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LIBRARY

Michigan Staff

Um v .1"- 2y

 

 

This is to certify that the
thesis entitled

THE INFLUENCE OF MECHANICAL IMPEDANCE
AND SHORT TERM ANOXIA ON RESPIRATION,
GROWTH AND STRUCTURE OF PHASEOLUS VULGARIS
L. ROOTS
presented by

 

Thomas Edward Schumacher

has been accepted towards fulfillment
of the requirements for

M.S. degree in Crop and Soil Science

 

 

Maj professor

Date la 7f

0-7639

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THE INFLUENCE OF MECHANICAL IMPEDANCE AND SHORT TERM
ANOXIA ON RESPIRATION, GROWTH AND STRUCTURE OF

PHASEOLUS VULGARIS L. ROOTS

 

By

Thomas Edward Schumacher

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

THE INFLUENCE OF MECHANICAL IMPEDANCE AND SHORT TERM
ANOXIA ON RESPIRATION, GROWTH AND STRUCTURE OF

PHASEOLUS VULGARIS L. ROOTS

 

Bv

J

Thomas Edward Schumacher

Investigations were carried out to study the effect of mechanical
impedance on shoot and root morphology, root oxygen uptake and short

term anoxia response of Phaseolus vulgaris. variety Seafarer. Seedlings

 

were grown in glass chambers containing 1 mm, 3 mm or no glass bead
matrices. Nutrient solution was passed through the root and matrix sys-
tem. Oxygen content of the solution was controlled by equilibrating it
with 0.2i, 0.10, 0.03, or 0.00 atm of oxygen. Oxygen uptake was measured
using a modified biological oxygen monitor. Primary root growth was
mechanically inhibited by both bead matrices. Lateral root growth appeared
to be limited the most by the smaller glass bead matrix. Porosity, dry
matter percentage, weight, length and volume of roots and several shoot
parameters were altered by mechanical impedance. Roots grown in the 1 mm
matrix had higher oxygen uptake rates per unit fresh weight. Shoot to
root ratios were highly correlated with root respiration for non-impeded
roots. Short term anoxia responses, when corrected for root system size,

were similar among matrix treatments.

ACKNOWLEDGMENTS

I wish to express my sincere appreciation to Louise Solomon Kolker
for the ethanol and amino acid analysis, to Karen Baker and June Mack
of the Electron Optics Laboratory for the transmission electron micro-
scope preparations and to Darrel Berns for laboratory assistance.

Special appreciation is due Dr. A. J. M. Smucker for his guidance
and encouragement during all phases of this thesis.

Acknowledgment is also due Dr. J. C. Shickluna and Dr. A. E.
Erickson of the Department of Crop and Soil Sciences and Dr. S. K. Ries

of the Department of Horticulture.

ii

TABLE OF CONTENTS

LIST OF TABLES
LIST OF FIGURES
INTRODUCTION
LITERATURE REVIEW .

Root Anoxia .
Root growth .
Root respiration
Root porosity .
Shoot growth
Root exudation
Ethylene .
Growth hormones
Root resistance
Ethanol production

Mechanical Impedance
Root growth .
Root respiration
Root porosity .
Shoot growth
Root exudation
Ethylene
Growth hormones
Root resistance .

Mechanical Impedance and Root Anoxia .
Root growth .
Root respiration
MATERIALS AND METHODS
Pyrex Root Chamber .
Plant Growth and Treatment Procedures

Description of Plant Growth System .

Description of Root Respiration Monitor

iii

Page

. vii

I.)

tn

k)
U ‘I

Measurement of Oxygen Uptake . . . . . . . . . . . . . . . . . 31
Transmission Electron Microscopy of Treated Roots . . . . . . 35
Measurement of Root Porosity . . . . . . . . . . . . . . . . . 35

Measurement of Shoot and Root Morphology and Growth . . . . . 36

Measurements of Waterlogged Treatments . . . . . . . . . . . . 38
RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 42
Oxygen Monitoring System . . . . . . . . . . . . . . . . . . . 42
Mechanical Impedance and Root Growth . . . . . . . . . . . . . 49
Mechanical Impedance and Shoot Growth . . . . . . . . . . . . 66
Mechanical Impedance Effects on Root Respiration . . . . . . . 73
Mechanical Impedance and Response to Short Term Anoxia . . . . 79

Potential Effects of Mechanical Impedance on Rhizosphere

Oxygen Utilization . . . . . . . . . . . . . . . . . . . 102
SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . 107
APPENDIX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . . 127

iv

Table

l\)

10.

11.

12.

13.

14.

LIST OF TABLES

Variables used in studies of mechanical impedance -
anoxia interactions

Relation of matrix treatments to pore size, surface
area and porosity

Influence of diurnal measurements on root respiration

Fresh and dry weights of Phaseolus vulgaris shoots and
roots on the day of transplanting to pyrex root chambers

 

Influence of matrix treatment upon root growth of 14 day
old Phaseolus vulgaris L. (var. Seafarer)

 

A comparison of lateral and primary root growth of beans
(var. Seafarer) for the three matrix treatments

Influence of matrix treatment upon root structural para-
meters of 14 day old Phaseolus vulgaris L. (var. Seafarer)

 

The effect of the three matrix treatments on the correla-
tion of root tissue and cellular densities with selected
root parameters

Effect of matrix treatment upon shoot growth of 14 day
old Phaseolus vulgaris L. (var. Seafarer)

 

Separate and combined correlation of leaf area with
selected plant parameters for the three
matrix treatments

Influence of matrix treatments upon selected plant para-
meters for Phaseolus vulgaris L. (var. Seafarer)

 

The effect of the three matrix treatments on the correla-
tion of shoot with root weight parameters

The effect of the three matrix treatments on the correla-
tion coefficients of selected root parameters

Effect of matrix treatments on microbial and root respira-
tion rates of Phaseolus vulgaris L. (var. Seafarer)

 

\7

Page

50

51

62

65

68

69

Table Page

15. Dry matter distribution ratios for roots used in
respiration studies . . . . . . . . . . . . . . . . . . . . 76

16. Separate and combined correlation of root
respiration with selected plant parameters for the
three matrix treatments . . . . . . . . . . . . . . . . . . 18

17. Means of matrix treatments at day 13 and 14, before and
after 24 hour oxygen treatment, averaged over all oxygen
levels . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

18. Analysis of variance for the change in oxygen uptake
rates of bean roots during 24 hour treatments with 4
oxygen levels . . . . . . . . . . . . . . . . . . . . . . . 85

19. The relationship of plant size to root growth rates and
the change in oxygen uptake rates (AOU) during the 0.210
atm p02 treatment . . . . . . . . . . . . . . . . . . . . . 87

20. The effect of 24 and 48 hours of anoxia on root and micro-
bial respiration for Phaseolus vulgaris plants grown in
three matrices . . . . . . . . . . . . . . . . . . . . . . . 91

 

21. The effect of 24 hour treatments of waterlogging (W) and
nitrogen gas equilibrated solution (N) on the oxygen
uptake of the three matrix treatments . . . . . . . . . . . 95

22. Ethanol root exudation by 14 day old Phaseolus vulgaris
plants subjected to a 24 hour waterlogging stress and
three matrix treatments . . . . . . . . . . . . . . . . . . 97

 

23. Amino acid exudation by Phaseolus vulgaris roots water-
logged for a 24 hour period . . . . . . . . . . . . . . . . 98

 

24. Solution pH and conductivity of matrix treatments after
waterlogging treatment . . . . . . . . . . . . . . . . . . . 100

25. Water uptake by 14 day old Phaseolus vulgaris plants sub-
jected to a 24 hour waterlogging stress and three matrix
treatments . . . . . . . . . . . . . . . . . . . . . . . . . 101

 

26. Percentage of root system located at various depths as
approximated from root length measurements . . . . . . . . . 103

27. Means of matrix treatments averaged over oxygen treatments
for 14 day old Phaseolus vulgaris root parameters . . . . . 123

 

28. Means of matrix treatments averaged over oxygen treatments
for 14 day old Phaseolus vulgaris plant parameters . . . . . 124

 

29. Means of matrix treatments averaged over oxygen treatments
for 14 day old Phaseolus vulgaris shoot parameters . . . . . 125

 

vi

LIST OF FIGURES

Figure Page
1. Pyrex root chamber construction . . . . . . . . . . . . . . l9
2. Diagram of the plant growth system . . . . . . . . . . . . . 24

3. An overview of the plant growth system in the growth

 

chamber . . . . . . . . . . . . . . . . . . . . . . . . . . 26
4. Diagram of the root respiration monitor . . . . . . . . . . 29
S. System used to monitor root oxygen uptake 32
6. Calibration curves for the root respiration monitor 33
7. Root length measurement grid 37
8. Comparison of methods for determining root volume . . . . . 39
9. Comparison of methods for determining root volume responses

to matrix treatments . . . . . . . . . . . . . . . . . . . . 40
10. The effect of solution flow rate on exit p02 and oxygen

uptake measurements . . . . . . . . . . . . . . . . . . . . 43
11. Relationship of exit p02 to treatment p02 for the root

respiration monitor . . . . . . . . . . . . . . . . . . . . 45
12. Relationship of oxygen uptake to treatment p02 for 14 day

old dry bean roots . . . . . . . . . . . . . . . . . . . . . 46
13. TEM micrographs of representative primary root tip cross

sections (x3200) . . . . . . . . . . . . . . . . . . . . . . 53
14. Root systems of the three matrices displaced on grid used

to measure root length and diameter . . . . . . . . . . . . S6
15. Comparison of the three matrices effect on root morphology S7
16. Root growth around a 3 mm glass bead 39
17. Root chambers containing 14 day old Phaseolus vulgaris

plants growing in different matrices . . . . . . . . . . . . 60

vii

Figure Page
18. TEM micrographs of cortical cell cross sections . . . . . . 61
19. Oxygen uptake by 14 day old Phaseolus vulgaris roots and

microbes as affected by matrixtreatmentsduring 2 hour
oxygen treatments . . . . . . . . . . . . . . . . . . . . . 80

 

20. Change in oxygen uptake rates (AOU) during 24 hour oxygen
treatments . . . . . . . . . . . . . . . . . . . . . . . . . 82

21. Change in oxygen uptake (AOU) during 24 hour oxygen treat-
ments of Phaseolus vulgaris roots for the three matrix
treatments . . . . . . . . . . . . . . . . . . . . . . . . . 84

 

22. Change in oxygen uptake (AOU) during 24 hour oxygen treat-
ments standardized against root fresh weight of Phaseolus
vulgaris for the three matrix treatments . . . . . . . . . . 86

23. Influence of 24 hour oxygen treatments on oxygen uptake per
unit fresh weight of Phaseolus vulgaris roots for the three
matrix treatments . . . . . . . . . . . . . . . . . . . . . 89

 

24. TEM micrographs showing the effects of a 24 hour anoxic
period on cortical cells located 1 cm above the lateral
root tip . . . . . . . . . . . . . . . . . . . . . . . . . . 92

25. TEM micrograph comparing cytoplasmic organization of 24
hour anoxic stressed and nonstressed 14 day old Phaseolus
vulgaris plants . . . . . . . . . . . . . . . . . . . . . . 93

26. Mechanical impedance-anoxia interactions from the study of
Gill and Miller (34) . . . . . . . . . . . . . . . . . . . . 113

27. Mechanical impedance-anoxia interactions affecting root
weight production from the study of Barley (9) . . . . . . . 114

28. Mechanical impedance-anoxia interactions affecting root
elongation from the study of Barley (9) . . . . . . . . . . 115

29. Mechanical impedance—anoxia interactions affecting root
elongation from the study of Aubertin and Kardos (4) . . . . 116

30. Mechanical impedance-anoxia interactions affecting depth
of root penetration from the study of Tackett and
Pearson (98) . . . . . . . . . . . . . . . . . . . . . . . . 117

31. Mechanical impedance-anoxia interactions affecting root
elongation from the study of Eavis (28) . . . . . . . . . . 118

32. Mechnical impedance-anoxia interactions affecting time to

reach 32P in compacted Commerce silty loam from the study
of Hopkins and Patrick (48) . . . . . . . . . . . . . . . . 119

viii

Figure Page

33. Mechanical impedance-anoxia interactions affecting root
penetration into compacted cores of Commerce silty loam
during a 14 day period from the study of Hopkins and
Patrick (48) . . . . . . . . . . . . . . . . . . . . . . . . 120

34. Influence of Hoagland's solution conductivity on the
oxygen uptake of 30 day old Zea mays roots . . . . . . . . . 121

35. Inhibition of 14 day old Phaseolus vulgaris root growth
caused by an acid nutrient solution (pH 2) . . . . . . . . . 122

 

 

36. Sequence demonstrating response of Phaseolus vulgaris to
conditions of low humidity (5% relative humidity) and a
negative pressure in the root chamber (~20 cm H20) . . . . . 126

 

ix

INTRODUCTION

Environmental stresses on crops contribute to the high degree of
variability in annual yield which plague many areas of the world. The
development of management practices and germ plasm to stabilize fluctu-
ating yields requires an understanding of the effects and mechanisms
of the various stresses.

Intensive cropping systems are being used more extensively as a
result of increasing demands for greater food production. Such systems
under conditions of poor soil management and marginal soils result in
compacted soils. These soils are associated with a soil microenviron-
ment which promote smaller root systems. A small root system in addi-
tion to imposing a possible stress on the shoot predisposes the plant
to damage from other environmental stresses.

Three major functions of the root are anchorage, absorption of
water and nutrients, and the synthesis of growth regulators (26). A
small root system has a reduced capacity to carry out these functions.
Plants with small root systems have lower thresholds for stress re-
sulting from excessive wind, drought and low soil fertility.

Plants with large root systems appear to have greater potentials
for absorbing water and nutrients due to the increased soil volume
available to plants. A deep root system is able to permeate into soils
containing greater quantities of water than small shallow root systems.

Deep and dispersed root systems may also provide greater anchorage.

Soil conditions interfering with root growth also appear to reduce
growth regulator export to the shoot (26). Consequently, smaller root
systems could directly reduce shoot development by inhibiting the
production of specific growth regulators.

Compacted soils are both physical and chemical barriers to root
growth. Root growth is mechanically impeded by compacted soils when
soil particles mechanically resist root elongation. A combination of
soil pores smaller than the mean root tip diameter and soil resistance
to deformation by the roots form the mechanical barrier.

A second barrier to root growth in compacted soils results from a
lowered oxygen supply available to roots. Root anoxia (oxygen deficiency
of root tissue) produces a reduction in aerobic respiration and a con-
comitant increase in anaerobic respiration. Subsequently, root growth
is reduced by toxic compounds, lower metabolic energy and changes in
hormonal relationships.

Mechanical impedance and anoxia frequently act together to limit
root growth and it is difficult to separate the effects these two fac-
tors have on root growth. Little is known concerning the interactions
of these two factors or their separate influences which limit root growth.

This study was designed to investigate the combined and
separate influences of mechanical impedance and anoxia on root growth.
Objectives of this study were:

1. To develop a system which could be used to separate and measure
the effects of mechanical impedance and anoxia on the whole root system

of dry edible beans (Phaseolus vulgaris L.) during the vegetative stage.

 

2. To determine the effect of mechanical impedance on root and

shoot morphological characteristics.

