .
LITERATURE REVIEW . . . . . . . . . . . . .
Exudation by Roots. . . . . . . . . . . .
Ethanol Accumulation. . . . . . . . . . .
‘Physiology of the Root Under Anaerobiosis
MATERIALS AND METHODS. . . . . . . . . . . . .
Seed Sterilization and Germination. . . .
Seedling Culture and Transplanting.
Root and Shoot Chambers . . . . . . . . .
Environmental Conditions . . . . . . . .
Measurement of Carbon Dioxide . . . . . .
Root Gas Treatments . . . . . . . . . .
Radioisotope Feeding of Shoots. . . . . .
Measurement of Root Respiration . . . .
Measurement of Root Exudates. . . . . . .
14

CO2 Fixation. . . . . . . . . . .

RESULTS AND DISCUSSION . . . . . . . .,. . . .,

Plant Growth 0 O O O O O O. O O O O O O O O

Carbon-l4 Activity in Shoot and Root.

iv

Page
vi

vii

10
10
11
13
16
16
17
18
21
22
22
24
24

28

Page
Carbon Dioxide Assimilated by Shoots . . . . . . . . . . . 30
Root Respiration . . . . . . . . . . . . . . . . . . . . . 30
Carbon-14 Exuded . . . . . . . . . . . . . . . . . . . . . 34
Ethanol Metabolism, Loss and Accumulation . . . . . . . . 42
Carbon-14 Partitioning . . . . . . . . . . . . . . . . . . 45
SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . 49

LIEMTURE CITED 0 O O O O I O O O O O O O O O O O I O O O O O 51

LIST OF TABLES

Table i Page

1. Fresh and dry weights of Seafarer and San Fernando
shoots, roots and entire plants on day 11 of the
experiment. Values are an average of 8 plants in
two separate experiments . . . . . . . . . . . . . . . . . 25

2. Sterile conditions of Seafarer and San Fernando root
systems as determined by plating 0.5 ml aliquots of
nutrient solutions from mist chambers onto Potato
Dextrose Agar (PDA) and Beef Lactose Agar (BLA) for
14 days. Each symbol represents the results of two
experiments containing 100-150 seeds each. . . . . . . . . 27

3. Influence of anaerobic stress and dry bean genotype
on the total fixation and translocation of 4C
assimilates. Each value represents the 14C activity
detected in chambers containing 4 plants . . . . . . . . . 29

4. Influence of anaerobic stress and dry bean genotype on
the 14C02 assimilated by four l7-day-old dry bean plants.
Each value represents the average of 2 experiments . . . . 31

5. Carbon-14 dioxide evolved by roots of four plants of
l7-day-old dry beans. Each value represents the average
of two experiments . . . . . . . . . . . . . . . . . . . . 37

6. Effect of anaerobic stress on the root exudation of 4
plants of dry bean varieties Seafarer and San Fernando.
Each value represents the average of two experiments . . . 39

7. Total 14C activity recovered per 4 plants and its
distribution between 14C fixed, exuded and respired. . . . 46

vi

LIST OF FIGURES

Diagram of glass culture vial for growing sterile
seedlings O O O O O O O O O O I O O I O O O O O O O 0

An overview of the aseptic mist root chamber and plastic
shoot chamber system for measuring the losses of 14C-
labelled assimilates by dry bean plants . . . . . . . . .

Diagramatic representation of system for carbon-14
dioxide flow through root chambers. . . . . . . . . .

Root systems displayed in pyrex glass root chambers . . .

Carbon dioxide assimilated per day for nonstressed
plants of Seafarer and San Fernando. Each point repre-
sents the average CO concentration for each chamber of
four plants taken between 0800 hours and 2300 hours from
day 1 through day 11 . . . . . . . . . . . . . . . . . .

C02 assimilated per day for anaerobically stressed plants
of Seafarer and San Fernando. Each point represents the
average CO concentration for each chamber of four plants
taken between 0800 hours and 2300 hours from day 1

through day 11. . . . . . . . . . . . . . . . . . . . . .

Carbon dioxide respired by individual aerobic Seafarer
and San Fernando plants. Each point represents the
average C02 concentration for each chamber of 4 plants
taken between 0800 hours and 2300 hours from day 1
through day 11.. . . . . . . . . . . . . . . . . . .

Carbon dioxide respired by individual aerobic Seafarer
and San Fernando plants. Each point represents the
average C02 concentration for each chamber of 4 plants
taken between 0800 hours and 2300 hours from day 1
through day 11. . . . . . . . . . . . . . . . . . . . . .

Carbon-14 accumulated per day in the mist chamber
nutrient solution by individual aerobically and
anaerobically treated Seafarer and San Fernando plants.
Each point represents the average measurements for each
chamber of 4 plants taken between 0800 hours and 2300
hours from day 1 through day 11 . . . . . . . . . . .

vii

Page

12

15

19

26

32

33

35

36

4O

Figure

10.

11.

Page

Ratio of carbon—14 assimilates detected in the mist

chamber nutrient solution to root respiration for

aerobic and anaerobic Seafarer and San Fernando plants.

Each point represents the average measurements for

each chamber of 4 plants taken between 0800 hours and

2300 hours from day 1 through day 11 . . . . . . . . . . 41

Ethanol accumulation in the mist chamber nutrient

solution by individual aerobic and anaerobic treated

dry bean varieties. Each point represents the average

of measurements for each chamber of 4 plants taken

between 0800 hours and 2300 hours from day 1 through

day 11 . . . . . . . . . . . . . . . . . . . . . . . . . 43

viii

INTRODUCTION

An ever increasing world population has led to a greater demand
for increased food production and increased tillage practices have
followed. However, soil compaction resulting from poor soil structure
and promoted by increased tillage practices has become one of the most
limiting components of crop yield.

Soil compaction and the resulting increases in soil density
decrease the diffusion of soil gases. Greater soil density reduces
the oxygen supplying capacity adjacent to plant roots resulting in
unfavorable conditions for root growth because of both increased soil
resistance and decreased gas exchange (Tackett and Pearson, 1964).

Plant roots consume large quantities of oxygen and at 25C may
consume 9 times their volume of oxygen gas each day (Greenwood, 1969).
Under oxygen stress, aerobic respiration and the production of plant
energy are reduced while anaerobic respiration increases. Anaerobic
respiration converts pyruvic acid formed during glycolysis into
ethanol which has a toxic effect on plant growth (Grineva, 1963).

Soil aeration is also necessary for the active absorption of water
and minerals by roots. .

Recent evidence suggests that soil compaction and the concomitant
periods of oxygen stress could increase root exudation of plants
(Grineva 1961, Smucker 1971, Barber and Gunn 1974). It also appears

that a large quantity of photosynthates produced by legumes may be

lost through root respiration and exudation, resulting in both reduced
plant growth and yields. Preliminary studies of dry beans suggests
that soil aeration stress promotes root exudation and that the loss of
photosynthates through root exudation may differ among genotypes.

