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INFLUENCE OF HYDROSTATIC PRESSURE
ON GAS BALANCE AND LACUNAR STRUCTURE
IN MYRIOPHYLLUM SPICATUM L.

presented by

Frederick Carroll Payne

has been accepted towards fulfillment

of the requirements for

Ph.D.

degree in Limnology

QMIIIJI

Dme May 12, 1982

MS U is an Affirmatiw Action/Equal Opportunity Institution

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be charged if book is
returned after the date
stamped below.

 

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6 H764.

INFLUENCE OF HYDROSTATIC PRESSURE
ON GAS BALANCE AND LACUNAR STRUCTURE

IN MYRIOPHYLLUM SPICATUM L.
By

Frederick Carroll Payne

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Fisheries and Wildlife

1982

ABSTRACT

INFLUENCE OF HYDROSTATIC PRESSURE
ON GAS BALANCE AND LACUNAR STRUCTURE
IN MYRIOPHYLLUM SPICATUM L.

By

Frederick Carroll Payne

In very clear lakes, non-lacunate hydrophytes grow to
great depths; their lower limits appear to be determined by
light quantity or quality. Lacunate hydrophytes are
restricted to much shallower regions of clear lakes.
Hydrostatic pressure may limit distribution of the lacunate
hydrophytes. A general model for gas balance in lacunae,
cell sap and surrounding water for submersed vascular hydro-
phytes showed that hydrostatic pressure restricts the devel-
opment of lacunar spaces as long as lacunar gas pressures
reflect hydrostatic pressure.

An experimental system was developed to allow indepen-
dent control of hydrostatic pressure and light intensity,
with gas levels maintained at atmospheric partial pressures.
Results of experiments on net oxygen efflux and lacunar
oxygen storage confirmed the general gas balance model, but
showed that hydrostatic pressure had no influence on shoots
of Myriophyllum spicatum L. The proposed mechanism for

hydrostatic pressure limitation required that lacunar gas

Frederick Carroll Payne

pressures be determined by the prevailing hydrostatic
pressure. This condition was not met by shoots of Myria-
phyZZum spicatum.

Anatomical studies revealed a lacunar arch system in
stems of Myriophyllum spicatum which was capable of expand-
ing the lacunae against external forces on the strength of
cell turgor pressure. Plants grown at 83 kPa hydrostatic
pressure had normal anatomy. The lacunar arch system
appeared strongest in young, apical shoots like those tested
in the net oxygen efflux and lacunar storage experiments.
Mature shoots collected from outdoor stock ponds had weaker
lacunar arch structures suggesting greater susceptibility
to external compression forces such as hydrostatic pressure.
The lysigenous lacunae of Myriophyllum spicatum roots had
no organized lacunar structure and therefore offer little
resistance to external crushing forces.

This research failed to demonstrate a mechanism for
hydrostatic pressure limitation of lacunate hydrophytes.

It did suggest a mode of lacunar expansion not described in
the literature. Exapnsion of the lacunar arch structure by
cell turgor pressure may explain the lack of hydrostatic
pressure effects on apical shoot sections. Weakening of the
lacunar arch system in older stems, as well as weakness of
the root lacunar system, remain as likely explanations for
the hydrostatic pressure limitation of lacunate hydrophytes

in lakes.

ACKNOWLEDGMENTS

My wife, Therese, is recognized by all as my
principle source of support throughout my graduate
programs. Her continuing love, encouragement and,
no least of all, financial support made my accomplishment
possible.

I sincerely thank Professor C.D. McNabb for his
guidance and assistance during his time as my major
professor.

My graduate guidance committee, Professors N.R.
Kevern, P.G. Murphy and R.G. Wetzel, was invaluable in
planning and discussing my research and reviewing this
dissertation. Special thanks go to Professor Wetzel
who provided me with the opportunity to study pressure
effects on seagrasses; this experience fundamentally
influenced my approach to macrophyte - pressure relation-
ships.

The experiments described here were made possible
through the assistance of fellow graduate student Jane L.
Schuette. Jane's knowledge of histological technique and
her professional laboratory presence cannot be overstated.

I also thank fellow graduate students Ted Batterson
and Robert Glandon fer many fruitful discussions which

preceded my experimentation.

ii

iii
I would finally like to thank Professor D.L. King
for his sincere and undying interest in my research program.
The research facility for this dissertation was
supported, in part, by funding from Project No. 959 of the

Michigan Agricultural Experiment Station.