3. To determine the oxygen uptake rates of mechanically impeded
and nonimpeded roots under selected oxygen concentrations.
4. To determine the effect of short term oxygen treatments on

root respiration and morphology.

LITERATURE REVIEW

Root Anoxia

 

Root Growth: Much literature is devoted to the adverse effects of

 

anoxia on root growth (41,97,61). Cell elongation and division are
retarded by anoxia. Elongation of cotton and soybean seedlings stopped
within 2-3 minutes after oxygen was removed from the media (49). Root
growth pressure was also reduced (29). Poorly aerated roots were shorter,
thicker, more branched and had fewer root hairs than well aerated roots
(28,60).

Root elongation was reduced at intermediate oxygen partial pressures
(p02) of 0.03 - 0.10 atm (28,49,66). Huck (49) found that roots gradu-
ally recovered their original elongation rates under continued exposure
to intermediate p02. Loepez-Soez et_al, (66) found that cell division

was more sensitive to p0 below 0.10 atm than was elongation. Eavis

2
(28), however, demonstrated that between p02 of 0.08 and 0.21 atm the
rate of cell division was unaffected while cell elongation decreased.

At a p02 of 0.03 atm the rate of cell division was also reduced.

Root Respiration: The loss of metabolic energy has been proposed

 

as a cause of root inhibition during anoxia (72). During anaerobiosis,
when oxygen is unavailable to accept electrons, organic acids from the
tricarboxylic acid cycle and other reduced compounds accumulate. This
prevents the oxidation of pyruvate in the mitochondria and the reoxida-

tion of cytoplasmic NADH formed during glycolysis. The cell must have

a mechanism to reoxidize NADH to continue anaerobic glycolysis. The
predominate pathway in most plants is through the decarboxylation and
reduction of pyruvate to ethanol. Anaerobic respiration yields two

ATP molecules, two carbon dioxide molecules and two ethanol molecules
from one glucose molecule. Aerobic respiration yields thirty-six ATP
molecules and six carbon dioxide molecules from the degradation of one
glucose molecule. Anaerobic cells attempt to adjust to an energy
imbalance by increasing the rate of glycolysis (Pasteur effect) (35,62).
The amount of energy available to the anaerobic root is 1% of that under
aerobic conditions (2).

As oxygen concentrations decline, oxygen uptake by the root declines
in a hyperbolic fashion. Oxygen concentrations at which oxygen uptake
began to decline, varied from just below 0.21 atm to below 0.05 atm
(2,89,110). Based on the Km for cytochrome oxidase and the concentra-
tion for oxygen in air-saturated water, oxygen consumption in the cell
should approach half the maximum velocity when cell pO2 approaches 3 x

10'5 to 4 x 10'3

atm (41). The former observations can be explained by
the slow diffusion of oxygen in water compared to that in air (97). The
diffusion of oxygen may be further reduced in living tissue to one fourth
of that in water (63).

Luxmoore 32 El: (71) concluded that the gas filled porosity of the
root system and the thickness of the water film around the root have
the largest influence on the oxygen relations of maize roots. Thickness
of the water film adjacent to roots is dependent on the soil water
potential and the distribution of roots in relation to soil pores.

Letey and Stolzy (65) calculated the following water film thickness as

being growth limiting: 400, 150 and 7 um for p02 of 0.20, 0.10 and 0.02

atm, respectively. Oxygen partial pressure gradients in water-saturated
aggregates can range from 0.21 to 0.00 atm in 1 mm if soil respiration
rates are high (42). Scotter e£_al, (89) concluded that decreases in oxy-
gen uptake of 50-60 day old dwarf peas grown in Ottawa sand at low oxygen
concentrations, were due to diffusion resistances inside the root and

not in the water film surrounding the root. Amoore (2) concluded that
respiration at low oxygen concentrations was limited by the slow diffu-
sion of oxygen through the tissue of pea root tips. Lemon (63) suggested
that oxygen uptake of stele cells could be decreased at a critical sur-
face oxygen partial pressure. Subsequent decreases in root surface pO2
resulted in an increase in the size of the oxygen deficient zone and

p02 dependent oxygen uptake rates. Critical levels increased with the
square of the radius of the root for any given respiration rate (64).

Root Porosity: Investigators have observed intercellular gas spaces

 

in roots since the late 19th Century (74). Tracer studies for several
1

species showed that 502 flow through the gas spaces fit a model of oxy-
gen diffusing down a long narrow porous tube (6,31,52,54). Root porosity
is directly related to the longitudinal oxygen diffusion coefficient

of roots and is simpler to measure (53). Generally, plants well

adapted to waterlogging have larger root porosities. Cultivars of

maize, wheat and rice with high root porosities appear to have improved
tolerance to anoxia (24,25,112). Simulation models demonstrated that

under appropriate conditions root porosity could contribute significant

amounts of oxygen to the root (70,110). Jensen et_al. (53) reported

o\°
o\°

the following root porosities: 4.2% :_.5%; 7.6 1 1%; 26.5 1 3% for

Phaseolus vulgaris L., Zea mays L. and Oryza sativa L., respectively.

 

 

 

The contribution of this pathway depends on the degree of root porosity.

respiration rate, length, diameter and cell permeability to oxygen (70).
Most plant species with root systems larger than that of a seedling may
not utilize this pathway to a great extent (41,78).

Limited oxygen content in the rooting medium induced higher root
porosities in several species (25,67,75,82,103), although this induction
was not found in maize and rice (68,102). High light intensity and'
temperature were also correlated with greater root porosities in wheat
and rice (67,96,102,103).

Shoot Growth: Shoot growth of many plant Species are inhibited by

 

root anoxia. Epinasty was usually the first symptom to appear (51)
followed by wilting, if conditions were favorable for high transpira-
tion rates (61). Continued anoxia resulted in the progressive yellowing
and death of older leaves (57).

Yields of sorghum, soybeans, cabbage, sweet corn and dwarf field
corn were drastically reduced when plants were subjected to long term
anoxic stress (108). Short term anoxia may also be very detrimental.
Pea and tomato yields were severely decreased when plant roots were
subjected to soil flooding for 24 hours during critical stages of growth
(30).

Root Exudation: Short periods of anaerobiosis increased the exuda-

 

tion of organic compounds (43,85,95). In comparison to controls, anoxic
corn and sunflower plants excreted larger amounts of the following amino
acids: alanine, aminobutyric acid, proline, glutamic acid, serine,
threonine, aspartic acid and histidine (43). In an aseptic mist chamber
study using peas, Smucker (95) reported that an atmosphere of 70% nitrogen
and 30% carbon dioxide increased the exudation of amino acids four-fold,
with alanine being the predominant amino acid exuded under anaerobic

conditions.

Ethylene: Ethylene is considered to be a factor in anoxic plant
damage (20,51,57,94). Root elongation in a wide variety of plant species
were inhibited by ethylene concentrations of 1 ppm or more (59,92).
Ethylene concentrations of 1 ppm or higher are frequently found in soils
when the p02 is below 0.02 atm (93).

Soil ethylene appears to be produced primarily by anaerobic bacteria
(20,91). Smith (91) purposed that ethylene is produced in anaerobic
microsites in the soil. The rhizosphere is likely to contain a high
prOportion of microsites because of the rapid utilization of oxygen
by plant roots and the rhizosphere bacteria (90).

Ethylene from the soil environment diffused to the shoot through
intercellular air spaces (50). The root also appears to transport
precursors or stimulators of ethylene formation to the shoot (51).

Growth Hormones: Waterlogging can effect the concentrations of

 

various growth hormones in the plant. Burrows and Carr (18) found
that flooding the root system of sunflower plants reduced the translo-
cation of gibberellin like substances. Phillips (81) found that flooded
plants contain three times the auxin content of control plants.
Resistance to anoxia in tobacco plants previously grown in saline
solution appeared to be caused by increased amounts of abscisic acid
in the shoot. Less damage also occurred in plants pretreated with
abscisic acid (76).
Cornforth (20) purposed that short term anoxic damage is the result
of ethylene interaction with growth regulators in the shoot. This
mechanism would be most critical during the reproductive phase due to

the important role of growth regulators in reproduction.

Root Resistance: Kramer (61) stated that flooding injury is

 

usually attributed to dessication resulting from increased root resis-
tance to water absorbtion. Resistance to water flow through root tissue
was greatly increased by removing oxygen from solution cultures (17,61,
105). Water uptake by tobacco plants was reduced by 50% when subjected
to a six hour anaerobic period (105). Shoot dehydration resulting from
root anaerobiosis was more prevalent under conditions promoting high
transpiration rates (61).

Ethanol Production: Plant damage during anoxia may be due to the

 

accumulation of ethanol. Many cultivated plant species subjected to
soil anaerobiosis produce ethanol as an end product of anaerobic metabo-
lism (21). Bolton and Erickson (l4) correlated ethanol content in the
xylem exudate with the degree of anoxic damage.

Crawford (22) concluded that the ability of a wide variety of
tissues and species to tolerate low oxygen environments was due to the
limitation of ethanol production. Ethanol production and movement in
the plant can be limited in various ways. Alternate pyruvate pathways
have been shown to occur in flood tolerant plants. In such cases, a
nontoxic compound (eg. malate) is formed from pyruvate in place of
ethanol (21). Studies have shown reduced alcohol dehydrogenase (ADH)
activities in flood tolerant species of maize and clover (32,72). How-
ever, other studies with barley and maize indicated that ADH activities
may be higher in flood tolerant cultivars (106,107). John and Greenway
(55) suggested that pyruvate decarboxylase (PCD) activities better
represent in_yivg_alcholic fermentation since the activity of PDC is
generally rate limiting. Rice adapted to anoxia by producing larger

amounts of ethanol (55). Crawford and Baines (23) suggested that this

10

could be explained by the ready diffusion of ethanol from the adventious
roots of rice. Plants which have a mechanism of removing ethanol may
be able to tolerate flooding. Intact roots have been shown to exude

large quantities of ethanol during short term anoxia (44,95).

Mechanical Impedance

 

Root Growth: Root elongation appears to decrease with increasing

 

mechanical impedance (ll). Pressures inhibiting root elongation were'
much smaller than pressures developed by the root. Soil pressures in
the range from 0.2 to 0.7 bar impeded root growth significantly (11,87).
This compared to root growth pressures of 5 to 25 bars produced by a
wide variety of plant species (29,33,99).

Greacen and Oh (40) postulated that the reduction of root elonga-
tion in mechanically impeded soil was due to a reduction in the ability
of the root to osmoregulate against an external force. Russell and
G055 (87) supported an alternate theory that elongation is controlled
by hormonal regulation of cell wall extension.

Root penetration into soil depends upon soil strength and the
diameter of soil pores (37). Accordingly, rigid (3,38,56,104) and non-
rigid (1,9,10,34,37) systems have been used to study mechanical im-
pedance. Rigid systems demonstrated that roots were unable to penetrate
pores smaller than the root tip diameter (37,104). Nonrigid systems
were used to determine the minimum pressure which inhibited root elonga-
tion (26).

Mechanical impedance generally stimulates the develOpment of
lateral roots near the root apex (87). If lateral root formation is
prolific, the total weight and ability of the root system to absorb

nutrients may not be affected (37).

ll

Morphologically, roots grown in a nonrigid 1 mm bead system had
longer root axes and fewer lateral roots per root axis than roots grown
in rigid systems. Barley axial roots penetrated up to 6 cm in a rigid
system of 1 mm glass beads before the root apex appeared to lose its
meristematic activity (36). Barley lateral roots grew twice as long
in the 1 mm bead matrix than in the 3 mm bead matrix. This compensated
for the lack of growth by the root axes (37).

Cortical cells of impeded roots were reduced in length but greatly
increased in width, resulting in a thicker root with essentially no
change in the cell volume (11,87). However, Wilson et_al: (109) ob-
served great variability in dimensions of cortical cells in impeded
barley roots. Radial expansion occurred near the root tip but overall
root volume and surface area were reduced. Radial expansion near the
root tip may reduce the stress on axial elongation (11).

Root Respiration: Very few studies have measured respiration rates

 

of mechanically impeded roots. Tackett and Pearson (98) found that
roots growing in a soil of high bulk density produced more carbon dioxide
per root dry weight than did roots grown in low bulk density soil. This
was true over a wide range of oxygen concentrations. However, a study
by Eavis (28) suggested that mechanically impeded roots need less oxygen
per unit root than nonimpeded roots. The larger root diameters of im-
peded roots (70) and the increased root surface area blocked by the
solid phase of the soil (41) could reduce the diffusion of oxygen
resulting in lowered oxygen uptake.

Oxygen uptake rates per unit root may not change for a mechanically
impeded root. Theoretical calculations based on the energy requirements

of an elongating root indicate that energy used in elongation is small

12

compared to the total amount produced. A root 1 mm in diameter
elongating at a rate of 1 mm per hour against a resistance of 10 bar
requires approximately 0.2% of the energy produced during respiration
(13).

Root Porosity: Although root porosities have not been measured

 

for mechanically impeded roots, there are indications that significant
changes can occur in impeded roots. Roots grown in the soil generally
have less porosity than those grown in nutrient solution (67). 6055
and Walter (38) observed distorted cortical cells in barley roots grow-
ing in a 1 mm rigid bead system. It appears that root responses to an
obstruction result in cell wall relaxation permitting turgor forces to
act externally causing cell distortion (12). Barley and Greacen (12)
suggested that cell distortion causes compression of the intercellular
spaces which decrease internal aeration. The decreased gas filled space
and perhaps greater tortuousity could reduce the diffusivity of gases
in plant tissues resulting in the accumulation of carbon dioxide and

volatile growth regulators, such as ethylene (ll).

Shoot Growth: Accurate observations on the effect of mechanical

 

impedance on shoot growth are difficult because a plant with an impeded
root system is prone to many other stresses. Laboratory investigations
which control water availability, nutrient availability and oxygen con-
centrations around the root indicate that mechanical impedance pgr_§3_
may not harm the shoot (26). Cannell and Finney (19) indicated that
mechanical impedance of the seminal roots of cereals grown in the field
was not harmful when an adequate supply of nutrients and water were

available.

13

Root Exudation: The loss of organic compounds was enhanced when

 

roots were grown in a solid medium (7,8). Barber and Gunn (7) grew
barley and maize in nutrient solutions with and without glass beads.
Exudation of amino acids and carbohydrates increased from roots grown
in the presence of 1 mm glass beads. Exudates from roots grown in 3
mm glass beads were greater than solution cultures and less than the
1 mm glass bead matrix. Hale et_al: (45) suggested that roots grown
in soil may exude more than those grown in solution culture, but
questioned the ability of investigators to remove roots from the soil
uninjured. Barber and Martin (8), in a soil study using 14C labelled
carbon dioxide, estimated root exudation to be at least 10% of the dry
matter increment of wheat seedlings. This value is much higher than
values obtained in nutrient culture studies.

Ethylene: Ethylene may be a contributing factor in the reaction
of roots to mechanical impedance. Kays et_al. (58) found that roots

of Vicia faba grown against a barrier produced six times more ethylene

 

than the control. Roots exposed to 100 ppm ethylene for 10-25 days
showed enhanced lateral root formation and inhibition of axial root
extension (92). This is similiar to responses seen in mechanically
impeded roots. Barley (11) purposed that endogenously produced ethylene
in mechanically impeded roots may cause elongating cells to become
shorter and wider resulting in the inhibition of root elongation.