Correlations have been found between root excretions and coloniza-
tion of the root surface by microorganisms (Rovira 1956, Rovira 1965a
and 1965b, Vancura and Hovadick 1965a). Root exudation appears to
influence the absorption of inorganic nutrients by plants (Barber,
1968). Although much work has been done on the composition of root
exudates little is known of the total quantity which is exuded by
dry bean roots and the differences in the total exudation losses among
varieties.

This study was designed to quantitatively measure the influence

of short periods of anaerobiosis upon photosynthate losses by roots

 

of Phaseolus vulgaris, L3 varieties Seafarer and San Fernando.
Objectives of this study were:

1. To develop axenic conditions for the growth of dry bean
seedlings in solution and mist cultures.

2. To develop a system which would allow shoots, completely
sealed from their roots, to receive 14CO2 gas. This system would
provide a method for sampling the sterile nutrient solution, which
continuously washed the roots, for root exudation of carbon-l4
assimilates.

3. To determine the effect of short term aeration stresses on
total root exudation, ethanol accumulation and 002 respiration.

4. To determine the difference in root exudation between two

dry bean varieties.

LITERATURE REVIEW

Exudation‘bzuggggg

Root exudation, the release of organic compounds from the roots
of intact plants, is affected by many factors. Soil aeration as well
as soil moisture (Katznelson et a1. 1954 and 1955), light (Rovira, 1959),
temperature (Husain and McKeen 1963, Rovira 1959, Rovira 1969, Schroth
et al. 1966, Vancura 1967), plant species (Vancura and Hovadik 1965,
Rovira 1969), age of plant (Rovira 1969, Shay and Hale 1973, Lespinat
et al. 1975) and plant nutrition (Rovira, 1969) affect root exudation.

Root exudation appears to be increased by soil anaerobiosis.
Plants grown under anaerobic conditions were found to accumulate
significant quantities of amino acids in their leaves and roots (Grineva,
1961). Anaerobic conditions enhanced the exudation of these accumulated
metabolites as well as sugars and organic acids. Excised roots of
barley lost organic acids, amino acids, K+ and Cl- during the first
fifteen minutes of anaerobic stress. Anaerobic conditions caused
organic and amino acids to leak from the roots to the bathing medium
(Hiatt and Lowe, 1967). Rittenhouse and Hale (1971) also found that
anaerobiosis increased exudation by peanut roots.

Alternate drying and wetting cycles of soybean, tomato and wheat
plants resulted in the greater liberation of amino acids from the roots
into the nutrient solution (Katznelson et al., 1954). Rovira (1959)
found that the light intensity at which plants grew affected the

amount as well as the type of root exudates. Clover grown at high

3

light intensity exuded more serine, glutamic acid and oralanine
than plants grown under 602 shade. Tomatoes produced lower levels
of aspartic acid, glutamic acids, phenylalanine and leucine in the
root exudates.

The temperature at which plants are grown also appears to
affect both the composition and quantity of exudation. Considerable
amounts of amino acids, alanine, serine, glutamine, glycine and
threonine were present in exudates from strawberry plants grown at
5C and 10C but were absent from strawberry plants grown at 20C and
30C (Russia and McKeen, 1963). This increase in root exudation
under lower temperatures could be explained by the effect of tempera-
ture on membrane permeability and cellular metabolism. It has been
postulated by Hale et a1. (1971) that lower temperatures reduce
metabolic energy causing assimilates to leak out of cells. However,
exudation of sugars and amino acids from germinating seeds of maize
- and cucumber increased with increasing temperature, but the exudation
of maltose and fructose was lower at 28C than at 19C (Vancura, 1967).
' The amount of exudation for germinating seeds of cotton and bean
remained constant from 15C to 33C but increased at 37C (Schroth et
a1. 1966). Rovira (1959) found that higher temperatures appeared to
increase the amounts of amino acids exuded by tomato and clover roots.

Qualitative and quantitative differences in exudates can some-
times be found to correlate with the age of the plant. The roots of
older maiae plants were observed to excrete more labelled carbon
than those of younger plants (Lespinat et al., 1975). In contrast,
root exudation from tomato, subterranean clover, and Phalaris was

reported to be greater during the first two weeks of growth than in

in the second two-week.period (Rovira, 1959). Vancura and Hovadik
(1965b) found that tomato and red pepper exuded tyrosine at fruiting
and cucumber exuded B-pyrazolylalanine only at the early seedling
stage. Vancura and Hovadik (1965a) found that amino acid excretion
by cucumber roots increased in parallel with growth while in late
fruiting phase it clearly diminished. They found three phases of
qualitative changes in root excretion during the growth of a cucumber
plant. The first occurred during the transition from seed and
cotyledon nutrition to primarily photosynthetic nutrition, the second
during flowering and the third during late fruiting. Hamlen et
a1. (1972) found a decreased concentration of total neutral carbohy-
drates released with increasing plant age.

Nutrient levels have been found to influence the exudation by
plant roots. Four times more sugar was exuded by peanut roots at
10 mg Ca+ than at 50 mg of Ca+ per liter (Shay and Hale, 1973). Shay
and Hale suggest that the low levels of Ca+ increased root cell membrane
permeability. Bowen (1969) found that phosphate-deficient seedlings

of Pinus radiata exuded more amino acids than control plants. He

 

postulated that increased exudation from phosphate-deficient plants
might be the result of doubling amino-nitrogen in the roots of the
seedlings rather than an increase in permeability.

The loss of organic metabolites from roots occurs primarily at
p (the growing apex, regions of elongation and root hair formation (Street,
1966). The region directly behind the root tip in healthy plants is
considered to be the major source of excreted substances (Pearson
and Parkinson, 1961). They also found that injury or stress caused

small quantities of substances to be released from any region of the

root. Van Egeraat (1975a) found that the tips of both the main

and lateral roots of young peas were important sites of exudation.

He also demonstrated that ninhydrin positive compounds were released
from the roots during the formation of lateral roots, a process

which damages the main root. McDougall and Rovira (1970) placed

wheat plants labelled with.carbon-l4 between moist sheets of filter
paper and found that exudation determined by spots of high radio-
activity occurred either from the root spices or from the points of
rupture where lateral roots emerged from the main root. Ayers and
Thornton (1968) suggested that root damage caused by experimental
methods may be responsible for a large proportion of organic materials
released by plant roots. However, there is evidence from other
experiments using nutrient solutions as the growth medium that organic
substances are lost from undamaged roots (Boulter et al. 1966, Hale
1969, Slankis et a1. 1964).