TABLE OF CONTENTS

LIST OF TABLES
LIST OF FIGURES

INTRODUCTION .
METHODS AND MATERIALS
RESULTS

Net Oxygen Efflux Measurements
Lacunar Oxygen Storage Estimates
Lacunar Structure

DISCUSSION
LITERATURE CITED
APPENDICES

Appendix I

Appendix II
Appendix III
Appendix IV

iv

PAGE

viii

19
36

36
45
48

59
71
75

75
80
84
97

TABLE

A-4

LIST OF TABLES

Summary of experiments conducted to deter-
mine the effect of hydrostatic pressure on
net photosynthesis in Myriophyllum spicatum.
Each group consisted of eight plants. For
each experiment, the simulated depth (2) is
given in meters; the range of irradiance
(PAR) is given in uE-m‘Z-sec'1 .

Results of preliminary experiments on the
influence of external oxygen concentration
on lacunar oxygen storage. Experiment 1:
all chambers were closed for oxygen accum-
ulation during the final three hours of the
light period, then were flushed to ambient
oxygen level . . . . . . .

Results of preliminary experiments on the
influence of external oxygen concentration
on lacunar oxygen storage. Experiment 2:
chambers 2, 3, 6 and 7 were closed for the
final 8 hours of the light period, then
were flushed to ambient oxygen concentra-
tion. The remaining 4 chambers flowed at
150 ml-min'l throughout the light period

Results of preliminary experiments on the
influence of external oxygen concentration
on lacunar oxygen storage. 'Experiment 3:
Chambers 2, 3, 6 and 7 were closed for the
final 8 hours of the light period, then
were flushed to ambient oxygen concentra-
tion. Chambers 1, 4 and 8 flowed at 150
ml-min'l throughout the light period;
chamber 5 flowed at = 5 ml-min'l through-
out the light period . .

Results of experiments on the influence of
light and hydrostatic pressure on rates of
net oxygen efflux (apparent photosynthesis)
in Myriophyllum spicatum. Values for PAR
are given as uE-m 2-sec‘1 ; values for net
oxygen efflux (NOE) are given as mgOz-h'l-
mg‘ Chl-a. The pressure level for each

PAGE

29

81

82

83

TABLE

A-6

A-7

A—lO

experiment is given in kPa above atmos-
pheric pressure

Fresh weight (FW), total chlorophyll
(Chl-t) and chlorophyll—a (Chl-a) for
shoots included in experiments on the
influences of hydrostatic pressure and
light on net photosynthesis in Myria-
phyZZum spicatum . . . . . .

Results of experiments on the influence
of ambient oxygen concentration on lacu-
nar oxygen storage at 67 kPa above atmos-
pheric pressure. Ambient oxygen concen-
tration (02a) is given in mg02-1"1 ;

hourly respiration rates (Rn) are given

in mgOz-h'l-l'1 measured in 300 ml sample
volume. Lacunar storage values (LS) were
calculated according to Equation 8

Results of experiments on the influence
of ambient oxygen concentration on lacu-
nar oxygen storage at 152 kPa above
atmospheric pressure. Ambient oxygen
concentration (02a) is given in mgOz-l'l;

hourly respiration rates (Rn) are given in

mgOz-h"1-l"1 measured in 300 ml sample vol-
ume. Lacunar storage values (LS) were cal-
culated according to Equation 8

Estimates of fresh weight (FW) and dry
weight (DW) for shoots involved in lacunar
storage experiments at 67 kPa above atmos-
pheric pressure

Results of experiments on the influence of
ambient oxygen concentration on lacunar
oxygen storage at 67 kPa above atmospheric
pressure. Ambient oxygen concentration
(02a) is given in mgOz-l‘1 ; hourly respir-

ation rates (Rn) are given in mgOz-h’l-g'lDW.

Lacunar storage values (LS) were calculated
according to Equation 8

Results of measurements of net oxygen efflux
following lacunar oxygen storage trials for
plants at 67 kPa above atmospheric pressure.
Hourly net photosynthesis values (Pn) are

vi

PAGE

85

89

91

92

93

94

TABLE

reported in mgOzoh‘I-g'lDW. Photosyn-
thetically active irradiance values (PAR)
are reported in uE.m'2-sec'1 for each
plant. Results were calculated from one

300 ml sample collected at hourly intervals.

No flushing was allowed between samples

vii

PAGE

95

FIGURE

LIST OF FIGURES
PAGE

The diffusion-controlled gas equilibrium
model for partitioning of oxygen in lac-
unar spaces, cell sap, and boundary water
for lacunate hydrophytes. AMB indicates
oxygen activity in water outside the
boundary region; this is the atmospheric
equilibrium level. Four conditions are
represented: A. darkness; B. early mor-
ning; C. mid-day with minimum circulation
of water surrounding the plant; D. mid-
day with high circulation of surrounding
water . . . . . . . . . . . . 10
Comparative values for equilibrium oxygen
concentration outside an oxygen-induced

gas space and net photosynthetic rate, rel-

ative to respective values at the water

surface. Henry's Law shows equilibrium

oxygen concentrations for submersed, oxygen-

induced gas spaces increase five-fold from

the water surface to 10 m depth. Decreases

in net photosynthetic rate lower the oxygen
concentration in boundary layer water, accord-

ing to Fick's First Law . . . . . . . . 13

Diagrammatic representation of steel pressure

chamber for control of hydrostatic pressure

in experiments on oxygen efflux and influx

for Shoots of Myriophyllum spicatum. The

tank accomodated 8 experimental enclosures.