Growth Hormones: The effects, if any, of mechnical impedance on

 

plant growth hormone regulation in the shoot is unknown. The root apex
has been shown to be a major source of auxins, cytokinins, gibberellins
and abscisic acid (86). 6055 and Ward (39) demonstrated that the root

apex was important in determining the rate of elongation. However, the

l4

relationship between the synthesis of growth hormones and root response
to mechanical impedance has not been investigated. Physical models of
root response to mechanical impedance are inadequate and there is in-
creasing speculation that root response to mechanical impedance is
mediated by growth hormones (87).

Root Resistance: Resistance to water movement by mechanically

 

impeded roots has not been measured. Mechanical stress resulted in
thicker cortical tissue and greater surface area per unit length of
root (109). If the main resistance to water flow is at the endodermis,
water uptake rates should be prOportional to root surface area (77).

An increased uptake of water and nutrients resulting from increased
root surface area may compensate partly for the smaller root system in
mechanically impeded roots. The increased tortuosity of cortical cells
of mechanically impeded roots (11) could result in an additional re-
sistance to water uptake if the cortical apoplast (free space) is

limiting water uptake.

Mechanical Impedance and Root Anoxia

 

Root Growth: Several investigators have studied the combined and

 

separate effects of mechanical impedance and anoxia on root growth (4,
9,27,28,34,48,84,88,98). Roots growing at 0.21 atm of 02 showed de-
creased growth rates with increasing mechanical impedance. Roots grow-
ing at minimum levels of mechanical impedance showed reduced growth
rates with decreasing p02. Data at intermediate levels of mechanical
impedance and oxygen partial pressures generally have been interpreted
as demonstrating an interaction between the two factors (12). Examina-

tion of the data indicates that the type of interaction taking place

varies widely.

15

A decrease in p02 from 0.21 to 0.10 atm resulted in varied root
growth rate responses for mechanically impeded and nonimpeded roots.
Most studies demonstrated a greater reduction of root growth rates in
nonimpeded roots (9,27,34,48). Other studies showed a greater decrease
in root growth rates for mechanically impeded roots (9,48); similar
increases in root growth rates (4) and no change in root growth rates
(98).

A reduction in p02 from 0.10 to 0.03 atm also resulted in discre-
pancies between studies. Studies demonstrated greater reductions in
root growth rates for nonimpeded roots (9,48); greater reductions in
root growth rates for mechanically impeded roots (4,48,98) and similar
reductions in root growth rates (29).

Part of the variance arises from the parameters used to measure
root growth. Barley (9) found a larger decrease in root growth at 0.03

atm pO with roots subjected to 0.5 bar pressure compared to 0.0 bar

2
pressure when using root weight as the parameter. When using root

length as the parameter, root growth decreased at the same rate at both
pressures. Discrepancies also arise from the methods and procedures used.
Table 1 outlines principal points from the various investigations. Graphs
in the appendix visually depict the interactions outlined previously,

Figures 26 to 33.

Root Respiration: Tackett and Pearson (98) measured the combined

 

influence of anoxia and mechanical impedance on carbon dioxide produc-
tion. Carbon dioxide production per dry weight of root, decreased
linearly with decreasing pO2 for roots grown in high bulk

density soil. Carbon dioxide production per root dry weight by roots

growing in soils of low bulk density were constant when p02 levels were

16

greater than 0.05 atm and then increased sharply at p02 levels less

than 0.05 atm. They concluded that root respiration was dependent on
p02 for roots grown in high density soils but not for roots grown in
low density soils. However, the microbial background was not measured.
Microbial respiration could have masked root response under conditions

of limited root growth.

 

 

 

 

 

 

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MATERIALS AND METHODS

Pyrex Root Chamber

 

A glass chamber was designed to hold the root medium and

provide a rigid support to the plant while allowing flow of aerated
nutrient solution past the root system, Figure l. Neoprene stoppers

were modified by removing the top quarter section and boring three holes
into the remaining section. The center hole served as an exit for the
circulating nutrient solution. The two plant ports were split diagonal-
ly on the side to facilitate the transfer of seedlings. The glass cham-
ber was flared at the top to accept the modified no. 10 stopper. Volumes
of the chambers were approximately 150 cm3. The lower inlet port was
fitted with a 6 mm septum through which aerated nutrient solution

entered the chamber.

Plant Growth and Treatment Procedures

 

Phaseolus vulgaris L., variety Seafarer, seeds were germinated in

 

an incubator at 25°C. Fifteen seeds were placed on each set of two
towels and saturated with 22 ml distilled water. On day five, when
hypocotyls were approximately 2.5 cm, seedling pairs of similar size
were transferred to the modified neoprene stOppers. Parawax, a 1:6
mixture (g/g) of paraffin and petroleum jelly, was used to support the
seedling in each plant port and prevent gaseous exchange and loss of

nutrient solution. Care was taken during the transfer process to

18

19

PLANT PORTS

   

EXIT PORT

 

 

 

 

T ¥ . MODIFIED NEOPRENE
T 3:" I'chl: STOPPER

 

15cm

i K? INLET PORT

Figure l. Pyrex root chamber construction.

 

 

 

prevent damage from dehydration and to minimize mechanical damage to
the root system.

Stoppers containing the seedlings were transferred to a small jar
containing nutrient solution and placed in the growth chamber. This
served as a means of assessing transfer injury before the introduction
of matrix treatments. Seedling pairs were again selected for uniformity
after 24 hours. The seedling and stopper were both fitted to the top
of a pyrex root chamber, filled with nutrient solution and a strip of
parafilm was used to seal the stopper to the glass chamber. Two rubber
bands aided in retaining the stopper and helped to prevent nutrient
solution loss.

Matrix treatments consisting of 1 mm glass beads, 3 mm glass beads
and no glass beads (control) were randomly chosen within each replica-
tion and introduced into the pyrex root chambers through the center port.
The glass beads were carefully added to the solution to minimize damage
to the root system and prevent the entrapment of air. DiSplaced solu-
tion was drained through a needle inserted in the bottom septum. Glass
beads were tightly packed by intermittent vibrations from 3 Cole Palmer
supermixer. Table 2 provides information for each matrix treatment.
Individual replications were randomly allocated positions within one
unit of the plant growth system.

Experiments having an objective of determining the effects of
mechanical impedance on shoot and root parameters were conducted at a
constant p02 of 0.21 atm. Plants were grown for 8 days in the plant
growth system, described in the next section, using a
randomized complete block design. Experiments designed to determine

the combined effects of mechanical impedance and anoxia on root growth

Table 2. Relation of matrix treatments to pore size, surface area and

 

 

porosity.
Matrix Matrix and
mean pore root chamber Matrix
diameter surface area porosity
Treatment (cm) (cmz) (%)
Control -- 180 100
3 mm matrix 0.045 1,950 40

1 mm matrix 0.015 5,870 36

 

were exposed to p0 below 0.21 atm for specific periods of time. One

2
experiment examined the effect of root exposure to p02 of 0.210, 0.105,
0.032 and 0.00 atm for one hour on oxygen uptake by roots. Selected

p02 were introduced in decreasing order to the treatments and oxygen up-
take monitored. The 0.00 atm pO2 treatment was followed by a return to
0.210 atm pO2 for two of the replications. These oxygen treatments

were introduced 14 days after placing seeds in the incubator. Another
experiment examined the effects of a 24 hour exposure to randomly
selected p02. Levels of p02 were the same as in the experiment described
above. The 0.00 atm p02 treatment was achieved by equilibrating the
circulating nutrient solution with nitrogen gas. The remaining p02
treatments were achieved by equilibrating the circulating nutrient solu-
tion with prepurified standards from Matheson. A waterlogging treatment
in which the nutrient solution was not circulated through the pyrex

root chambers was also applied and analyzed separately. Gas treatments
were introduced on the 13th day. Oxygen uptake measurements were made
before and after the 24 hour treatment period. The order of measurement
among treatments was determined randomly within each replication.

Limitations of space and time resulted in the application of the
various oxygen treatments separated by time. A split plot design
was used. The main treatments consisted of oxygen levels and the sub-
treatments were two replications of the matrix levels.

The inability to measure other parameters before treatment and the
variability between treatment periods precluded the use of other para-
meters as indicators of effects of short term anoxia. An exception was
the waterlogging treatment in which root exudation (ethanol and amino
acids) andwater uptake data werealso taken during the 24 hour treatment

period.

23

Description of Plant Growth System

 

The objective of the plant growth system was to maintain a control-
led environment for the shoot and root systems. The differing environ-
ments of the shoot and root required separate control systems.

The shoot environment was controlled by a Warren-Shearer model
CEL-36-10 growth chamber. The growth chamber light source consisted of
eight 75 watt:incandescent bulbs and twelve 39 watt fluorescent bulbs
which produced 680 uEinsteins sec.1 m-2 for full light. Incandescent
light produced quanta of 86 uEinsteins sec.1 m- as measured by a Lambda
LI-l70 quantum/radiometer/photometer. The 15 hour photoperiod consisted
of 13 hours of full light preceded and followed by 1 hour of incandes-
cent light only. Temperature was maintained at 20°C for 11 hours during
the dark period and at 30°C for 13 hours during the light period. Rela-
tive humidity in the growth chamber was maintained at 40—60%. Pyrex
root chambers were clamped on a stand with the top of the stopper 45 cm
from the light source.

The root environment was controlled in part by pumping aerated
nutrient solution past the root system, Figure 2. The circulation sys-
tem consisted of 3 units each with a separate nutrient solution reservoir
and pump channel. Each unit had the capacity to service 6 pyrex root
chambers (two replications of matrix treatments). Temperature of the
root system was slightly lower than the shoot temperature.

A variable speed Masterflex pump continuously circulated a modified
half-strength Hoagland's solution at a flow rate of 15 to 25 ml/min.
Nutrient solution from a common 6 l reservoir was pumped into the pyrex
root chamber through a syringe needle at the base of the chamber and flowed

past the root system. Nutrient solution in the reservoir was vigorously

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25

aerated with acid washed compressed air flowing through cindered gas
dispersion tubes. Oxygen treatments less than 0.210 atm p02 were intro-
duced through gas dispersion tubes from the appropriate gas tanks for
the experiment involving 24 hour oxygen treatments. A photograph of

the actual plant growth system is shown in Figure 3.

The pH and conductivity of the nutrient solution were monitored
daily. The pH was maintained at 6.5 :_0.5 pH units by the addition of
either 0.1 M H2804 or 0.1 M NaOH. Nutrient solution was replaced every
seven days. All tubing, glass chambers and reservoir flasks were
covered with aluminum foil to prevent algal growth.

The primary difficulty of this system was the need to closely
monitor the system for leaks. Tubing in the peristalic pump needed to
be replaced every three to four days. The system could be improved by
installing an air entrapment device between pump and the pyrex root

chambers. This would prevent air entry into the root chamber and

oxygen monitor system.

Description of Root Respiration Monitor

 

Root oxygen uptake measurements were made using a modified YSI
model 53 biological oxygen monitor. The system is based on polarographic
oxygen sensors. Other investigators of root respiration have incorporated
the use of polarographic oxygen sensors in their studies. A brief sum-
mary of their studies follows.

Scotter et_al. (89) determined, using polarographic methods, the
oxygen concentration in gas circulated through a sand column containing
pea roots. Root oxygen uptake was measured in a closed circulating

system by monitoring the change in gaseous oxygen concentration with

26

 

Figure 3. An overview of the plant growth system in the growth chamber.

27

time and measuring the gas pressure increase when a known amount of
gas was added to estimate the gas volume. Willey(105) used a similar
method in a mist chamber study. An additional oxygen electrode was
used to determine if equilibrium existed between the misting solution
and the treatment gas phase.

Higgins et_al, (47) employed a polarographic oxygen sensor to
measure the respiration rate of excised root tips in stationary solu-
tion. Boogie (15) measured the oxygen concentrations of aliquots of
nutrient solution using a polarographic oxygen sensor for a root growth
study.

The requirements of the present study determined the need for a
system which could monitor the oxygen concentrations in solution before
and after flowing past a root system. The respective lack and presence
of a solid medium in the control and bead treatments prevented the use
of a mist chamber or a gas permeated solid medium study. Inability to
control the water film thickness around the root between the different
matrix treatments prevented the use of directly supplying oxygen, as a
gas, to the roots. The supply of oxygen to the root is dependent upon
the distance oxygen must diffuse through water to the root (65). This
study required a method of minimizing differences in oxygen gradients
around the root surface arising from the different matrix treatments.
The use of a solution flowing past the root system to supply oxygen,
water and nutrients was an attempt to minimize gradient differences of
these components among the matrix treatments.

The designed root respiration monitor measured the difference in
oxygen partial pressures in solutions before and after passage

through the root system. Root oxygen uptake was calculated using the

28

differential oxygen concentration and solution volume determined from
the nutrient solution flow rate.

A diagram of the root respiration monitor used to measure the root
oxygen uptake is shown in Figure 4. A four liter flask modified as a
Mariotte flask (a) served as the reservoir for the one-half strength
modified Hoagland's solution. The siphon from this flask delivered
solution to a 500 ml thermos bottle serving as the gas equilibrium
chamber (c). Treatment gases from bottled gas (b) were equilibrated in
the nutrient solution by vigorous mixing through gas dispersion tubes.
A magnetic stirring bar aided in bringing the solution to gas equili-
brium. The gas equilibrated solution was pumped (d) from the gas
equilibrium chamber to a smaller chamber (e) within the water bath (i)
housing the oxygen probe chambers. This chamber allowed the solution
to reach temperature equilibrium with the water bath while gas was
dispersed through the solution. Siphons (not shown in diagram) were
connected to this chamber from the gas equilibrium chamber as well as
the two chambers containing the oxygen probes. The siphons maintained
the level of solution to the level of the air inlet tube of the Mariotte
flask. Temperature equilibrated solutions were pumped through the oxy-
gen probe chamber I (f) via access holes (2.4 mm) which were drilled
through the acrylic oxygen probe plunger. The chamber contained 5 ml
of solution. A magnetic stirring bar at the base of the chamber pre-
vented oxygen diffusion gradients from developing around the electrode
membrane. The solution was then transferred to the glass chamber con-
taining the root system (j). Solution exiting the root chamber flowed
through a vertical float type flow meter (1). Temperature of the re-

turning solutions were equilibrated by a glass thermal exchange coil (m)

29

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in the oxygen monitor water bath. The solution next flowed

through the oxygen probe chamber II (o) as described for the first probe.
This chamber had a slight positive pressure preventing the entry of
solution from the siphon tube. The solution was then pumped into a
reservoir for waste or sampling.

A small line (g)(ID lnmfl served as a shunt between (j) and (c) to
minimize pressure build up within the root chamber. This line also
served to trap air bubbles introduced into the system. A three-way
valve (h) was also positioned near this line providing the possibility
of obtaining solution from another source in the event of air introduc-
tion in the first half of the system. A Y tube (n) fitted with a septum
was placed between the oxygen probe chamber II and glass thermal exchange
coil and served as an air entrapment device. A similar tube (not shown
in the diagram) was placed between the temperature equilibrium chamber I
and oxygen probe chamber 1.