The cultural conditions for roots is very important during
exudation studies. McDougall and Rovira (1965) found that 0.12 of
carbon-l4 assimilated by wheat plants was exuded by the roots into
the nutrient solution in which they were growing. But if the roots
were immersed in distilled water rather than plant nutrient solution
during the course of the experiment, the amount of exudate was
increased. Amino acids liberated into the culture medium by the roots
of pea seedlings were found to be much greater in sand compared to
liquid culture medium (BOulter et al., 1966). Cereal plants grown
under sterile conditions in solution culture were found to exude more
when their roots grew between glass ballotini than unrestricted (Barber

and Gunn, 1974). Martin and Barber (1974) found 6.1% of the carbon-l4

assimilated by wheat roots was exuded under sterile conditions.
However, more was exuded under nonsterile conditions, suggesting a
stimulative effect by soil organisms on the loss of carbon by roots.
Vancura (1965a) stated that during plant development, root
exudates together with other factors determine the size and composi-
tion of the bacterial population in the rhizosphere. Consequently,
these losses affect the life processes of the plant, promote the
susceptibility or resistance of the plant to pathogenic organisms
and possibly influence the relationships between Rhizobia or between
saprophytic fungi and plants. Several workers have observed these
correlations between root excretions and the colonization of root
surfaces by microorganisms (Rovira 1956, Roviera 1965a, Vancura 1965).
Currier and Strobel (1976) demonstrated that Rhizobium spp. are
attracted to root exudates of both legumes and non-legumes. However,
Van Egeraat (1975a) found that the roots of young pea plants, in‘
addition to growth-stimulating substances, exude inhibiting compounds.
These exuded materials, whether growth stimulating or inhibitory, are
important in the establishment and maintenance of the rhizosphere

population of young plants.

Ethanol Accumulation
Ethanol and other reduced metabolites (e.g. DPNH) replace
oxygen as the final electron acceptor during anaerobic metabolism.
The pyruvic acid formed from the anaerobic decomposition of sugars
is decarboxylated forming C02 and acetaldehyde. Acetaldehyde is
reduced further leading to the formation of ethanol. The accumulation

of ethanol in plants under anaerobic conditions makes it an indicator

of the extent of damage caused by anaerobic conditions (Fulton and
Erickson, 1964). There are many cases where ethanol has been found

to accumulate in plants under such.stress conditions (Kenefick 1962,
Grineva 1963, Bolton and Erickson 1970, Leblova et a1. 1969). Under
anaerobic conditions, ethanol production by excised apical root
tissues was five times greater than for non-stressed plants (John

and Greenway, 1976). Ethanol was not found to be produced under
aerobic conditions with either the stressed or non-stressed plants.
The use of alcohol dehydrogenase (ADH) activity as a test for flooding
tolerance is a practical possibility. Prolonged flooding was found

to increase the acvitity of ADH more in roots of flooding sensitive
cultivars than in roots of flooding tolerant cultivars (Crawford

1967, Crawford and McManmon 1968, Francis et a1. 1974, John and Green-
way 1976). Under anaerobic conditions it has been found that alcohol
accumulates in the roots of corn, sunflower and peas, which.is

excreted into the external environment (Grineva 1963, Smucker and

Erickson 1976).

Physiology gf_the Root Under Anaerobiosis

Oxygen is the final electron acceptor in respiration. If oxygen
is limiting, electron transfer and respiration are limited and
fermentation occurs. Anaerobic respiration of one mole of glucose
results in the production of 2 moles each of CO2 and ethanol with.the
release of 54 kcal per mole of glucose. Aerobic respiration yields
686 kcal' per mole of glucose and 6 moles of C02 and H20 (Crable, 1966).
Therefore, fermentation produces only a fraction of the useable energy

produced by respiration. Fulton and Erickson (1964) showed that

flooding of tomato roots quickly inhibited respiration and metabolism
in all plant parts and inhibited the Krebs citric acid cycle in roots.
Respiration in plants is necessary for mineral and water uptake
and the growth and maintenance of plants. Many workers have shown
‘that poor soil aeration will inhibit root growth and elongation (Gill
and Miller 1956, Kramer 1965, Geisler 1965). Letey et a1. (1962)
found that shoot as well as.root growth was reduced under low oxygen
conditions at early stages of development. Oxygen levels of 11 to
10; caused an 802 reduction in the rate of cell division of Xigig
£223 after a 24-hour treatment (Williamson, 1968). Anaerobiosis has
also been found to inhibit water uptake of tobacco roots by 502 or
more (Willy, 1970). Inadequate aeration appears to reduce the
absorption of water by reducing the size of the root system which would
limit the surface area for absorption of water. Low oxygen also
decreases the permeability of roots to water (Glinka and Reinhold, 1962).
Root morphology and physiology appear to be modified by anaerobiosis.
ROot hair development may be suppressed decreasing nutrient uptake
(Daubenmire, 1959). When root penetration is restricted, lateral roots
occur (Greenwood, 1969). Roots growing in deficient oxygen conditions
are usually shorter, thicker, more branched and have fewer root hairs

than those which are sufficiently aerated (Kramer, 1965).

MATERIALS AND METHODS

Seed Sterilization and Germination

Phaseolus vulgaris L., varieties Seafarer and San Fernando, were
used in this experiment. Seeds were surface sterilized by dual sterile
soakings as follows. Seeds of each variety, 150, were selected and
washed in Alconox and water and rinsed six times with tap water. The
seeds were then surface sterilized in 100 m1 of a solution of 12
sodium hypochlorite containing two drops Tween 20, for 5 minutes in
a 200 m1 Erlenmeyer flask. The seed-slurry was thoroughly agitated,
by hand, during the sterilization period. Seeds were rinsed six times
with sterile distilled water and then allowed to soak for 4 minutes
in sterile distilled water. The water was replaced by 100 m1 of
0.54% sodium hypochlorite, seeds were shaken by hand for 2 minutes
and then rinsed six times with sterile distilled water. Sodium
hypochlorite concentration was reduced because of increased penetra-
tion into the seed during the second soaking period resulting in a
reduction of viable seeds. With this surface sterilization technique,
98% San Fernando seeds germinated with 502 of these seeds aseptic.
For Seafarer, 602 of the seeds germinated of which 90% were aseptic.

Under a sterile Environmental Air lamina flow transfer hood,
five seeds were placed in a petri dish containing 2 sheets of Whatman
#1 filter paper and 6.0 ml distilled water. A correlation between

germination of Seafarer and water added to each petri plate was

10

11

determined. The percentage of seeds germinated ranged from 35.0%
when 5 m1 of water was added to each dish to a maximum of 60.02
germination when 6 ml were added to the petri dishes. San Fernando
was more tolerant of the amount of water added than Seafarer, and
germination remained constant at all water levels. The petri dish,
two sheets of Whatman #1 filter paper, and the water were sterilized.
by autoclaving and allowed to cool before transfer of sterilized seeds.
The glass petri plate cover was secured in place with masking tape.
Seeds were germinated in an incubator at 27C for 2 days. Selected
germinated seeds were transferred, under sterile conditions, to a
sterilized vial (Figure 1) with forceps, sterilized in 702 ethanol

and flamed. Seeds remaining in each plate were cultured on Potato
Dextrose Agar (PDA), pH of 5.6, and Beef Lactose Agar (BLA), pH of
6.8, to detect surface contamination. Aliquots of 0.5 ml of the
nutrient solution was removed from each vial with a syringe, employing
sterile techniques, 1 day after seed transfer into the vials. The

solution.samples were cultured on PDA and BLA to detect surface

contamination.