Flow through the oxygen monitoring system was regulated with an
eight channel Miniplus II HP8 peristalic pump. Flow rates from 6 to 12
ml/min were achieved. Oxygen partial pressures were recorded on a
Linear Instruments Corp. Model 282/MM recorder from either of the two
oxygen probe chambers by means of a switch on the oxygen monitor control
unit (p). Temperature was maintained in the oxygen probe chambers at
20 :_0.05°C and the glass chamber at 21 :_0.1°C by a Lauda K-2/R circu-
lating water bath. A mercury halide lamp (k) provided 680 uEinsteins
sec“1 111.2 of light for the seedlings at 70 cm from the light source.

The photosynthetically active radiation at this distance was similar to

that in the growth chamber, although the radiation intensity gradient

was three times greater.

31

All measurements were made in a constant temperature room maintained
at 20 :_2°C. All of the solution carrying lines and the oxygen monitor-
ing system except for the part containing the glass chamberifinmashielded
from the light source. Photographs of the system used to monitor oxygen

uptake by roots are shown in Figure 5.

Measurement of Oxygen Uptake

 

The oxygen monitoring system was calibrated daily with circulating
0.210 atm oxygen saturated nutrient solution. 'Calibration curves based
on standard oxygen concentrations indicated similar linear responses for
static and circulating systems, Figure 6. A slight deviation from the
linear for the circulating system was noted at the lowest oxygen concen-
trations. During the initial calibration procedure the current output -
applied voltage relationship of the oxygen probes were investigated.
Sensor performance was determined to be satisfactory if the relationship
indicated a departure from linearity of less than 0.5% (111). The probe
membranes were inspected daily for air bubble inclusion. Abnormalities
were corrected before proceeding with measurements. Calibration with
the gas used in the measurement was carried out before and after each
measurement. After calibration, the pyrex root chamber was connected
to the system. An initial drop in oxygen concentration was followed by
a recovery until equilibrium was reached. This was caused by the con-
sumption of oxygen occurring during the time of transfer from the growth
chamber to the oxygen measuring system. Measurements were continued at
least fifteen minutes after reaching apparent equilibrium as indicated
by a steady flat trace on the recorder. Measurement time generally took

from 1 to 2 hours. The time to reach equilibrium was variable and

.mpomzem :omxxo u~zL=puopn~oz ucmcwmucco sums noun: .0 .possmzo woos o:u w:_:dmu:ou

nun; hope: .3

.Eoumxm ozu mo zow> fiquo>o :< .c

.oxnuz: :owxxo woos headcoe ow com: scumxm

.m ewzwwu

 

1(30

1

9C)-

8(1-

7(1-

SC}

I

1% of full scale)

SC)-

0|!

lespanse

40-

ISO

Recorder

2(1

\

- a static system
A dynamic system (probeI)
10 _ 0 dynamic system (probe 111

 

 

o aé.oé.05.oé 10 12 1h 16 15.55.12
002 (atm)

Figure 6. Calibration curves for the root respiration monitor.

34

depended on solution flow rate, time taken to transfer, and oxygen sink
of the rhizosphere.

Samples of nutrient solution which had passed the root system were
taken at the termination of the measurement period. Samples were placed
into 10 ml vacutainers acidified with sulfuric acid to a pH of 2 to
prevent microbial growth and release carbon dioxide from the solution.
These samples were stored in a refrigerator for possible analysis at a
future time. Solution flow rates were also taken at the termination of
the experiment by determining the time for 6 to 8 ml of solution to
collect in a pipette. The pyrex root chamber was either placed back in
the growth chamber or the plants were harvested depending on the experi-
ment conducted.

Oxygen uptake per hour was determined by the following equation:

x = (6.234)(r)(60)(d)
where x is the oxygen uptake perlwnu7(u1 O2 hr-l); d is the differential

l

P0 r is the flow rate (cm3 min' ); and 6.234 is the solubility of

2;
oxygen in solution at 21°C.

Microbial respiration was estimated by removing the root system,
replacing the solution and matrix treatment and measuring the resulting
oxygen uptake. Chambers with root systems removed were stored at 4°C
for 1 day before microbial respiration could be measured. Comparisons
of experimental units before and after refrigerated storage indicated

that the period of storage had little or no effect upon respiration

rates .

35

Transmission Electron Microscopy of Treated Roots

 

Roots were removed from each of the three matrix treatments at 14
days. Four to six 3 mm sections were taken from representative axial
root tips, lateral root tips and 1 cm above the lateral root tip. The
sections were placed in vials containing 3% gluteraldehyde in 0.1 M
Sorenson buffer. The vials were placed in an ice bath and carried to
the Center for Electron Optics at Michigan State University for further
processing. The samples were stained with osmium, embedded, sectioned
and placed on a grid for observation under the transmission electron
microscope. Samples were taken from roots treated with 24 hour treat-

ments of 0.210 atm p07 and 0.00 atm pO2 (nitrogen gas saturated solution).

Measurement of Root Porosity

 

Root porosity was determined by the procedure outlined by Jensen
§£_al: (53). The root porosity was determined using the following formula:

root porosity = 100(Wh-Wr+w)/(Ww+Wr-Wr+w)

where Wh = weight of homogenated root and pycnometer
wr+w = weight of root, water and pycnometer
Wr = fresh weight of root
ww = weight of water and pycnometer

The denominator when multipled by the density of water at 20°C is the
fresh root volume. The numerator when multiplied by the density of
water at 20°C is the volume of air spaces in the root. The fresh root
weight and root volume were used to calculate the root tissue density.
Root porosity determinations were tedious especially during tissue homo-
genation. At times mishaps would occur resulting in the loss of homo-
genate. This was noted and the root porosity determinations for these

samples were discarded.

36

Measurement of Shoot and Root Morphology and Growth

 

Slides were taken of the intact plants in the pyrex root chambers
before harvesting. Plants were removed from the root chambers by re-
moving the shoot at the base and carefully removing the stopper of the
chamber. Leaf area was measured using a Lambda LI-3000 leaf area meter.
Shoot fresh weights and dry weights were also measured. The roots were
carefully tapped out of the chamber and removed from the matrix. The
roots were then washed and carefully laid out on a grid in atxwu'contain-
ing water, Figures 14 and 15. The roots were separated and photographed
on the grid. The grid consisted of squares with lengths of 0.75 cm.
Slides were used to document root morphological differences among treat-
ments, and to measure root length and diameter.

Root length was measured from the slides by projecting them onto
a translucent screen and tracing roots with an inch counter (83).
Measurement of root length was made in square areas consisting of 10
grid squares per side (100 grid squares). The measurement areas were
given a vertical and horizontal vector designation. Figure 7 designates
the measurement areas used. Root length was determined using a scaling
factor based on the known length of the grid squares. Total root length
was determined by summing the root lengths of the measurement areas.

Root diameter was determined by randomly selecting at least 30
roots per measurement area and measuring the diameter from the projected
slide with a ruler. A scaling factor was also used to obtain actual
diameters. The length and diameters of primary roots were measured
separately.

Two methods were used in this study to measure root volume. Root

volume was estimated by the calculation method using the root length (h)

Vertical Vector

37

r1111
3
{P3

 

 

252cm

 

 

on" 4:."

_ ills.
'1
as)
at”

.4.

2112 ‘_

{\t x 4
m its
Y, OWN;

x. x2 x3 x4 x5

Horizontal Vector

 

 

 

 

 

 

 

 

 

 

Figure 7. Root length measurement grid.

38

and diameter (d) measurements. Estimates were made by approximating
the roots to a cylinder (69). The volume of a cylinder = n(d/2)2h.
Root volume was also determined by water displacement from the root
porosity measurements (53). This method was a more precise and less
time consuming method than by measurement and calculation. These two
methods were highly correlated, Figure 8. Root volume measurements
tended to be lower for the calculation method. The differences between
these methods of estimating root volume were similar among matrix
treatments, Figure 9. A major source of error in the calculation method
is the use of representative root diameters. This results in the treat-
ment of the root system as a uniform cylinder. Any error in the
diameter representation is magnified when finding the volume due to
the squaring of the radius.

Root systems were measured for fresh weight and placed in a drying

oven. Roots dried at 70°C were weighed to obtain root dry weights.

Measurements of Waterlogged Treatments

 

Waterlogging involved a 24 hour treatment period during which
nutrient solution was not circulated past the root system. Since nutri-
ent solution remained in the root chamber this treatment lent itself to
a number of measurements which could not be made on the other treatments.
Measurement was made of water uptake and root exudation of ethanol and
amino acids during the 24 hour period of waterlogging.

Water uptake was measured by connecting the exit siphon from the
pyrex root chamber to a parafilm sealed test tube containing 40 m1 of
nutrient solution. The amount of water removed from the test tube was

measured after the 24 hour period.

 

39

 

 

m b
E
O
3.
CD
:5: :1 1mm matrix
'6 ' A 3mm matrix
>
0 Control 0
H2- 0
o
o
a:
“U
.‘1.’
.“2
:11-
0
"c6
0
C) i I i l i 1
O l 2 3

Figure 8.

Displacement Root Volume (cm3)

Comparison of methods for determining root volume.

.
«:1- Displacement method
1:- Calculation method
A 2-
CT) 1
E
3
(D
E
.2
O -
>
‘5
o
1::
1t

 

c3—1

1 mm 77mm control
Matrix Treatment

Figure 9. Comparison of methods for determining root volume responses
to matrix treatments.

41

Samples of solution were taken from the chamber at the end of the
24 hour period, stored in a freezer, and analyzed for ethanol and amino
acid contents. Ethanol content was measured by a GC2-A Beckman gas
chromatograph equipped with a hydrogen flame detector. Samples, 1 ul,
were eluted through a 6 foot x 1/16 inch stainless steel column con-

taining Porapak 08, 100/120 mesh. The carrier gas, N flowed 80 ml/min

2,
through the column at isothermal conditions of 180°C. Ethanol content
of the nutrient solution was determined by comparing the area under
sample peaks with the area under peaks produced by injections of ethanol
standards.

A modified Durrum HPLC amino acid analyzer was used to analyze
solution for amino acid composition. A 0.3 x 25 cm column containing
Durrum DA-X4-ll resin was used for amino acid separation. A Durrum
femto - buffer under a column pressure of 300 to 330 psi and flowing
at a rate of 12 ml/min was used to elute amino acids from the column.
The column temperature was programmed to change from 45°C at 27 min to
65°C at the end of 49 minutes. Fluoropa (o—phthalaldehyde) was used

as the detection agent. Amino acid amounts were determined by com—

paring standard chromatograms to the sample chromatogram.

RESULTS AND DISCUSSION

Oxygen Monitoring System

 

The oxygen monitoring system provided a means of continually
monitoring oxygen uptake by roots. Root response could be accurately
measured to 1.5 pl 02 hr-l. Over short intervals this measurement
accuracy could be greatly increased by expanding the recorder scale
through the use of an offset zero and increased recorder attenuation.

Preliminary measurements were made to determine the effects of

nutrient solution flow rate on oxygen uptake by Phaseolus vulggris roots.

 

A root chamber containing two bean plants grown in nutrient solution

for 17 days were used as the experimental unit. Oxygen uptake by this
experimental unit was greater than those in succeeding experiments which
used 14 day old plants. Figure 10 depicts the effect solution flow rate
has on the change which occurs in the p02 of the nutrient solution after
passing through the root chamber (exit p02). Also shown is the effect
of solution flow rate on the oxygen uptake rate of the bean roots.
Decreasing the nutrient solution flow through the root chamber resulted
in lowered exit p02 for both the 0.210 and 0.105 atm p02 treatments
(treatment p02). The slower the flow rate the greater was the differ—
ence in p02 between the lower and upper parts of the root system. The
largest oxygen gradient which developed for a treatment p02 of 0.210 atm

resulted in an exit p07 of 0.067 atm and this apparently did not affect

the oxygen uptake of the root system.

42

43

Exit pOZfor:

-°- 0.210 atm p02
‘0' 0.105 atm p02
Oxygen Uptake at:
-o- 0.210 atm pO2
-I- 0.105 atm p02

 

 

 

 

 

 

E 16 9
+-' F' <
a? «8008
"’ ‘er 700 E;
CDN g
Q 8 ii:
-/\‘\‘ 41600 g-
15 4.. '9:
l L. . . . . :1: r"
06" ' 6 7 8 9 10 0

Solution Flow Rate (ml min‘11

Figure 10. The effect of solution flow rate on exit p02 and oxygen up-
take measurements. Results were obtained from the roots of

two 17 day old Phaseolus vulgaris plants.

 

44

At a treatment p02 of 0.105 atm, the root system became more sensi-
tive to changes in solution flow rate. Compared to the same root system
exposed to a flow rate of 10 cm3 min-1, a solution flow rate of 6.7 cm3
min.1 resulted in an exit p02 of 0.006 atm and a decrease in the oxygen
uptake rate of approximately 60-80 pl 02 hr-l. This is small in compari-
son to the decrease of approximately 150 pl 02 hr—1 in root oxygen uptake
caused by a change of the treatment p02 from 0.210 to 0.105. This is
expected since the whole root system is subjected to a substantially
lower oxygen partial pressure when the aerating gas is changed from
0.210 to 0.105 atm. When the flow rate is altered only a small change
in oxygen partial pressure results to the whole root system. However,
at low oxygen partial pressures the flow rate may have an effect on
oxygen uptake because of the hyperbolic relationship of oxygen uptake
to oxygen partial pressure (110). As the oxygen partial pressure
approaches zero, small changes in the gradient can cause a greater
amount of inhibition than at higher oxygen partial pressures.

A preliminary study was conducted to determine the influence of
p02 on oxygen uptake. Oxygen gradients encountered when measuring oxy-
gen uptake of 14 day old bean roots indicate that there was very little
loss of oxygen in solution when running the system without roots, Figure
11. A comparison of the response without plants and with plants indi-
cates a substantial uptake of oxygen by the root system. The tendency
of the two lines to converge is an indication that less oxygen is being
used at low p02. Figure 12 indicates that oxygen uptake rates are

lower for the lower treatment p0 The large range for each treatment

2.

p02 includes response due to different sized root systems, different

45

'°" Response without plants
“A- Response with plants

21

(10"2 atm)
C0 16

Exit p02
on

 

 

L l

L g I I I
O 3 6 9 12 15 18 21
Treatment p02 (10"2 atm)

Figure 11. Relationship of exit p07 to treatment p07 for the root

respiration monitor.

46

8

Iant")

3

(111 hr'1 1)

 

ate
5

 

N
8
1;

Oxygen Uptake R

 

O 3 l l 1 1
Treatment p02 (lO'zatm)

Figure 12. Relationship of oxygen uptake to treatment p0, for 14 day old

dry bean roots. Brackets indicate the range of response.

47

levels of microbial background, difference among matrix treatments and
experimental variability.

The form of the curve in Figure 12 indicates a hyperbolic relation-
ship for p02 levels below 0.21 atm which is similar to other reports
(110). When interpreting data resulting from oxygen treatments it is
important to note that the oxygen treatment represents a p02 gradient
within the root chamber. Treatment of 0.210, 0.105 and 0.032 atm of
oxygen produced exit p02 as low as 0.140, 0.030 and 0.000 atm of oxygen,
respectively. Comparisons of oxygen uptake with flow rate demonstrated
that the 0.210 atm p02 treatment did not have oxygen uptake rates which
were dependent on the oxygen gradient within the chamber, Figure 10.

As a result, measurements were made using an entry solution equilibrated
with 0.210 atm p07. In addition to minimizing the effects of an oxygen
gradient in the chamber, this removed the recorder response from the
non-linear region of the calibration curve, Figure 6.

The effects of ionic strength on oxygen uptake by roots of corn
seedlings were measured using nutrient solutions. There was a trend for
an increase in oxygen uptake with solution conductivity, Figure 34.
Consequently, all measurements were made using one-half strength Hoagland's
solution which was passed by the root system once and then discarded.

Diurnal changes in oxygen uptake were negligible during the period

from 9:00 am - 9:00 pm, Table 3.

48

 

 

Table 3. Influence of diurnal measurements on root respiration.
Values are average of six replications.
Oxygen uptake Relative Coefficient
rate per oxygen of
root dry wt uptake rate variation
Time (In 02hr 1g 1) (%)
10:00 AM - 3:00 PM 5503 1.00 37
3:00 PM - 8:00 PM 5527 1.01 37
8:00 PM - 1:00 AM 5922 1.09 35
LSD .10 N.S. N.S.

 

49

Mechanical Impedance and Root Growth

 

Data from Tables 4 and 5 demonstrate that during the eight days
of treatment root systems grew extensively for all matrix treatments.
Figure 35 gives an indication of the size of the root system at the
initiation of the matrix treatments.

Matrix treatments differentially affected the rate and form of
growth. Roots grown without glass beads accumulated fresh weight at
twice the rate of the 1 mm bead matrix, 18 mg h"1 compared to 9 mg h-1
during the treatment period. Roots grew at an intermediate rate of 15
mg h.1 in the 3 mm bead matrix. Root growth rates were less in the
matrix treatments as shown by the reduced root weight, length, volume
and surface area for these treatments, Table 5. Table 6 compares pri-
mary root growth with lateral root growth. Primary root length was
impeded by the 3 mm and 1 mm matrices. Primary root tip cells were
plasmolysed for roots grown in 1 mm matrices but not for the other
matrix treatments, Figure 13. This suggests that the primary roots
grown in 1 mm matrices have lost meristematic ability while the primary
roots grown in 3 mm matrices have retained this ability. Root growth
appears to continue at slow rates if meristems subjected to high external
pressures arerwn:injured (86). This has important implications for roots
growing through hard pans. Presumably a slow growing root could eventu-
ally penetrate the pan if mechanical impedance was not so great as to
damage the meristem.

Total lateral root length was also reduced by the glass bead matrix
treatments, Table 6. Comparisons of lateral root length per unit primary

root length indicate a direct relationship between relative lateral root

50

 

 

 

Table 4. Fresh and dry weights of Phaseolus vulgaris shoots and roots
on the day of transplanting to pyrex root chambers. Values
are an average of six replications.

Root Root Shoot Shoot
fresh dry fresh dry
weight weight weight weight

(g/plant)
Mean 0.19 0.013 0.66 0.145

Standard deviation 0.05 0.003 0.13 0.030

 

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m m m on we m_ m.eueeeuieee

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oas~o> camcofi noun oommusm +oasfio> H3 sue a: :mopw

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52

 

 

 

 

 

 

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v.mfi mo.c wc.o ed ow cow chwca EE _

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m.m~ eo.o mc.c v~ we cma fiocpcou

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mesa eoemuzm woumEme Heuoamww spu:e_ woos :umce_ newcoa
woos fieuou woos aces thamum woos woos
mo owmucoocoa Hmuoumq xumawam zuwcofi woos Ahmafisa Hmhoumfi
xpmsfiaa Museumfi ~muok Hope?

 

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. WHCQEHQQHH

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53

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.m_ ee:u_e

 

54

growth and matrix pore space, Table 6. The reduction in lateral root
growth per primary root length accounts for 22 and 29% of the lateral
root length reduction for the 3 mm and 1 mm matrices, respectively.

The remaining reduction in lateral root length results from the smaller
primary roots being unable to initiate or support as many lateral roots
as a longer primary root. Compensatory lateral root growth, reported

by 6055 with barley (37), was not found in this study. These differences
could be due to the different root diameters of the two species, reported
in these two studies.

Phaseolus vulgaris root diameters, Table 6, were found to be twice

 

as large as barley roots. A comparison of lateral root diameter and
the matrix pore diameter, Table 2, indicate that both lateral and pri-
mary bean roots should encounter a considerable mechanical impedance to
growth by the 1 mm matrix treatment and less for the 3 mm matrix treat-
ment. Wiersma (104) demonstrated that the rigidity of a matrix system
decreases as the width of the chamber used increases. Lateral roots
growing in the 1 mm matrix treatments may have been able to elongate

by deforming the matrix.

Primary root diameters were unchanged by matrix treatment since
primary root growth was limited by both the 3 mm and 1 mm matrices.
Lateral root diameters increased for the glass bead matrix treatments.
This increase in root diameter agrees with other reports (86) relating
root responses to mechanical impedance. Ethylene may be responsible
for the increased root diameters caused by mechanical impedance (11).

The contribution of primary roots to the total surface area is

shown in Table 6. Roots grown in the 1 mm matrix appeared to have

a greater percentage of surface area from primary roots than

55

the control and 3 mm matrix treatments. Lateral root growth in
the 1 mm matrices also differed considerably from the other treatments.
Lateral roots from the 1 mm matrix were considerably shorter in length
but more numerous than for the other matrix treatments, Figure 14.
Lateral roots grown in the 1 mm matrix were initiated closer to the
root apex and were clumped together more than the other treatments. A
close up of a primary root with associated lateral roots depicts the
contrast between treatments, Figure 15a. Increased lateral root clump-
ing for the 1 mm matrix treatment appears to be the result of increased
lateral root initiation, smaller roots and the formation of secondary
and tertiary lateral roots. Figure 15b compared representative lateral
roots from the three treatments demonstrating secondary lateral root
formation on the 1 mm matrix lateral root. Three mechanisms have been
postulated to explain the increased formation of lateral roots close
to the root apex. these are: i. increased promoter from mature tissues
(73), ii. decreased inhibitor production in the root apex (100) and
iii. ethylene stimulated root formation (100). Tissue maturing closer
to the root apex resulting from the slower growing roots (86) could have
resulted in the greater production of a promoter. Damage to the root
apex may have reduced inhibitor synthesis. Wounding from mechanical
impedance (58) and bending around obstructions (i.e. glass beads) (73)
could have resulted in ethylene formation. All three mechanisms may be
acting together to increase lateral root formation in the 1 mm matrix
treatment.

Influence of the matrix treatment on lateral root formation is
apparent in the amount of curvature and distortion observed, Figure 15b.

Flattened regions were formed on the lateral roots grown in glass beads.

56

.H

.xflsuca EE H .u .xmcume as m .s .

ouoammv c:m :uwcoH poop euzmeoa ou pom: vacw :o pouefiammv moofluuee conga use we we

Houu:ou .:

oumxm ace:

.3 Bi:

 

57

.xdo>wuoogmou .mucoaueocu fiopucou

vze xLAumE 55 m .xfiuums EE _ ozu uzomosmos uzwws cu umo~ seam muoos assuam some :_ .mucos

annoys; .n .muoos xsmE_u; .m

.waHOELLOE Hoes :o uoomwo moowuums mouse ecu mo mesmwsezsou

 

.2 95E...

58

Trouse (101) observed similar flattened regions in roots grown in dense
soil. He suggested this may occur when roots move into a fracture and
the elongating cells are allowed to expand ahead and parallel to the
fracture but not perpendicular to the fracture. The effect this has on
the association of the roots with the glass beads is demonstrated in
Figures 16 and 17. The 1 mm beads were tightly associated with the root
system and many of the beads could only be removed through the careful
use of small tweezers. Presumably, the root surface area in contact
with beads was unavailablelknfnutrient and gas exchange with the sur-
rounding solution. Greenwood (41) suggested that such contact acts in
soil to reduce oxygen availability to the root.

Distortion of lateral roots was also observed at the cellular level,
Figure 18. The cortical cells from the control were spherical in shape
and surrounded by abundant intercellular spaces. Cells from the 3 mm
matrix treatment appeared to be intermediate in character. Cells from
the 1 mm matrix treatment were distorted and generally appeared smaller
than the other two treatments.

These observations agree with 6055 and Walter (38) who observed
distortions in older cortical cells of barley roots growing in a matrix
of 1 mm glass beads. Barley and Greacen (12) suggested that cellular
distortions could lead to a compression of intercellular spaces result-
ing in the loss of internal aeration.

Root porosity determinations were carried out to determine if the
compression of intercellular spaces resulted in significant losses of
internal air space. Matrix treatments significantly affected root
porosity, root tissue density and root dry matter percentage, Table 7.

The 1 mm matrix treatment significantly lowered root porosity, increased

59

 

Figure 16. Root growth around a 3 mm glass bead.

60

.xmsuaa as «

 

o

 

.o .x_suma EE m

.2 ._oLu:ou .:

.2 83:...

 

61

.xfipume 25 H .u .xduuna 55 m .; ._osuccu .m .Achme mucoaumeuu stuea Hg: cow ;_u

woos Heuou6_ use o>onw 56 M :oxmu ope: meHLEmm

.mcofluoom mmouu fifioo Hmo_uuoo mo ngeswocofls 22%

.w_

ou:m_;

 

62

Table 7. Influence of matrix treatment upon root structural parameters

of 14 day old Phaseolus vulgaris L. (var. Seafarer).

 

 

 

Roott Root Rooti
tissue Root dry matter cellular
density porosity percentage density

Treatment (mg/cma) (%) (%) (g/cm )
Control 994 3.41 5.3 1.026
3 mm matrix 993 3.61 5.1 1.023
1 mm matrix 1010 2.17 6.2 1.030
Number of

replications 10 8 13 8
L50 .01 12 1.16 0.7 N.S.
LSD .05 9 0.83 0.5 N.S.

 

tBased on root volume measured by the water displacement method and root
fresh weight.

iCalculated by subtracting root air volume from total root volume.

root dry matter percentage and increased density of root tissue. These
parameters were similar for the 3 mm matrix and control treatments be-
cause the majority of new growth of these treatments occurred as the
result of relatively unimpeded lateral root growth. The 3 mm matrix
primary root was mechanically impeded and the amount of new growth from
this source was less than 14% of the total new growth (primary roots
accounted for 14% of the total root surface area, most of which occurred

before treatments were installed).

Root porosity is an important factor in the internal aeration of
roots. Decreased root porosity could increase tortuosity and interrupt
interconnecting intercellular spaces resulting in reduced diffusion to
the root from the shoot (31) and reduced radial aeration from the media
surrounding the root. Diffusion of oxygen through a water film at the
root and soil interface and into intercellular gas spaces could eliminate
much of the very slow diffusion of oxygen through living tissue (64).

It is suggested that the reduced root porosity of 1 mm matrix treatments
reduced the availability of oxygen to internal root cells.

Reduced root porosity may also lead to greater accumulations of
ethylene or other volatile growth regulators within the root tissue (11).
Since mechanically impeded roots may produce six times more ethylene
than nonimpeded roots (58), high local ethylene concentrations could
develOp. Decreased root porosity could result in the accumulation of
ethylene within the root and change growth regulation patterns. Greater
concentrations of localized ethylene could increase lateral root forma-
tion (73,87), change cortical cell growth (11), and inhibit root elonga-

tion (92).

64

The higher root tissue density for the 1 mm bead matrix treatment
could be a result of decreased air volume of the root and/or an increase
in the protoplasmic density of the root. Since overall root volume is
composed of solid, liquid and air volumes a decrease in root air volume
would result in an overall decrease in root volume. This would result
in a decrease in the denominator of the root tissue density term (g/cm3)
and an apparent increase in root tissue density. An increase in solute
concentration in the cell cytoplasm could also account for an increase
in root tissue density through increasing the numerator much more than
the denominator of the density term.

Root tissue density correlations with other root parameters, Table
8, indicate that the decrease in root air volume may be a greater factor
than an increase in root dry matter percentage. One might expect the in-
creased dry matter percentage to result in an increased root tissue densi-
ty. The correlation coefficient for root tissue density with root dry
matter percentage indicates some degree of association for combined
treatments and for the 1 mm matrix, but not nearly as much as root poro-
sity. The root tissue density with root porosity correlation coefficients
indicate a highly significant association of increasing root tissue
density with decreasing root porosity (i.e. decreasing air volume) for
all matrix treatments. The nonsignificant relationship of root tissue
density with root fresh weight is indicative of root tissue density
(and root porosity) association with all other root growth parameters.

Relative contributions of the two factors contributing to the de-
crease in root tissue density was more closely examined by calculating
the density of the solid and liquid portion of the root (root cellular

density). The volume of the solid and liquid portion of the root was

65

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.w eases

66

found by calculating the air space volume from the root porosity and
total root volume data and subtracting this from the total root volume.
The root cellular density was found by dividing the root fresh weight
by this volume.

Results shown in Table 7 indicate that the root tissue density
increase for the 1 mm matrix treatment resulted from the reduction in
air space volume. There is a trend suggesting increased root cellular
density of the 1 mm matrix roots. Most of the difference in root dry
matter percentage (70-90%) can be accounted for by the differences in
root cellular density. The increased dry matter percentage for roots
subjected to 1 mm matrix treatments could be the result of an increase
in the proportion of cell wall material per cell. Tackett and Pearson
(98) inferred from their thin section analysis of mechanically and non-
mechanically impeded roots that the increase of root dry matter percent-
age was due to an increase in the percentage of root volume occupied by

cell wall material.

Mechanical Impedance and Shoot Growth

 

Table 4 provides data on shoot fresh and dry weights before begin-
ning the matrix treatments. The primary leaves were beginning to appear
when matrix treatments were allocated to the bean plants. Figure 17
illustrates the shoot growth at a plant age of 14 days (8 days after
beginning the matrix treatment). A considerable amount of shoot growth
occurred in the 8 days of treatment. Generally, by the end of the treat-
ment period the first trifoliate had appeared and was rapidly increasing

in area.

67

The 1 mm matrix caused significant modification in shoot fresh
weight and dry matter percentage, Table 9. Leaf area was related to
the shoot fresh weight, Table 10, but the differences between treatments,
Table 9, were not significant until the 10% probability level.

Total plant dry weight and leaf area ratio (LAR), Table 11, indi-
cate that mechanical impedance had little or even a slight positive
effect on the photosynthetic capabilities of the leaves. There was a
significant shift in the distribution of plant photosynthates as shown
by greater shoot to root ratios, Table 11. The lack of change in shoot
dry weight, Table 9, between treatments indicates that photosynthate
is being diverted from the root system to the shoot for the 1 mm matrix
treatment. Aung (5) reviewed evidence demonstrating the interdependence
of root and shoot growth on photosynthate supply. Studies were cited
which demonstrated that as the root growth rate was slowed, photosyn-
thate accumulates in the leaf. This is a possible explanation of the
relatively high dry weight level in the shoot and the higher shoot dry
matter percentage for the 1 mm matrix treatment.