Seedling Culture and Transplanting
Glass storage vials, 25 ml, each fitted with a ground-glass
plant port supported sterile seedlings for the first 4 days of growth
(Figure l). A modified Hoagland nutrient solution, 23 ml of 1/2
strength, was added to each glass storage vial. Vials were wrapped
in aluminum foil to prohibit light penetration. A ground glass plant

port, 18 X 20 mm, the base of which contained a small opening

approximately 6 mm in diameter, and a thin section of polyurethane

12

Q ‘--GLASS BEAKER
' ~PLANT PORT

 

 

' e

 

' --.STORAGE VIAL

 

 

Figure 1. Diagram of Glass Culture Vial for growing sterile seedlings.

13

stopper was fitted onto each vial. The stopper was provided to
support the hypocotyl in early growth stages. To ensure sterility,
a 10 ml pyrex glass beaker was secured over the top of each vial
with a 2 X 6 cm strip of aluminum foil. The vial, filled with
nutrient solution, glass plant port, stopper and beaker were auto-
claved for 30 minutes at 15 lbs pressure and 121C and allowed to cool
before sterile transfer of terminated seeds. Seedlings were grown
in vials for four days after which they were transferred frOm.the
vials to the sterilized glass root chambers. Each plant port was
filled with sterilized parawax, a 1:8 g/g mixture of hard paraffin
and petroleum jelly. The parawax provided a medium which enabled
the seedling to expand during growth but prevented gaseous exchange

and contamination by microorganisms.

Root and Shoot Chambers

Sixteen plants were grown in modified pyrex glass mist chambers
designed by Smucker and Erickson (1976). The brass nozzles were
replaced by 0.94 cm stainless steel nozzles attached to the glass
access tube of the reservoir with quick-drying epoxy cement. The
inline cellulose ester filter, used to filter circulating solution
for plant debris which would clog the brass nozzles, was removed.
The larger orifice of the stainless steel nozzles allowed flow through
the nozzle despite accumulation of plant debris in the nutrient
solution. This system was especially advantageous for the study of
root responses to changes in rhizosphere gaseous composition. The
entire system could be autoclaved, and aseptic conditions maintained

throughout the experiment.

14

Each glass reservoir was filled with 200 ml of a sterilized
1/2 strength modified Hoagland's nutrient solution. The reservoir
was connected to the root chamber with a ground glass joint. A
plexiglas shoot chamber, 20.6 cm in length.and 11.5 cm in diameter,
with a wall thickness of 1.3 cm, was fitted over each pyrex mist
chamber and secured in place with.modelling clay. A strip of foam
rubber weatherstriping, l X 32 cm, was secured to the base of each
plexiglas chamber using Dupont Duco cement. The weatherstripping
along with modelling clay-created an air tight seal at the junction
of plexiglas shoot and glass mist chambers. Gas inlets and outlets
each with a 5 mm orifice, were located on opposite sides of the
plexiglas chamber, 3 cm from the tap and bottom. A thermometer
extending halfway into the plexiglas chamber, sealed by a swage
fitting, was located approximately 5 cm below the gas inlet.
Additional features of the sterile plant chamber system may be
observed in Figure 2. After the condenser and condensate separator
were attached, the system was carefully transferred to the growth
chamber approximately 15 minutes after transfer of seedlings from
the glass growth vials to the pyrex mist chamber. A variable speed
Gilson minipuls pump continuously recirculated the sterile nutrient
solution at a flow rate of 25 m1 min-1. Gas flowing into the root
chamber through the gas inlet was sterilized by a Millipore filter
having a mean pore size of 0.2u. Flow through the gas inlet of

1

each chamber was 30 ml min. t 5 m1 min-1. Plants were grown in the

mist chambers for 13 days.

15

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16

Environmental Conditions

The environmental conditions were controlled by a Warren-
Shearer model CEL-36-10 growth chamber. The light source in the
growth chamber consisting of eight lOO-watt incandescent bulbs and
twelve 39-watt General Electric High Output warm White fluorescent
bulbs produced 800 uEinsteins sec:1 in.2 for full light at the plant
surface as measured by a Lambda LI-170 quantum/radiometer/photometer.
The photoperiod was 16 hours consisting of 14 hours of full light
preceded and followed by 1 hour of incandescent light only. The
growth chamber was 70% relative humidity while the humidity in the
sealed plexiglas shoot chambers was 1002 as measured by a Bacharach
Industrial relative humidity detector. Temperatures were 18C during
the dark period and 21C.during the light period in the growth chamber.
However, due to heat absorption by the plexiglas shoot chambers,
temperature within these chambers was approximately 220 during the

dark period and 26C during the light period.

Measurement gf_Carbon Dioxide

 

 

Carbon dioxide concentrations of the root and shoot chambers
were monitored by the rapid method of CO2 analysis described by Clegg
et a1. (1978). The method involved injecting a 10 ml or less gas
sample with a 10 ml syringe through a modified glass tube fitted
with a rubber septum into the flowing carrier gas that passed through.
the infrared analyzer. The flow rate of the nitrogen carrier gas
was maintained at 600 m1 min-1. The gas sample passed through a
1.5 cm X 11 cm tube of Hammond drierite before entering the analyzer

to remove any moisture in the sample. A strip chart recorder,

17

Linear Instruments Corporation Model 261, was attached to the infra-
red analyzer so that peak response was obtained which was proportional
to the carbon dioxide concentration. The sample and reference outlets
were each connected to glass tubes containing 50 m1 ascarite which
was used to trap 14C0 contained in the gas sample.

2

Root Gas Treatments

 

Experiments were duplicated and repeated twice. Two of the
root chambers received two 48 hour stress treatments of 20% carbon
dioxide with the balance of nitrogen. The two control chambers at
all times received air delivered by bottled gas with a C02 concentra-
tion of 340 ppm. The first 48 hour stress treatment was introduced
3 days after transplanting and again on day 8. Between the two
stress treatments, the two root chambers received air from the same
gas source as the aerobic controls. To maximize air displacement by
the treatment gas at the beginning of each stress period, flow of
the treatment gas was introduced into each of the two root chambers
at the rate of 275 mu min.1 for 20 minutes. At the end of each 48
hour stress period, air flow into each treatment root chamber was
increased to 275 ml min.1 for 20 minutes before returning to the normal
flow of 30 ml min“1 i 5 m1 min-l.. Gas displacement rate in the root
chambers was determined by Smucker and Erickson (1976). Gas flow
through the control root chambers was maintained at 30 ml min-1

: 5 ml min“1 throughout the experiment.

18

Radioisotope Feeding of Shoots

A diagram of carbon dioxide flow to the shoots is shown in
Figure 3. On day 3, the four plexiglas shoot chambers were connected .
with a series of t-tubes and tygon tubing, 0.63 cm I.D., to a 20 liter
polypropylene carbon (A) which served as a gas mixing vessel. A
small electric fan (B) attached to the bottom of the carboy by epoxy
glue, mixed the 14C02, having a specific activity of 5.47 millicuries
per 137 liters, with air in the carboy. The 14CO2 was added through
injection port (C). Injection and sample ports were modified glass.
t-tubes fitted with a rubber septum.

At 0800 hours on day 3, all carbon dioxide was removed from the
carboy by pumping air in the carboy through bypass (D) and through a
tube of ascarite (E). A diaphram pump (F) replaced peristalic pump
(G) during carbon dioxide removal from the carboy. Carbon dioxide
was depleted to less than 5 ppm within 10 minutes. Air was pumped
from the carboy and through the ascarite tube at a rate of 10 liters
min-1. Gas samples were removed through port (H) and injected into
the infrared gas analyzer to determine carbon dioxide concentration
in the carboy. When carbon dioxide concentrations were less than 5 ppm,
the minimum detection limit, the ascarite bypass was closed. A 10 ml
sample of 14C02 was removed from the bottled radioactive gas and

injected into port (C) to bring the concentration in the carboy to

340 ppm CO The gas was recycled through the carboy for 5 minutes

2.
to allow sufficient time for thorough mixing. Gas samples were

removed through port (C) and injected into the infrared analyzer to

monitor CO2 concentration in the carboy throughout the experiment.

‘19

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20

When the desired 14CO2 concentration had been reached in the
carboy, the diaphram pump was replaced with.the variable speed
peristalic pump (G) which.was used for the duration of the experiment.
Line (D) was once again closed and gas was pumped from the carboy at
a rate of 130 ml min“1 t 8 ml min-1 into the four plexiglas shoot
chambers (I-L) and back into the carboy giving a distributed rate of
33 ml min.1 per chamber. Several times per day gas flow rates into
the four chambers was determined by redirecting flow through line (M)
which was connected to a flow meter and closing flow into the carboy
at valve (N). The flow meter was attached to an erlenmeyer flask
containing the solution of ethanolamine and methanol used to trap
14602 lost during flow rate determination. This measurement took
approximately 30 seconds, line (M) was closed and valve (N) opened to
resume flow into the carboy.

Gases circulating through the shoot chambers were sampled several
times daily and the CO2 concentration monitored. When the CO2 concen-
tration approached 340 ppm, valves (0 and N) were closed, line (D)
opened, and gas in the.carboy was recycled by passing the four shoot _

l4

chambers. Between 5 and 10 ml CO ‘were injected into port (C) to

2

bring the CO2 concentration in the carboy to 340 ppm. The gas was
.recycled within the carboy for 2-3 minutes to ensure thorough mixing
of the added gas. At the end of this time, a 10 m1 gas sample was
removed through.port (H) and injected into the infrared analyzer.
When a gas equilibrium was reached, line (D) was closed and valves
(0 and N) opened. This procedure was followed whenever injection of
14

CO2 was required; the time and amounts being quite variable. Gas

samples, 10 ml each, were removed through ports (P-S) and injected into

21

the infrared analyzer to determine the rate of carbon dioxide used
by the shoots in each chamber at 0800, 1200, 1600, 2000, and 2300

hours on day 1 through day 11.

J

Measurement 2: Root Respiration

 

 

Carbon dioxide respired by the roots for all treatments was
measured every 4 hours during the light period from day 1 through.day
11 using the following procedure. Tygon tubing; 0.63 cm I.D., approxi-
mately 76 cm in length, each containing a sampling port, were attached
to the four glass condensate separators. The four tygon lines were
connected to two 500 ml erlenmeyer flasks each containing 300 ml of
the solution of ethanolamine and methanol, used to trap any 14CO2
respired by the roots (Kobaysashi and Maudsley,l969). Gas samples,

10 ml each, were removed from the sampling ports and injected into the
analyzer to determine carbon dioxide respired by the roots at 0800,
1200, 1600, 2000 and 2300 hours. Also, during these sampling periods,
the four tygon tubes were disconnected from the two 500 m1 erlenmeyer
flasks and each was attached to one 20 ml erlenmeyer flask containing
the solution of ethanolamine and methanol. Gas-respired from the roots
of each root chamber flowed into the appropriate flask for 15 minutes
after which the four tygon lines were reconnected to the larger, 500
ml, erlenmeyer flasks. Solution, 1 ml aliquots, was sampled from the
20 ml erlenmeyer flasks and together with 9 mls of Beckman Ready-Solv
Ep Scintillation fluid, was added to scintillation vials. The vials
were then shaken for 30 minutes on a Forma Scientific model 2563

shaker. Carbon-14 activities were determined by counting each vial in

a Beckman 8100 System Scintillation Counter for 10 minutes. The

22

remaining solution in the 20 ml flasks was replaced with fresh solution

every sampling period.

 

Measurement 9; Root Exudates

At 0800, 1200, 1600, 2000, and 2300 hours on day 1 through day
11, 2 ml samples were extracted from each reservoir through the access
port by a 3 ml syringe and needle. Each access port was fitted with
a rubber septum and covered with aluminum foil to avoid contamination.
The aluminum foil was lifted only during sampling. Of the extracted
sample, 1 ml was immediately injected into 3 m1 glass vacutainer tubes
and frozen for analysis of ethanol using a Beckman GC 72-5 gas chromato-
graph. The remaining sample was added with 9 ml Beckman Ready-Solv
EP scintillation fluid to a scintillation vial. The vials were shaken
'for 30 minutes to ensure proper mixing of sample and scintillation
fluid. Carbon-l4 activity was determined using a Beckman Scintillation
counter. Each sample was counted once for ten minutes. (Every other
day, after sampling, modified l/2 strength, sterilized Hoagland's
nutrient solution was injected into each reservoir via the access port
to bring the level to 200 ml.

To test for axenic conditions of each reservoir, 0.5 ml samples
of the nutrient solution were removed from each reservoir through the
access port on days 1, 4, 7, 10 and 11. PDA and BLA were used to test
for aseptic conditions.

14

C02 Fixation

On day 12 after initiation of 14C02, the gas mixing carboy was

closed and gas from the four shoot chambers was cycled through the

23

ascarite tube and back into the shoot chambers at a rate of 130

ml min.1 t 8 ml min"1 for approximately 2 hours. This was done to
remove carbon-l4 in the shoot chambers before the chambers were
removed. Gas samples, 10 ml, were extracted periodically during the
two hours from port (H) and injected into the infrared analyzer.
When carbon dioxide could no longer be detected, plants were removed
from the chambers. The shoot and root of each plant was separated,
weighed, and dried for 5 days at 70C.