Another explanation of the higher shoot dry matter percentage and
shoot fresh weight is the effect of a small root system on water uptake.
Brouwer (16) showed that leaf growth can be inhibited by the removal of
a small part of the root system with only a small effect on the rate of
transpiration. This explanation is given some credence by the correla-
tion data in Tables 10, 12 and 13. Leaf area is highly correlated with

root surface area and root dry weight for the 1 mm matrix treatment,

68

Table 9. Effect of matrix treatment upon shoot growth of 14 day old

Phaseolus vulgaris L. (var. Seafarer). Values are means of

 

eleven or more replications.

 

 

Shoot Shoot Shoot
fresh dry Leaf dry matter
weight weight area percentage
Treatment (g/plant) (g/plant) (cmZ/plant) (%)
Control 2.91 0.337 82 11.8
3 mm matrix 2.51 0.331 77 13.1
1 mm matrix 2.27 0.341 66 14.9

LSD .01 0.59 N.S. N.S. 1.0

 

69

Table 10. Separate and combined correlation of leaf area with

selected plant parameters for the three matrix

 

 

 

treatments.
Treatment r values

Parameters Combined
correlated Control 3 mm matrix 1 mm matrix r values
Leaf area with

root dry weight .62* .30 .80** 57**
Leaf area with

root surface area .64 .02 .98** .73**
Leaf area with

shoot fresh weight .93** .94** .98** .98**

 

*,**Significant at .05 and .01 levels of probability, respectively.

7O

 

I'l'lllllllll'l

 

 

 

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mma cov.e om.m mn.m He.m amuusa EE H
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wwfi wme.c am.e Ae.m ~m.~ aesuceu
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Auzwaez uzwfloz uzwwez peeu\uee:m eeeH\uee:m
xue ucmmm xse :meww
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m<3
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.se>u .4 mwumwfis> msfleemmsg we» mseueamhmm ucmfim peueefiem some maceEumesu weaves we 66:6:HmcH .HH efinmh

71

Table 12. The effect of the three matrix treatments on the correlation

of shoot with root weight parameters.

 

Treatment r values

 

 

Parameters Combined
correlated Control 3 mm matrix 1 mm matrix r values
Shoot dry weight with

root dry weight .55 35 .49 .36
Sheet fresh weight with

root fresh weight .50 - 22 77** 53**
Sheet dry matter

percentage with root

dry matter percentage .32 .53 .40 .53**

 

*,**Significant at .05 and .01

levels of probability, respectively.

Table 13. The effect of the three matrix treatments on the correlation

coefficients of selected root parameters.

 

Treatment r values
Parameters Combined
correlated Control 3 mm matrix 1 mm matrix r values

 

 

Root fresh weight with
root dry weight .80** .81** .94** .90**

Root length with

root dry weight .93* ,93* .96* 93**
Root surface area with

root fresh weight .94* .92* 1,00** .97**
Root volumet With

root fresh weight l.00** l.00** l.00** l.00**

 

*,**Significant at .05 and .01 levels of probability, respectively.

tMeasured by displacement method.

\J
(A

Table 10. The correlation with root surface area is especially signifi-
cant since a larger root surface area can result in less resistance to
water uptake (77). The correlation of shoot and root fresh weight,
Table 12, is probably due to the association of shoot fresh weight with
leaf area, Table 10, and root fresh weight with root surface area, Table
13. The shoot dry matter percentage was apparently not related to the
the root dry matter percentage for individual experimental units, Table
12.

A third possible explanation is that growth hormone production is
disrupted or changed by the matrix treatments. The resulting change in
growth hormone concentrations may trigger a shoot response which results

in a higher dry matter percentage.

Mechanical Impedance Effects on Root Respiration

 

Oxygen uptake rates were measured at a plant age of fourteen days.

Measurements were made on Phaseolus vulgaris roots grown in nutrient

 

solution equilibrated with a p02 of 0.210 atm, all data reported in this
section are for the three different matrix treatments replicated five
times.

Microbial oxygen uptake rates were measured by removing the root
system and measuring the resulting oxygen uptake by the media without
roots. Microbial oxygen uptake rates were increased in the bead matrices
compared to the control, Table 14. Microbial populations could be greater
in the bead matrices due to increased root exudates of mechanically im-
peded roots (7). The larger microbial populations of the bead matrices
may reflect the increased surface area of the matrices. Table 2 indi—
cates that the surface area available for microbial attachment was 30 and

10 fold the control for the 1 mm and 3 mm matrices, respectively.

74

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Cd. 2%:

 

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mmm wo.c mm.v chm mmm mam «OH xfiwuea as m
Nmm mm.c mm.m mmvm cum mom mo~ fieupcou
amEev Aaov amsov Awe Amy nucmfizv mucmaav acesumeus
easfie> ueec :uwCoH cope eomwssm u;wwe3 psmmez ucmmm :ewempwzmes
pagedsofimu peoz poem zap zmecu dawnewowz
poem weez

 

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a H

 

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.eH efinwk

75

Oxygen uptake rates for Phaseolus vulgaris roots grown in the three

 

matrix treatments are reported in Table 14. There were no differences be-
tween oxygcn uptake rates per unit dry weight for all treatments.

Oxygen metabolism was apparently unchanged by mechanical impedance.
Observed differences in per plant oxygen uptake among treatments should
be a function of the amount of dry matter found in the root systems.

The amount of dry matter found in the root system was dependent on the
size of the root system, the percentage of dry matter of the root system
and the supply of photosynthate to the root system.

The larger oxygen uptake rates per plant found for the control
roots was a result of the larger root system associated with nonimpeded
roots. Standardization of the oxygen uptake rates for root system size
results in oxygen uptake rates which are generally greater for roots
grown in the 1 mm matrix. The difference in oxygen uptake rates was
partly the result of different distributions of dry matter in the root,
Table 15. The 1 mm matrix roots contained relatively greater amounts of
dry matter for the root dimensional parameters as well as for the fresh
weight parameter.

The increased diameters of the matrix grown roots, Table 5, also
contributed to the differences in oxygen uptake rates. Average fresh
weight per unit length of root was 2.7 g cm—1 for the 1 mm and 3 mm
matrix treatments and 2.2 g cm.1 for the control treatment. This indi-
cates a contribution from the changed root geometry corresponding to
the larger diameters of matrix grown roots. A larger diameter results
in more root tissue per unit length and per unit surface area of root.
This resulted in a trend in which the 3 mm matrix treatment oxygen uptake

was of an intermediate character for these two root parameters.

 

 

 

Table 15. Dry matter distribution ratios for roots used in respiration
studies. Values are means of five replications.
Root dry matter
Surface Fresh
area_ Length Volume weight

Treatment (mg cm 2) (mg cm 1) (mg cm 3) (mg g 1)
Control 0.711 0.117 82 49
3 mm matrix 0.739 0.124 74 50
1 mm matrix 1.043 0.221 126 65

LSD .05

N.S. 0.082 N.S. 10

 

77

Dimensional root respiration parameters best describe root inter—
action with the microenvironment surrounding it. Oxygen uptake per root
dry weight does not take into account any of the root structural para-
meters which affect the intensity of oxygen uptake in the immediate
vicinity of the root. In this study, mechanical impedance apparently
resulted in root structural changes which increased the oxygen needed from
the microenvironment surrounding the root. Root fresh weight was highly
correlated with volume and other dimensional root parameters, Table 13.

Another factor influencing root respiration may be competition for
photosynthate. Hatrick and Bowling (46) demonstrated a complete depend-
ence of root respiration on photosynthate flow to sunflower roots. They
concluded that for young plants root metabolism was immediately dependent
on sheet metabolism. A source-sink relationship can be postulated to exist
between the shoot and root. Photosynthates are rapidly utilized in the
root for the synthesis of complex compounds for the cell wall, cytOplasm
and organelles. Sucrose and other simple organic compounds are kept at a
low level and if not used, are converted to starch and stored in the meri-
stem (79). In this study, shoot to root ratios were greatest for the 1 mm
matrix and lowest for the control treatment. Previous mention has already
been made of possible accumulation of photosynthates in 1 mm matrix shoots
resulting from the smaller root sink. The oxygen uptake per unit fresh
weight rates were found to be significantly correlated with shoot to root
ratios for the control and 3 mm matrix, Table 16. For both treatments
a larger proportion of shoot compared to root should result in a
larger supply of photosynthate being made available per unit root.

An increase in photosynthates for a rapidly growing root system which

is serving as a strong assimilate sink should result in the

78

Table 16. Separate and combined correlation of root respiration
with selected plant parameters for the three

matrix treatments.

 

Treatment r values

 

I,

Parameters 3 mm 1 mm Combined
correlated Control matrix matrix r values

 

1 02 hr" 8‘1 (FW) with

 

Root length -.82 —.66 -.52 -.58*
Root fresh weight —.90* -.87 -.62 —.73**
Root dry weight -.87 -.62 —.67 —.66**
Sheet dry weight -.14 .08 -.69 -.39
Sheet fresh weight —.05 .74 -.79 -.33
Leaf area -.24 .38 -.67 -.43
Shoot/root (fresh weight) .90* .96* .55 .76**

 

*,**Significant at .05 and .01 levels of probability, respectively.

79

stimulation of oxygen uptake. Roots in the 1 mm matrix grew at a reduced
rate and constitute a weaker assimilate sink. The larger shoot to root
ratio for the 1 mm matrix roots indicates a relatively larger source of
photosynthate for the root system. The greater source and weaker sink

for the 1 mm matrix roots could have resulted in the reduced association
of oxygen uptake rates with shoot to root ratios. Large root systems

were associated with smaller rates of oxygen uptake per root fresh weight,
Table 16. This may be due to a decrease in the respiration rates of

older root tissues. Root respiration rates have been shown to vary with
distance from the root apex with greater respiration rates associated

with the elongating root tip region (69). Large root systems have smaller
proportions of the roots consisting of apical or meristematic tissue which
may result in lower oxygen uptake rates per unit root parameter. The
noncorrelation of shoot parameters with oxygen uptake rates results be-
cause supply of photosynthate was a function of both sink (root) and

source (shoot).

Mechanical Impedance and Response to Short Term Anoxia

 

A preliminary experiment was conducted in which root respiration
was measured concurrently with selected oxygen treatments. Figure
19 represents oxygen uptake rates of the entire rhizosphere at selected
oxygen levels. Two hour treatments of 0.105 atm and less p02 lowered
oxygen uptake rates. Little interaction between the matrix and oxygen
treatments were noted. The Pasteur effect upon root systems was
measured by returning the root systems to 0.210 p02 after a two hour
treatment at 0.00 p02. Oxygen uptake rates were increased 10%, 15% and

1% for the 1 mm matrix, 3 mm matrix and control treatments, respectively.

80

O)
O
O

t-

'0- 1mm matrix

'e' 3mm matrIx
-0- Control

8‘
9:

VI

2001

Oxygen Uptake per Unit Fresh Weight 1111 hr'1g'11

 

 

C) I L. L I 1 -l I
O 3 6 9 12 15 _ 18 21
Treatment p02 (10 2 atm)

Figure 19. Oxygen uptake by 14 day old Phaseolus vulgaris roots and

 

microbes as affected by matrixtreatments.during 2 hour

oxygen treatments.

81

Oxygen uptake rates were difficult to measure at the lower pO2 for rea-
sons discussed in the oxygen monitor section.

The remaining experiments reported in this section consisted of a
24 hour treatment with randomly selected oxygen treatments given in the
growth chamber. All oxygen uptake measurements were made at 0.210 p02
before and after the 24 hour treatment period.

Figure 20 represents the change in oxygen uptake rates (AOU) which
occurred over the 24 hour treatment period. The decrease in AOU with
lowered oxygen treatments reflects the lowered oxygen uptake rates ob-
served in Figure 19. The Pasteur effect was not observed after 24 hours
of anoxia. Oxygen uptake rates per plant were greater for the larger
control roots when measured on day 13 and 14, Table 17. The increase in
oxygen uptake between day 13 and 14 results from root growth which
occurred during the 24 hour treatment period. Microbial respiration
followed trends observed in the last section.

Figure 21 represents the AOU which occurred per plant during the
24 hour treatment period for each matrix treatment. The matrix systems
interacted with the oxygen treatments, Table 18. When compared to the
other treatments the control roots had the greatest increase in oxygen
uptake rates at 0.210 atm p02 and the greatest decrease at 0.00 atm p02.
Much of the matrix differences in AOU were due to the size of the root
system. Figure 22 corrects for root system size resulting in response
curves which are very similar for the three matrix treatments. Table 18
indicates that when corrected for root system size no interaction occurs
between matrix and oxygen treatments.

Table 19 compares AOU and root growth rates for the 24 hour period

of 0.210 atm p02. Root weights on day 13 were calculated assuming

ZOO 1'

(plhru1 plant")

 

AOU

 

 

 

-1000 s e 9 12 15 18 21
Treatment p02 (lO'Zatm)

Figure 20. Change in oxygen uptake rates (AOU) during 24 hour oxygen

treatments. Values are averages of the three matrix treatments.

Table 17. Means of matrix treatments at day 13 and 14, before and after

24 hour oxygen treatment, averaged over all oxygen levels.

 

Root respiration rate pert

 

 

Microbial Plant at Plant at
respiration day l3_ day l4_
(ul 02 hr 1/ (ul 02 hr 1/ (pl 02 hr 1/
Treatment plant) plant) plant)
Control 85 434 461
3 mm matrix 127 329 358
1 mm matrix 176 263 295
LSD .05 18 118 105

 

tMicrobial oxygen uptake is subtracted from each root respiration
measurement.

84

 

 

 

    

 

 

 

01mm matrix
«b- 3mm matrix
[ '0' Control
300k
E, - 443
‘3 2001-
.—°'
‘5
E 100» 4
D
O —
<1 0 '7
_1—Matrix Oxygen
150.05 -'-' 7O LSD.05:168
'100l- ——
'200r ‘

 

O 3 6 9 1 15 18 21
Treatment p02 (10'2 atm)

Figure 21. Change in oxygen uptake (AOU) during 24 hour oxygen treat-

ments of Phaseolus vulgaris roots for the three matrix

 

treatment 5 .

00
U1

Table 18. Analysis of variance for the change in oxygen uptake rates

of bean roots during 24 hour treatments with 4 oxygen levels.

 

 

AOUt AOUt
per plant per root

Source of (pl 02 hr 1/ fresh weight
variation df plant) (pl 02 hr 1 g 1)
Replications l 6580 864
02 levels 3 148659* 41803*
Error 1 3 7453 1613
Matrix levels 2 1191 967
Matrix x O, 6 15050** 816
Error 2 8 1764 1704

 

*,**P < 0.03 and 0.01, respectively.

tAOU signifies change in oxygen uptake rate.

86

 

 

 

 

 

'0' 1mm matrix
aa- 3mm matrix
'0' Control
.. 1501-
lm E
'5
3 100+ '
E .
.E’
0)
3 501- _
.c: . 7
g Oxygen
u.
a . LSD =130
'E O!- . .05
D
8
o.
D .
o '50
.4
'1 (yL_A 1 l I 1
O0 3 6 9 15 15 18 21

2

Treatment p02 (10’ atm)

Figure 22. Change in oxygen uptake (AOU) during 24 hour oxygen treat-
ments standardized against root fresh weight of Phaseolus

vulgaris for the three matrix treatments.