Carbon-l4 activity in the shoots and roots was determined by
homogenizing the entire dried plant sample with.4 mls of distilled
water. Of the resulting homogenate, 3-0.35 ml aliquots were each
added to 1.5 ml Beckman BTS—450 and allowed to digest over night..
Aliquots of 15 ml of Ready:Solv—EP was added to the digested material.
The sample was shaken in the scintillation for 1/2 hour and carbon-14
activity was determined using a Beckman scintillation counter.

A 2 X 2 factorial was used. The main effects were varieties X
aeration. Plants were replicated 4 times and physiological measurements
(i.e. DPM, ethanol, respiration, etc.) were measured per chamber. Each

experiment was run twice.

RESULTS AND DISCUSSION

Plant Growth

 

Shoot and root growth appeared to be good during the 11 days of
treatment. However, plants showed visual symptoms of slight wilting
and chlorosis, mainly on the older leaves, after 6 days that persisted
throughout the remainder of the experiment. Humidity in the shoot
chambers approached saturation 4 days into the experiment. Although
concentrations of gases other than CO2 were not measured, these
symptoms may have resulted from the accumulation of oxygen in the shoot
chambers as well as the high humidity.

Roots of all treatments appeared healthy during the first 3 days
of the experiment and displayed extensive branching (Figure 4A).
Aerobically treated roots remained white throughout the experiment
as did anaerobic Seafarer roots. San Fernando roots subjected to
anaerobic treatments turned dark 2 or 3 days after the initiation of
the first 48-hour anaerobic stress and displayed very few white tipped
branches upon termination of the experiment (Figure 43). Fresh and
dry weights of shoots and roots for all treatments were not signifi-
cantly different (Table 1).

Root chambers were monitored after introducing 14CO2 on days 1,
4, 7, 10 and 11 for root contamination of bacteria or fungus (Table 2).
Sterile conditions were maintained for 80% of the experiments. Aerobic

Seafarer control appeared to become contaminated between day 7 and day

24

25

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nu

26

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27

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28

10. Multiple samplings of the mist chamber nutrient solution or
addition of nutrient solution could have caused the detected contami-
nation. Microorganisms have been reported to increase the carbon-l4
assimilates found outside of the roots (Barber and Martin 1976,
Vancura and Hovadick 1965, Martin 1976). However, in this experiment,
there was no significant change in carbon-l4 assimilates detected in
the nutrient solution of anaerobic Seafarer controls between days 7

and 10.

Carbon-l4 Activity in_Shoot and Root

 

Total fixation of 14C by dry bean plants was determined by
measuring the radioactivities of roots and shoots of Seafarer and
San Fefnando. Neither anaerobic stress nor variety had a significant
effect at the .05 level on total fixation of 14C (Table 3). Aerobic
Seafarer contained 412 and 272 more 14C activity per unit dry weight
of shoot and root, respectively, than aerobic San Fernando. Anaerobic
Seafarer contained 21% more 14C activity per unit dry weight shoot than
anaerobic San Fernando. Both anaerobic stressed varieties contained
approximately the same amount of 14C activity per unit dry weight of
root.

14C activity found per unit dry weight shoot was greater than
that contained in a unit dry weight of root for each of the treatments.
Between 3 and 5 times more 14C activity was found in the shoots than
the roots. Martin (1976) also found a difference in the amount of 14C
activity detected in the shoots compared with the roots of the same
plant. Rye grass, wheat and clover had 2.4, 4.8, and 5.6 times more

14C activity in the shoots than roots, respectively. It is possible

29

Table 3. Influence of anaerobic stress and dry bean genotype on
the total fixation and translocation of 14C assimilates.
Each value represents the 14C activity detected in
,chambers containing 4 plants.

 

Treatments Shoot Root

 

DPM/gram dry weight
Aerobic controls
Seafarer 8,347,461A 2,246,605A

San Fernando . 4,927,910A 1,650,979A

Anaerobic stress

 

Seafarer 6,859,312A 1,352,632A

San Fernando - 5,385,917A . A 1,533,421A

 

Those values within columns having different subscripts represent
statistical difference at the .05 level according to Duncan's Multiple
Range Test.

30

that carbon compounds translocated to the roots were lost by root
exudation or respiration. Grineva (1961) found that organic compounds
typical of root tissues were excreted by the roots into the growth

medium.

Carbon Dioxide Assimilated by_Shoots

 

1«C02 fixed in the light by the shoots of each treatment was

measured several times daily throughout the experiment. Both aerobic

and anaerobic Seafarer plants fixed more 14CO2 than San Fernando on

day 11 (Table 4). Seafarer plants of aerobic and anaerobic treatments

fixed 802 to 1002 and 2502 to 5502 more l['COZ than San Fernando, respec-

tively. Both aerobic treatments (Figure 5) and anaerobic treatments

(Figure 6) experienced a diurnal fluctuation in CO fixed with a

2
decreasing trend. It would be expected that as the plants grew and

leaf surface area increased, more carbon dioxide would be fixed per

unit leaf area. The deteriorating condition of plants due to a possible
oxygen toxicity in the shoot chambers could explain the decrease in

CO2 fixed by the shoots.

Root Respiration

 

14CO2 evolved from the roots of the 4 treatment plants was

determined at 0800, 1200, 1600, 2000, and 2300 hours from day 1
Ithrough day 11. Carbon-l4 dioxide respired by the roots of aerobic
San Fernando plants ranged from 3.5 ul C02[p1ant/minute on day 3 to
1.0 ul COZ/plant/minute on day 11 (Figure 7). Aerobic Seafarer plants
showed a gradual decrease in 14C evolved by its roots, with a range

of approximately 2.3 ul/plant/minute on day l to less than 0.5 ul/plant/

31

 

 

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minute on day 8. Daily carbon dioxide respired by San Fernando roots
was approximately twice that of Seafarer (Figure 7). It is possible
that the declining condition of the shoots affected Seafarer more
adversely than San Fernando which would explain the decrease in

4CO2 evolved throughout the experiment. 14CO2 respired by the roots
of anaerobic San Fernando ranged between 2.5 ul/plant/minute on day 2
to approximately 3.8 ul/plant/minute on day 10 (Figure 8). Anaerobic
Seafarer plants respired between 0.8 ul/plant/minute on day 7 to a
high of 3.5 ul/plant/minute on day 10.

On day 11, roots of the variety San Fernando evolved 1.6 times

and 1.4 times more 14CO2 per unit dry weight root than Seafarer for

the aerobic and anaerobic treatments, respectively (Table 5). Anaerobic
San Fernando and Seafarer respired approximately 2.0 and 2.5 times
more 14CO2 per unit dry weight root, respectively, than the aerobic
controls. Oxygen stress enhances anaerobic respiration which could
account for the increase in 14CO2 evolved by the oxygen stressed plants
in the experiment. Street and Cockburn (1972) have found that between
1 to 9% oxygen there is a minimum rate of carbon dioxide evolved and

below this range, the CO evoluation rises steeply due to glycolytic

2

fermentation.