87

 

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.QH ofiamk

88

similar oxygen uptake per unit fresh weight rates on day 13 and 14.
Absolute root growth rate during the 24 hour period was over three
times greater for the control root system compared to the 1 mm matrix
root system. Root growth rates and AOU were dependent on the size of
the root system as shown by the relative root growth rates (root growth
rate per unit root fresh weight) and AOU. Table 19. The relative root
growth rate was lower for the 1 mm matrix even though relative AOU was
similar for all matrix treatments.

The 1 mm matrix root system was restricted by both its initial size
resulting from the primary root impedance and by a slower growth rate
of the lateral root system as demonstrated by the lower relative root
growth rates. The reduction in initial size from the primary root
impedance was the major factor in the impedance of the 3 mm matrix root
system as shown by the similar relative root growth rates of the 3 mm
and control treatments. The lateral root growth reduction indicated by
lower lateral root length per unit primary root length. Table 6. could
have been the result of lag in adaptation to the new environment.

The effect of root size on root growth may explain impedance—anoxia
interactions. found in earlier studies, of greater reductions in root
growth by nonimpeded compared to impeded root systems. Nonimpeded root
growth should be affected more because of the greater number of rapidly
growing roots even if impeded and nonimpeded roots respond similarly
to low oxygen partial pressures.

The lowered AOU resulting from decreasing p02 treatments was caused
by a decrease in root growth and a reduction of the oxygen uptake rates
per unit fresh weight. Figure 23 demonstrates that oxygen uptake rates

per unit fresh weight for all matrix treatments generally appeared to

89

.p
O
O

1. 1L

 

00
O
O

    

-0-1 mm matrix
«o-Bmm matrix
-°-Control

Oxygen Uptake per Unit Fresh Weight (ul hr'1g '1)

 

OT I L J l t l l I
o 3 6 9 12 15 18 21
Treatment p02 (1o-2atm)

Figure 23. Influence of 24 hour oxygen treatments on oxygen uptake per

unit fresh weight of Phaseolus vulgaris roots for the three

 

matrix treatments.

90

decrease in a similar pattern to lowered p02. The 1 mm matrix treatment
appeared to have higher oxygen uptake rates per unit fresh weight com-
pared to the other matrix treatments at all levels of p07. Root and shoot
parameters measured on day 14, after the oxygen treatment, indicate similar

results as found in the previous sections, Tables 27, 28 and 29.

An experiment was conducted to measure the effect of two consecu-
tive 24 hour periods of anoxia on oxygen uptake rates. The majority of
the reduction of root respiration appears to occur during the first 24
hours of anoxia, Table 20. A large part of the differences between
matrix treatments is accounted for by the more rapidly growing root
systems of the control and 3 mm matrix treatments. The increase in
oxygen uptake noted for the 1 mm matrix treatment was probably due to
an increased microbial population. The microbial oxygen uptake was
larger compared to results found in Table 17. The larger surface area
of the 1 mm glass beads provided a greater area for microbial growth.

TEM micrographs were used to observe the root ultrastructure of
samples taken at()andl cm from the root tip of roots subjected to the
24 hour 0.00 p02 treatment. TEM micrographs demonstrate the apparent
plasmolysis of cells and a general disorganization of the cytoplasm for
all matrix treatments, Figure 24. The granular nature of cells in
Figure 25 indicates the marked change which occurs during a 24 hour
period of anoxia. The change in cytoplasmic structure occurring during
short term anoxia likely results in changed cellular activities as indi-
cated by the observed decrease in oxygen uptake per unit fresh weight
during this stress period.

A waterlogging treatment containing 0.00 atm p02 was imposed by

ceasing the circulation of the nutrient solution through the root

91

 

 

 

 

Table 20. The effect of 24 and 48 hours of anoxia on root and microbial
respiration for Phaseolus vulgaris plants grown in three
matrices. Values for microbial respiration represent one
replication; all others are means of two replications.

Change in 02 uptake
Microbial
First Second respiration
24 hr period 24 hr period after 48 hr

Matrix (pl 02 hr 1/ (pl 02 hr 1/ (pl 02 hr 1/

treatment plant) plant) plant)

Control —266 - 2 131

3 mm matrix -155 -41 184

1 mm matrix

R)

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+
H
\J
to
1..
U1

 

92

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Figure 25.

93

 

TEM micrograph comparing cytoplasmic organization of 24 hour
anoxic stressed and nonstressed 14 day old Phaseolus vulgaris
plants. The root cross sections are taken from control

matrix plants 1 cm above the lateral root tip. a. Nonstressed
cortical cell (x4500), b. 24 hour anoxic stressed cortical

cell (x10,000).

94

chambers. This treatment enabled us to complete several measurements
which could not be made on previous treatments. In this treatment it
was assumed that volatile and nonvolatile metabolites accumulated in a
manner similar to a field situation. When possible, results from the
waterlogging treatment (N) were compared to the nitrogen gas equili-
brated treatment (N).

Table 21 indicates that the reduction in oxygen uptake during the
24 hour treatment period tended to be greater for the N treatment. A
comparison of microbial oxygen uptake rates also indicates a lower value
for the waterlogging treatment, Table 21. A possible explanation is the
production of a toxic compound (e.g. ethylene) during the waterlogging
treatment. Ethylene has been shown to be a powerful inhibitor ofi
microbial growth (90).

The control treatment resulted in the greatest absolute and rela-
tive reductions in oxygen uptake for both the N and W treatments. The
response of the nonimpeded root may have been a response of its rela-
tively faster growing root system and a lack of storage carbohydrates.
Since the root growth relative to root weight was higher for the nonimpeded
root system its response to anoxia could also have been proportionally
as severe. Less storage carbohydrate may have been available in the
control root system compared to the other matrix treatments. Anoxia
around a sink for photosynthates (e.g. root) has been shown to reduce
photosynthate translocation (80). Oxygen uptake after the anoxic period
could be much lower if the control root was low on storage carbohydrates
because of its rapid growth before an anoxic period. The control root
may also be low on storage carbohydrates because of loss through exuda-

tion.

95

 

 

 

 

 

Table 21. The effect of 24 hour treatments of waterlogging (W) and
nitrogen gas equilibrated solution (N) on the oxygen uptake
of the three matrix treatments.

AOUT/root Microbial
AOUi/plant fresh weight respiration
W N_ W N W N-
(pl 02_hr 1 _ _ (ul 02_hr 1

Treatment plant 1) (pl 02 hr 1 g 1) plant 1)

Control ~162 -152 -94 -86 19 77

3 mm matrix - 95 - 43 -6O -31 76 153

1 mm matrix - 64 - 33 -84 -41 83 192

 

tAOU signifies change in oxygen uptake rate during the 24 hour treatment

period.

96

Ethanol exudation data indicate that the control matrix exuded more
ethanol than the other matrix treatments, Table 22. This was true even
when the data was expressed on a per root weight basis. Amino acid
exudation data shows a similar pattern, Table 23. The confinement of
the larger control root system to a limited volume may have resulted in
the formation of anoxia earlier than the other matrix treatments. This
could also have contributed to the greater exudation observed in the
control root system during the waterlogging treatment.

Large starch granules were observed in the 3 mm matrix primary root
tip, Figure 13. The 1 mm matrix root system has been shown in the previ-
ous section to have a greater dry matter percentage and this may be due
to increased storage of carbohydrates. The low Pasteur effect reported
earlier for the controlixxnzsystem may have been the result of low
levels of carbohydrate reserves.

The difference in oxygen uptake rates are not necessarily related
to root damage or the ability of the root to recover. However, the
generally lower AOU for the W treatment are indicative of a more severe
treatment.

During the period of waterlogging the humidifying system malfunc-
tioned resulting in relative humidity values of 10%. All waterlogged
plants wilted, however, those plants not subjected to waterlogging but
maintained at 0.210 p07 did not wilt. In contrast only one plant wilted
during the N treatment when the relative humidity was at 40—60%.

The wilting occurred with different degrees of severity. The most
severely wilted plants resulted in chlorophyll degradation in the pri-
mary leaves, leaving irregular yellowish patches. All plants eventually

resumed turgidity. The most severely wilted leaves had portions of

97

Table 22. Ethanol root exudation by 14 day old Phaseolus vulgaris

 

plants subjected to a 24 hour waterlogging stress and three

matrix treatments. Values are means of two determinations.

 

Ethanol exuded

 

 

Root Root
fresh dry Leaf
Plant weight weight area _
Treatment (umol/plant) (umol g 1) (umol g 1) (umol cm 1)
Control 33 20 479 492
3 mm matrix 15 10 234 236

1 mm matrix 12 16 274 238

 

 

 

 

 

Table 23. Amino acid exudation by Phaseolus vulgaris roots waterlogged
for a 24 hour period. Data for the 1 mm matrix treatments
are the means of 3 replications. All others represent 1
replication.
Total amino acid exudation per
Root Root
fresh dry
Plant weight weight
Treatment (nmol/plant) (nmol g 1) (nmol g 1)
Control 2,110 1,310 32,560
3 mm matrix 180 110 2,580

1 mm matrix

690 880 16,440

 

99

their blade which were structurally damaged and could not regain turgidity.
Generally, those plants showing the greatest wilting damage were of the
control treatment. The one plant which wilted during the N treatment

was from the control treatment.

The regaining of turgidity may have been simply the result of sto-
matal closure resulting from severe leaf dehydration. A cycle of loss
and gain of turgidity was observed on a plant grown at 0.210 atm p02,
whose root system was under tension as a result of an improperly connected
plant chamber. Figure 36 illustrates a sequence which repeated itself
every forty minutes over a period of eight hours.

An examination of the pH and conductivity data indicates a trend
to decrease the pH and increase the concentration of the nutrient solution
during the N treatment, Table 24. The pH appears to be decreased most
by the control treatment. The larger root system would be expected to
excrete more organic acids than the smaller root systems of the bead
matrices.

Water uptake by the waterlogged plants appears to be controlled by
leaf area, Table 25. The 1 mm matrix treatment appeared to absorb more
water through its root system per unit weight of root. This was probably
due to the increased root surface area per unit weight of root resulting
from the increased diameter of the impeded roots and the relatively
greater demands for water from the shoot.

Generally, a similar pattern of response to low p02 was observed
when results were corrected for size of the root system. There was a

trend for the control treatment to have lower AOU than other matrix treat-

ments when exposed to 0.00 atm pO7 even when corrected for root size.

100

 

 

 

Table 24. Solution pH and conductivity of matrix treatments after water—
logging treatment. Each treatment mean represents two measure-
ments.

Treatment Half
Control strength
3 mm 1 mm at 0.21 atm Hoagland's

Parameter Control matrix matrix p02 solution

pH 6.30 6.43 6.60 6.88 6.60

Conductivity

(umhos) 1043 1043 1043 1001 991

 

101

 

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.mm oacmh

102

Low storage carbohydrate, greater exudation and confinement of the root

system may have contributed to this response.

Potential Effects of Mechanical Impedance

 

on Rhizosphere Oxygen Utilization

 

The rhizosphere is an oxygen sink which can cause the formation of
anaerobic microsites (91). Several results indicate that the rhizosphere
of a mechanically impeded plant may be a greater oxygen sink than that
of a nonimpeded unconfined rhizosphere. The unconfined nonimpeded root
system simulates root growth under ideal soil physical conditions. The
mechanically impeded root system simulates root growth in relatively uni-
form compacted soil. The compact morphology, slower growth rate, in-
creased oxygen uptake per root dimension, increased root diameter, de-
creased root porosity and larger water films around the root could result
in a more rapid development of anoxia in the field to mechanically impeded
I‘OOtS .

The morphology of mechanically impeded roots could enhance oxygen
depletion in the rhizosphere. Mechanically impeded roots were found
to have a denser formation of lateral roots along the length of the
impeded primary root. This resulted in a smaller more compact root
system in which roots are closely associated with each other and compet-
ing for rhizosphere oxygen.

Table 26 indicates the vertical distribution of the root system.

The impeded root systems are more compact and a greater prOportion of

the root system was located in the top 7.5 cm of the media. Under field

Table 26.

103

Percentage of root system located at various depths as

approximated from root length measurements.

 

”o-.—

 

Percentage of root system located at

 

 

0-7.5 cm 7.5-15 cm 15-22.5 cm 22.5-30 cm
'Treatment (%) (%) (%) (%)
Control 44.1 40.7 12.7 2.7
3 mm matrix 58.9 35.9 5.3 O

1 mm matrix

 

104
conditions in which anoxic conditions are beginning to form the impeded
root system could enhance the formation of anaerobic microsites in the
root microenvironment because of the increased demand for oxygen associ-
ated with its rhizosphere. Since the impeded root system is confined to
a smaller portion of the soil, localized anoxic zones could affect a
greater proportion of the root system. The damage done to the plant
may also be proportionally greater since impeded root systems could be
limiting plant growth.

The increased oxygen uptake per unit root dimension of the mechanical-
ly impeded root system should create steeper oxygen gradients around the
root. This could compound the effect of relatively larger water films
surrounding the root which occur in compacted soils (65). Solid con-
tacts surrounding the root are also increased and represent reduced area
available for oxygen exchange.

The slower growing root tip of the impeded root could increase
Opportunities for microbial colonization near the root tip. This could
add an additional demand on oxygen in the root environment. Microbial
colonization of dead root tip cells would also have a similar effect.

Root porosity may also adversely affect anoxia survival under
certain conditions. The root length (L) which could be supported by
plant aeration alone was calculated using a simple plant aeration model

developed by Wooley (110) for nonaerated conditions:
1

 

L = 7Q2c0a)(w(1-a)b)’
where C equals concentration of oxygen (.206 ml/ml); D equals the diffu—
sion coefficient of oxygen in air (.178 cm2/sec); b equals oxygen uptake
rates per root fresh weight; W equals root tissue density and a equals root

porosity. Root lengths that the 1 mm, 3 mm and control matrices could

105

theoretically support were, respectively, 6.0, 8.9 and 8.4 cm. Assuming
oxygen diffusion from the shoot is not limiting and that oxygen will
diffuse first through the primary roots, 60-90% of the primary roots
could be supplied with oxygen by plant aeration. The 1 mm and 3 mm
matrix treatments could supply a larger percentage of their primary roots
because the primary roots are not as long as the control roots.

These estimates of plant aeration are likely to be high because
tortuousity of the intercellular spaces is not included in the calcula-
tions (54). Tortuousity may be of greater importance for the 1 mm
matrix and 3 mm matrix roots than the control roots. A comparison of
electron micrographs, Figure 18, indicate that the intercellular spaces
appear more distorted in the bead matrix treatments.

The larger root diameters of mechanically impeded roots could
result in larger critical oxygen concentrations. Critical oxygen con-
centration is defined as the p02 at the root surface which produces a
p02 of 0.00 in the center of the root (63). Calculations were made
using Lemon's model for radial aeration:

' 2
cR = qF /4Di

where cR is the critical oxygen concentration; q is the oxygen uptake

per unit root volume; R is the root radius; and Di is the root diffusivity

(9.0 x 10'6 cm2 sec—1). Diffusivity estimates were taken from Lemon's

paper (64). The calculations indicate a critical p0 of 0.23. 0.14 and

2
0.15 for the 1 mm matrix, 3 mm matrix and control root systems, respec-
tively. The greater critical oxygen concentration was due to the greater
diameter and oxygen uptake rate of the 1 mm matrix root.

During short term anoxia, the reduction of 07 uptake per unit fresh

weight tended to be greater for roots subjected to mechanical impedance

106

indicating a possible greater CR. for the matrix treatments when com-
pared to the control, Figure 23. Figure 19 indicated a trend for a
greater reduction in oxyoen uptake during the oxygen treatment which in-
cludes the microbial respiration. A treatment of 0.150 p02 would pro-
vide a better test of this model since 0.105 p02 was past the predicted
critical p02 for all treatments.

Mechanical impedance can be predicted to enhance oxygen demand in
the rhizosphere and increase the number of anaerobic microsites which
may synergistically increase the detrimental affects of mechanical
impedance upon plant root growth in the field. Mechanical impedance-
anoxia interaction in the field probably results from the increased
oxygen demand which occurs in an impeded rhizosphere resulting in anoxic
conditions occurring earlier and to a greater percentage of the impeded
root system compared to a nonimpeded unconfined root system. The greater
part of the interaction is probably not due to a synergistic physiological
effect.

The ability of a nonimpeded root system to withstand anoxia in the
field should decline as the root system is confined. Confinement of
roots to a small volume of noncompacted soil in the field could result
when there is a plow sole or hardpan and heavy inter-row traffic. Under
conditions of confinement individual roots of the nonimpeded root system
will be growing densely in a localized area. This should result in an
increased oxygen sink per volume of soil and increased damage from
localized anoxic zones. In the case of a confined nonimpeded root system
anoxia may be more severe than for a mechanically impeded root system

growing under similar conditions.

SUMMARY AND CONCLUSIONS

A system was designed to grow plants and monitor root respiration
under conditions in which the chemical and physical environment of the
root could be controlled independently of each other. The system was
used to determine the effect of mechanical impedance and short term
anoxia on root respiration. A comparison of static and dynamic calibra-
tion curves indicated that the circulation of solution had little effect
on the measurement of oxygen. Root response could be measured to :_5 pl
02 hr-l. Flow rate had no effect on root respiration at 0.210 p02. The
root respiration monitor could be adapted for more detailed studies of
intact root system response to changes in the root chemical environment
and the shoot environment.

The 1 mm matrix severely impeded the primary and lateral root sys-
tems resulting in an overall growth rate 50% of the control. The 3 mm
matrix impeded the primary root but unlike the 1 mm matrix the root tip
remained intact. The size, morphology and structure of the mechanical
impeded root systems contrasted sharply with the control. The 1 mm matrix
root system was smaller with lateral roots clumped along the primary root.
All lateral roots of the 1 mm matrix were distorted. Secondary lateral
roots were only formed in the 1 mm treatment. The localized nature of
the severely impeded root system could predispose plants to damage from
transient localized soil conditions such as low water and oxygen avail-

ability. The smaller slower growing root systems result in less uptake of

107

108

immobile nutrients in the soil. As a result the efficiency of banding
fertilizer relative to broadcasting should be increased in compacted soils.

Root system growth depended on the size of the root system. A
large root system could grow faster because of the greater number of
growing roots. Impedance of the primary root or a temporary inhibition
of root growth in the field may result in a much greater overall loss
of root growth because of the smaller root system.

The 1 mm matrix resulted in distorted cortical cells and reduced
intercellular air spaces reflected in lower root porosity values. The
reduced root porosity has two effects on the oxygen relations of the
root. The lowered root porosity results in a smaller contribution of
oxygen from the shoot. This result of compaction may have more signifi-
cance in a plant species such as rice which acquires a major portion of
its oxygen from the shoot. A second effect of reduced root porosity is
possible decreased radial diffusivity of oxygen and ethylene. Wound-
ing caused by cellular distortion from mechanical impedance could be
the stimulus for ethylene production and combined with reduced radial
diffusivity could result in changes in root morphology. The effects of
plasmalemma distortion and possible damage to plasmadesmata on nutrient
and water uptake is an area of possible investigation.

The 1 mm matrix root system had larger root dry matter percentages
and tissue densities than the other matrix treatments. The increased
root tissue density was largely due to the decreased root porosity. A
greater proportion of cell wall material or storage of surplus photo-
synthate could have resulted in increased root dry matter percentage.

It may be worthwhile to investigate the feasibility of using the root

109

dry matter percentage or root tissue density as a field measurement of
mechanical impedance since the entire root system does not need to be
recovered for these measurements.

Shoot fresh weight but not dry weight was decreased by mechanical
impedance. Leaf area tended to be reduced but photosynthetic capabilities
appeared to not be reduced. The association of leaf area and root surface
area for the 1 mm matrix indicated that the reduced leaf area may have re-
sulted from the limitation of a small root system on water uptake. A
build up of a surplus of photosynthate in the leaf may produce the in-
creased dry matter percentage found in the shoot. This illustrates that
under conditions of severe mechanical impedance shoot growth may be affected
under otherwise near optimal growth conditions. Further investigations
are needed to study these effects in greater detail over the life of the
plant.

Shoot to root ratios were larger for 1 mm matrix grown plants. Root
respiration rates were correlated with shoot to root ratios of the con-
trol and 3 mm matrix plants indicating that the availability of photo-
synthate may control root respiration. The 1 mm matrix root system may
be less correlated because of surplus photosynthate. The relationship
of the shoot to root ratio with root respiration suggests that any factor
which increases photosynthesis in the shoot could also result in an in-
crease in root respiration. This could be a partial explanation for in-
creased plant damage from anoxia under high light conditions. Another
factor affecting shoot and root relationships could be the changes in
growth hormone distribution.

Oxygen uptake rates per unit dry weight were similar for all matrix

treatments indicating that metabolism of photosynthate in nonimpeded and

110

mechanically impeded roots uses similar amounts of oxygen. The differen—
‘tial distribution of dry weight between matrix treatments resulted in
larger oxygen uptake rates per unit fresh weight for the 1 mm matrix root
system. This relationship held true for root dimensional parameters as
well. Under field situations, the capacity for oxygen deficiences to
develop in the mechanically impeded root may be increased by the larger
uptake of oxygen per unit dimension of root. The resulting lower oxygen
partial pressure in the rhizosphere may change the character of the micro-
bial pOpulation near the root and could increase possibilities of patho-
gen infection.

Short term anoxia reduced oxygen uptake rates per root system the
most for control roots. Root system size and rate of growth was primarily
responsible for the control response. Correcting the change in oxygen
uptake for root weight resulted in no statistically significant differ-
ences between matrix treatments in response to short term anoxia. There
appeared to be a trend for the control root to have the greatest decrease
in oxygen uptake even when corrected for the size of the root system.
This may indicate a proportionally greater reduction in relative root
growth than the other treatments or a slower recovery rate due to low
supply of carbohydrate. Root growth relative to root weight was highest
for the control root and a period of 0.00 atm p02 should result in a
larger decrease in oxygen uptake even when corrected for root weight.

The control root because it was growing the fastest probably had the
lowest supply of carbohydrates at the initiation of the period of 0.00
atm p02. The control root also exuded the greatest amount of root exu-
dates during the waterlogging treatment. The control root may have been

depleted of carbohydrate upon ending the 0.00 atm p05 treatment.

111°

Waterlogging generally tended to result in greater decreases in
oxygen uptake than circulating nitrogen equilibrated solution. The
microbial respiration was also much reduced during waterlogging. Both
results may be explained by the accumulation of volatile toxic sub-
stances particularly ethylene.

The majority of inhibition of oxygen uptake occurred during the
first 24 hours of 0.00 atm p02.

The overall response to short term anoxia indicated that there was
little physiological differences in response between the matrix treat-
ments. Mechanical impedance-anoxia interactions observed in the field
or in other laboratory studies which indicate relatively greater inhibi-
tion of mechanically impeded roots may have resulted from a combination
of factors which could cause more rapid depletion of oxygen around
mechanically impeded roots compared to nonimpeded roots. The smaller
and denser root system, greater oxygen uptake rates per unit root
dimension, the probability of increased microbial uptake, decreased
root porosity and thicker water films surrounding the root in compacted
soils. all could contribute to a more oxygen deficient zone around a
mechanically impeded root. These characteristics of mechanically
impeded roots are proposed to predispose the impeded root system to
anoxia under marginally oxygenated field environments.

The mechanical impedance—anoxia interaction in compacted soils
could be reduced, i. by reducing the oxygen deficiency around the root.
ii. by modifying the characteristics of the mechanically impeded root
which cause its greater oxygen uptake. iii. by increasing the tolerance
of the root system to anoxia. or iv. by improving the compacted nature

of the soil. Scott and Erickson (88) used the first strategy to overcome

112

the mechanical impedance-anoxia interaction by using calcium peroxide

as a source of oxygen. The application of calcium peroxide to a compacted
field in sufficient quantities to alter the root environment could entail
considerable expense. Concerning the root modification strategies, very
little is known of the physiological genetics which pertains to root
response to mechanical impedance. Considerably more is known about the
mechanisms and genetics of root response to anoxia and presumably this
knowledge could be used to develOp plant varieties which are less sensi-
tive to anoxia. Ideally, the best strategy for a compacted soil is to
improve soil structure through improved soil management techniques. In
lieu of reducing soil compaction, the most practical strategy for over-

coming the mechanical impedance-anoxia interaction would be the develop-

 

ment of a Phaseolus vulgaris variety which is more tolerant of anoxia
than varieties currently in use.

Solution studies have not taken into account the changes which
occur in roots when growing in a solid medium. Conclusions from solution
studies should be analyzed to determine if changes in root morphology
and structure observed in roots grown in solid media could affect

results observed.

APPENDIX

1.6

."
N

(Cm/12 hr)

Root Elongation
_o
on

Q
is

 

 

Figure 26.

-o- Opsi
-o- 5psi

 

 

1 1 1 1
3 6 9 12 213 18 21

p02 (10' atm)
Mechanical impedance-anoxia interactions from the study of
Gill and Miller (34).

,5?
L- r
‘3 0.2F
CD
E
.C
H
E
- 0.1-
CD
4...;
O
0
CE
0
Figure 27.

114

CT
0)
q

NT‘T‘QP
00000010

.0.
1&-
'CP
.o-
1t-

 

. /

 

l l l

 

4.. , '—_L.
O 3 6 9 12 15 18 21

p02 (10'2atm)

Mechanical impedance-anoxia interactions affecting root

weight production from the study of Barley (9).

115

+1111
8

(10
l

Root Elongation N (mm hr'1)
\\

 

 

C’0'“ 3 6 9“_‘§"‘"‘—"—"'1 13 18 21

F302 (10.2 atm)

Figure 28. Mechanical impedance—anoxia interactions affecting root

elongation from the study of Barley (9).

116

-1:1- Rigid System

.0- Nonrigid System

(cm hr'1l

Root Elongation

é a 1'2 1'5 18 21
p02 (10.231111)

 

O
0.31-

Figure 29. Mechanical impedance-anoxia interactions affecting root

elongation from the study of Aubertin and Kardos (4).

117

80*-

__A

.43 o

\1
C?
T

_‘l\

Bulk Density
-0- 1.3
1.

0')
0

0'1
9
+1111
«N;

is
g

Depth of Root Penetration (mm)
(<13
[J

 

N
O

 

8
I

/ /

4| ‘1'

 

 

068 8 8111811821
p02 (10 atm)

Figure 30. Mechanical impedance-anoxia interactions affecting depth of

root penetration from the study of Tackett and Pearson (9S).

118

Gram Wt.
-°- 20
*- 4O
'0- 60
-°- 80

;_A
U"
l

r‘
O
T

Root Elongation (mm hr")

0.5 -

 

I l l i I ' I I
O 3 6 9 12 15 18 21
[302 (10-2 atm)

Figure 31. Mechanical impedance-anoxia interactions affecting root

elongation from the study of Eavis (28).

119

Ft- lb in'3
«a- 1.5
1. 4.0
o 10.0

 

(mm hr'1)
A" (1° 4?
1
1

Root Elongation

_I
I

 

o 8 8 8 12151821
p02 (10'2atm)

0

Figure 32. Mechanical impedance-anoxia interactions affecting time to
32 . .
reach P in compacted Commerce Silty loam from the study of
. . . 32
Hopkins and Patrick (48). *Based on time to reach P at a

depth of 4.5 cm.

' 120

5' Ft-lbin’3
o 1.5
4:1- 4.0
5- «10.0

 

w 1:-

N
l

Root Penetration (9)

 

 

 

o__.sr- . /
O 3 6 9121518 21
Figure 33. Mechanical impedance-anoxia interactions affecting root

penetration into compacted cores of Commerce silty loam during

a 14 day period from the study of Hopkins and Patrick (48).

121

1000'-

(D
O
O
T

Oxygen Uptake (ul hr'1plant'1)

 

0'1: 1 . 1 . .
O 2 4 6 8 10
Solution Conductivity (10'5mmhos)

Figure 34. Influence of Hoagland's solution conductivity on the oxygen
uptake of 30 day old Zea mays roots. Each value is the mean

of two replications.

122

 

Figure 35. Inhibition in 14 day old Phaseolus vulgaris root growth
caused by an acid nutrient solution (pH 2). Root size is

similar to that at time of transplantation to root chamber.

123

Table 27. Means of matrix treatments averaged over oxygen treatments

for 14 day old Phaseolus vulgaris root parameters. Values

 

are means of ten measurements.

 

 

Root Root Root
fresh fresh dry weight
weight weight percentage

Treatment (g/Plant) (g/plant) (%)
Control 1.74 0.081 4.7
3 mm matrix 1.49 0.068 4.6
1 mm matrix 0.94 0.056 6.2
LSD .01 0.41 0.019 0.6

LSD .05 0.29 0.014 0.4

 

124

Table 28. Means of matrix treatments averaged over oxygen treatments

for 14 day old Phaseolus vulgaris plant parameters. Values

 

are means of ten measurements.

 

 

Plant Plant
Shoot/root Shoot/root fresh dry
weight weight
Treatment (fresh weight) (dry weight) (g/plant) (g/plant)
Control 1.83 4.51 4.88 0.443
3 mm matrix 1.95 5.07 4.34 0.410
1 mm matrix 2.68 5.67 3.38 0.368
LSD .01 0.40 N.S. 1.16 N.S.

LSD .05 0.28 0.92 0.81 0.072

 

125

 

 

 

Table 29. Means of matrix treatments averaged over oxygen treatments
for 14 day old Phaseolus vulgaris shoot parameters. Values
are means of ten measurements.

Shoot Shoot Shoot
fresh dry Leaf dry matter
weight weight area percentage

Treatment (g/plant) (g/plant) (cmZ/plant) (%)

Control 3.14 0.362 87.8 11.5

3 mm matrix 2.85 0.342 80.4 12.2

1 mm matrix 2.44 0.313 67.6 13.1

LSD .01 0.55 N.S. 17.3 N.S

LSD .05 0.44 N.S. 14.0 1.4

 

126

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LITERATURE CITED

10.

11.

LITERATURE CITED

Abdalla, A. M., D. R. P. Hettiaratchi and A. R. Reece. 1969.
The mechanics of root growth in granular media. J. Agric. Engng.
Res. 14:236-248.

Amoore, J. E. 1961. Dependence of mitosis and respiration in
roots upon oxygen tension. Proc. Roy. Soc. (London), ser. B,
154:109-129.

Aubertin, G. M. and L. T. Kardos. 1965. Root growth through
porous media under controlled conditions: I. Effect of pore size
and rigidity. Soil Sci. Soc. Amer. Proc. 29:290-293.

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