Carbon-14 Exuded

 

Total 14C losses by stressed and control roots were determined by
taking daily measurements of the 14C accumulation in the nutrient
solution. Roots of both Seafarer and San Fernando exuded some 14C-
labelled assimilates even under aerobic conditions. However, anaerobic

conditions greatly enhanced the root exudation of both varieties

35

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38

(Table 6). San Fernando and Seafarer exuded 30 and 19 times more
14C-labelled materials per unit dry weight root, respectively, than
the aerobic controls for each variety. These results agree with
others who have reported enhanced exudation under oxygen stress
(Grineva 1961, Hiatt et a1. 1967 and Rittenhouse and Hale 1971).

Under anaerobic conditions, an increase in 14C assimilates,
detected in the mist chamber nutrient solution, were found to be
directly related to the time of treatment for both Seafarer and San
Fernando (Figure 9). As much as 19 X 103 disintegrations per minute
(DPM) were detected in the nutrient solution during the anaerobic
stress for Seafarer and 7 X 103 DPM for San Fernando. After cessation
of the first 48-hour stress period, between days 2 and 4, 14C activity
decreased in the nutrient solution to approximately 7 X 103 DPM for
anaerobic Seafarer and l X 103 DPM for anaerobic San Fernando; repre-
senting a 2.5 and 7 fold decrease for Seafarer and San Fernando,
respectively. The root exudates could have volatilized or were
respired off by the roots. Since volatile l4C assimilates may or may
not have been trapped by the ethanolamine, there can be no definite
conclusions. Another possible explanation is that material exuded
during oxygen stress is reabsorbed upon return to aerobic conditions.
Cossins and Turner (1959) have shown that previously exuded ethanol
was reabsorbed by the root tissue.

Exudation increases during anaerobic conditions appear to be
varietal dependent. Anaerobic Seafarer exuded 42% more 14C labelled
assimilates than San Fernando. Figure 10 demonstrates the ratio of

4 . . . .
C assimilates which accumulated in the nutrient solution to root

respiration throughout the experiment. The ratio for anaerobic

39

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DPM PLANT" x 10'3
01

 

4O

-a-AEROBIC SEAFARELAND
SAN FERNANDO

-x- ANAEROBIC SEAFARER

-O- ANAE ROB I C SAN
FERNANDO

 

‘23: Z 3 6 7‘ 3 91‘0”
TIME (DAYS)

Carbon-14 accumulated per day in the mist chamber nutrient
solution by individual aerobically and anaerobically
treated Seafarer and San Fernando plants. Each point
represents the average measurements for each chamber of

4 plants taken between 0800 hours and 2300 hours from

day 1 through day 11.

41

A AEROBIC SEAFARER
x AEROBIC SAN FERNANDO

 

 

19"nANAEROBIC SEAFARER
‘7"oANAERosIc SAN
0 F
c? 15 ERNANDO
2 13
"‘ 11+
U
3 7
E” 5
3
1* ‘Am _.--
115,54 5 (3 i s 6 lO 1‘]

TIME (DAYS)

Figure 10. Ratio of carbon-14 assimilates detected in the mist
chamber nutrient solution to root respiration for
aerobic and anaerobic Seafarer and San Fernando plants.
Each point represents the average measurements for each
chamber of 4 plants taken between 0800 hours and 2300
hours from day 1 through day 11.

42

Seafarer is considerably higher each day than for anaerobic San
Fernando. The greater exudation for anaerobic Seafarer could
explain the higher ratio in Figure 10. Another possibility is that
the respiration rate of Seaferer could be lower than for San
Fernando throughout the experiment. The last day's measurements
indicate that anaerobic San Fernando respired 1.4 times more 14CO2
per unit dry weight root than stressed Seafarer roots. There appears

to be a varietal difference in the rate of exudation to root respira-

tion between anaerobic Seafarer and San Fernando plants.

Ethanol Metabolism, Loss and Accumulation

 

A trace of ethanol (0.25 to 0.29 ppm) was detected in the
nutrient solutions of 4 day old seedlings. The accumulation of ethanol
suggests that the oxygen content may have been depleted by the
growing roots during the four day period. There were no significant
differences in ethanol concentrations found between varieties. How-
ever, since there were no significant differences between plants of
either variety, the differences in ethanol accumulation measured
during each experiment are attributed only to the stresses imposed.

Ethanol loss and accumulation by roots of plants in the mist
chambers were determined at 0800, 1200, 1600, 2000 and 2300 hours in
the reservoir nutrient solution. Ethanol accumulation in root exudates
during the first 48-hour anaerobic stress was 55% and 86% greater than
the accumulation of ethanol during the second 48-hour oxygen stress
for Seafarer and San Fernando, respectively (Figure 11). Part of the
root system may have been destroyed after imposing the first stress

(Smucker et al., 1978) and the decrease in surface area caused by root

43

-l- Aerobic Seafarer and
San Fernando

  
  
   

'C'Anaerobic San Fernando .

-x- Anaerobic Seafarer

' .0009

 

Mg ETHANOL/PLANT x 103

I
~
I
9

b
1
q
.

TIME (DAYS)

Figure 11. Ethanol accumulation in the mist chamber nutrient
solution by individual aerobic and anaerobic treated
dry bean varieties. Each point represents the average
of measurements for each chamber of 4 plants taken
between 0800 hours and 2300 hours from day 1 through

day ll.

44

tissue death could account for the decrease in ethanol lossed by

the roots and the accumulation in the nutrient solution. Another
possible explanation is that between the first and second stress

an aromatic alcohol dehydrogenase was activated resulting in greater
root suberization. Davies et a1. (1973) have postulated that the
aromatic alcohol dehydrogenase functions in the metabolic route from
cinnamic acid to lignin. The postulated route involves the reduction
of p—hydroxycinnamic acid, ferulic acid and sinapic acid to the
corresponding alcohols and subsequent incorporation into lignin. If
the activity of an aromatic alcohol dehydrogenase increases then it
could lead to an increase in lignin formation and suberization,
resulting in a decrease in root exudation. A decrease in root exuda-
tion could account for the decrease in ethanol lossed through the
roots during the second 48-hour anaerobic stress.

Ethanol did not accumulate throughout the duration of the
experiment in the nutrient solution of aerobically treated Seafarer
or San Fernando (Figure ll). A slight oxygen stress was probably
imposed on the plants during seedling development in the vials which
would account for the initial ethanol concentrations found in the
nutrient solution of the mist chambers. In contrast to the controls,
ethanol accumulated in root assimilates under anaerobic conditions.
As much as 8 X 10..3 mg/plant and 7 X 10-3mgblant was detected at one
time for anaerobic stressed Seafarer and San Fernando, respectively.
Similar results have been reported by Smucker and Erickson (1980),
Smucker and Erickson (1976) and Bolton and Erickson (1970). The
increased production of alcohol could indicate an increase of glycolysis

during anaerobiosis. Grineva (1961) has postulated that oxygen

45

deficiency causes an increase in membrane permeability. An increase
in permeability could account for the increase in ethanol detected
for Seafarer and San Fernando under anaerobic stress.

Ethanol accumulation in the root assimilates under anaerobic
conditions was greater for Seafarer than San Fernando throughout the
experiment which suggests a varietal difference in ethanol exuded.
Noor (1979) found that alcohol dehydrogenase (ADH) activity which
controls ethanol production was higher in Seafarer roots than in
San Fernando. This greater ADH activity could explain the greater
ethanol concentrations in Seafarer exudates.

Ethanol accumulation in the mist chamber nutrient solution for
both Seafarer and San Fernando was found to be in direct proportion
to the time of anaerobic treatment (Figure 11). Nordheim (1961)
also found that ethanol lossed through the roots coincided with the
time of treatment. The decrease in ethanol accumulation between stress
periods indicates that ethanol may have been reabsorbed by the root
tissue and/or respired by the roots. Cossins and Turner (1959) also
showed that in a variety of germinating pea seedlings previously
accumulated ethanol was reabsorbed by the tissues and by providing
ethanol-Z-C14 (Cossins, 1962) to pea cotyledons, they were able to
show extensive conversion to a variety of products, including acetal-

dehyde, acids of the tricarboxylic acid cycle and amino acids.

Carbon-14 Partitioning

 

Carbon-l4 contained in the treatment plants ranged between

81.3% for aerobic Seafarer to 65.7% for anaerobic San Fernando of the

14CO2 fixed (Table 7). The aerobic controls, Seafarer and San Fernando,

46

 

 

 

 

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47

contained 25% and 12.5% more 14C, respectively, than the anaero—
bically stressed treatments.

Anaerobiosis also had a considerable effect on 14C assimilates
lost by roots. A total of 1.52% and 1.3% of the 14C fixed was exuded
by the anaerobic treatment plants, Seafarer and San Fernando, respec-
tively. This was 35% and 25% more assimilates detected in the nutrient
solution of anaerobic Seafarer and San Fernando, respectively, than
for each of the aerobic control plants. McDougall and Rovira (1965)
found that approximately 0.1% of 140 fixed was exuded by the roots.
Martin (1974) found that wheat plants exuded 6.1% and 8.3%. 14C
assimilated under sterile and nonsterile conditions, respectively.

If these values were included with the carbon dioxide respired by the
roots, 7.6% and 15.7% of the total 14C assimilated was lost through
the roots. Minchin and Pate (1973) found that 47% of the total carbon
fixed by peas was lost through the roots.

Respired 14C, interpreted for the 11 days of the experiment, is
shown in Table 7 to be between 18.6% of the 14C fixed for aerobic
Seafarer and 33% for anaerobic San Fernando. The exudation potential,

4C assimilates detected outside of the roots at any one time which
included 14C assimilates accumulated in the nutrient solution and CO2
respired, for 11 days, ranged between 18.8% for aerobic Seafarer and
34.3% for anaerobic San Fernando. Aerobic Seafarer and San Fernando
had exudation potentials of 18.8% and 24.9% of the 14C fixed, respec-
tively. Anaerobic stressed Seafarer and San Fernando demonstrated
exudation potentials of 33.3% and 34.3%, respectively. The exudation

potentials for both anaerobically stressed varieties, Seafarer and

San Fernando, increased under anaerobic conditions and were 1.8 and

48

1.4 times greater, respectively, than either their aerobic controls.

Improved soil management techniques would be the most important
step in reducing soil compaction which may cause reduced growth and
yield. Plant breeding programs designed to develop varieties more
tolerant of anaerobic conditions are also needed. The exudation
potential could be used to indicate the degrees of tolerance dry bean
varieties have to root anaerobiosis. Improved soil management
techniques as well as appropriate breeding programs must be implemented
before yields of dry beans are to be improved on fine textured soils
in Michigan.

Additional research utilizing 14C labeling techniques should
be conducted which would be designed to fractionate the organic
components (e.g. volatile, soluable, etc.) exuded by roots. Automated
sampling procedures as well as a large number of experimental replica-

tions would be needed for conclusive results.

SUMMARY AND CONCLUSIONS

A system was designed to determine short-term aeration stresses
on total root exudation, ethanol accumulation and 002 respiration under
axenic conditions employing 14C feeding to plant shoots. The system
allowed independent changes in the gaseous composition of the roots and
shoots as well as multiple sampling of the sterile nutrient solution
for 14C activity. The system was effective in maintaining sterile
conditions of the root systems. Sterile conditions were achieved for
80% of the experiments.

Aerobic Seafarer was found to contain 41% and 27% more 14C
activity per unit dry weight of shoot and root, respectively, than
aerobic San Fernando. Anaerobic Seafarer contained 21% more 14C
activity per unit dry weight shoot and approximately the same 14C
activity per unit dry weight of root than anerobic San Fernando. 14C
activity found per unit dry weight of shoot was 3 to 5 times greater
than 14C activity contained in the roots for each of the treatments.

C assimilates translocated to the roots may have been exuded into
the nutrient solution or respired by the roots.

Oxygen stress enhances respiration. Anaerobic conditions
increased CO2 evolved by the roots of Seafarer and San Fernando.
Anaerobically stressed Seafarer and San Fernando respired 2.5 and 2.0
times more CO2 per unit dry weight root, respectively, than the

aerobic controls.

Roots of dry bean varieties Seafarer and San Fernando exuded

49

50

C assimilates even under aerobic conditions. However, anaerobic
stress enhanced root exudation of both dry bean varieties. San
Fernando and Seafarer exuded 30 and 19 times more 14C assimilates
per unit dry weight root, respectively, than the aerobic controls.
Anaerobic Seafarer exuded 42% more 14C-labelled assimilates than
anaerobic San Fernando. Anaerobiosis as well as varietal differences
seem to influence total root exudation.

Aerobic controls did not exude ethanol into the nutrient solution.
However, ethanol accumulated in root assimilates in response to
anaerobiosis with a greater accumulation for Seafarer than San Fernando.
Ethanol exudation as well as production by dry bean roots also seems
to be varietal dependent.

Of the 14C fixed, 35% and 25% more assimilates were detected for
Seafarer and San Fernando, respectively, under anaerobiosis than in
aerobic conditions. Aerobic Seafarer and San Fernando had an exudation
potential of 18.8% and 24.9%, respectively. Anaerobic stressed Sea-
farer and San Fernando demonstrated exudation potentials of-33.3% and
34.3% respectively . The exudation potentials for both anaerobic
varieties Seafarer and San Fernando increased under anaerobiosis and
were approximately 1.8 and 1.4 times greater, respectively, than for
either of their aerobic controls.

Anaerobic conditions promoted by soil compaction appear to enhance
the root exudation potential of dry beans which may cause a reduction
in growth and yield. Improved soil management techniques, as well as
plant breeding programs designed to develop varieties more tolerant
of anaerobic conditions, are needed if yields of dry beans are to be

improved on fine-textured soils in Michigan.

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