Water was removed from each enclosure through
sampling tubes which led to the exterior of

the tank . . . . . . . . . . . . . 20

Enclosing chamber and base unit for measure-
ment of oxygen efflux and influx for shoots
of Myriophyllum spicatum . . . . . . . . 24

Net oxygen efflux for Myriophyllum spicatum

shoots as a function of photosynthetically

active radiation (PAR) at 10, 67, 101 and

152 kPa above atmospheric pressure. . . . . 37

viii

FIGURE

10

11

12

13

PAGE

Logarithmic regression curves for net

oxygen efflux in Myriophyllum spicatum as

a function of photosynthetically active

radiation (PAR) at 67, 101 and 152 kPa

above atmospheric pressure . . . . . . . 39

The time lag for achieving steady net

oxygen efflux following changes in light

level during the treatment sequence I-B-

I-C-I-D . Values shown are treatment means

plotted at the mean light level; bars indicate

90% confidence limits. Similar patterns were
observed in sequences I-C-I-D-I-E , II-C-

II-D-II-E and IV-B-IV-C-IV-D . The solid line
indicates the logarithmic curve for the 67

and 152 kPa experimental groups. . . . . . 41

Estimates of lacunar oxygen storage as a

function of dissolved oxygen concentration

in the water surrounding shoots of Myria-

phyZZum spicatum at 67 and 152 kPa above

atmospheric pressure . . . . . . . . 46

Development of lacunar spaces in the meristem

region of Myriophyllum spicatum. A. Apical

cells, 0.04 mm below shoot apex; B. Transverse

stem cross-section, 0.4 mm below shoot apex;

C. Transverse stem cross-section, 0.7 mm below

shoot apex; D. Detail of C, showing septum

cells . . . . . . . . . . . . . . 49

Transverse cross-section through stem of
Myriophyllum spicatum. Section taken 10
cm below shoot apex . . . . . . . . . 52

The lacunar arch system in the cortex of
Myriophyllum spicatum stems . . . . . . . 54

Stem cross-section of Myriophyllum spicatum

taken 2 m below the apex. Examined under

scanning electron microscope at the Center

for Electron Optics at Michigan State

University. . . . . . . . . . 57

Magnitude of the oxygen gradient outside leaf-

lets of Myriophyllum spicatum, as a function

of boundary layer thickness. A. Values calcu-

lated from Equation 18, using oxygen efflux

values at 150 - 170 uE-m‘Z-sec‘1 PAR. B. Val-

ues calculated from Equation 19 using estimated
oxygen efflux at 1,000 iiE-m'Z-sec'1 . . . . 66

ix

FIGURE
A-l

PAGE

Spectral characterization of photosyn-
thetically active radiation (PAR) for four
sources: 1. Clear-sky terrestrial solar
curve at East Lansing, Michigan, at noon

on the summer solstice; 2. Lake George,

New York, at 10 m depth under clear sky;

3. Lake Michigan off Grand Haven at 10 m
depth under high overcast; 4. Lawrence Lake,
Barry County, Michigan, at 10 m depth during
July, under clear sky. Each curve is
adjusted to a total PAR of 1, 000 uE m "2-sec'1
for comparison. . . . . . . . . . 76

Spectral characterization of photosyn-

thetically active radiation (PAR) emitted

by high intensity discharge (HID) sodium

lamps. The curve is adjusted for total

PAR of 1, 000 uE m 2-sec"1 . . . . . . . 78

Typical leaf structure in MyriophyZZum

spicatum. A. Transverse cross-section

through rachis; lacunae are seen as clear

areas in the mesophyll. B. Transverse
cross-section through leaflet; lacunae are

seen as small gaps between mesophylly cells . 98

Typical configuration of a node in the stem
of Myriophyllum spicatum, demonstrating the
maximum constriction of the stem lacunar
system. A. Transverse cross-section through
node showing leaf traces; notice that rachis
lacunae are also visible but do not join stem
lacunae. Leaf traces decrease lacunar cross-
section by 40%. B. Transverse cross-section
above node at the point where adventitious
roots exit the stem. Lacunar cross-sectional
area is reduced 60-70% in this section. No
lacunae are visible in the root. C. Longi-
tundinal cross-section through node, showing
leaf traces crossing through lacunae . . . 100

Transverse cross-sections through Myriophyllum
spicatum roots. A. Mature primary root, show—

ing large, lysigenous lacunar spaces. Notice

that the lacunar arch structure visible in stem
sections and, to a lesser extent, in the rachis
sections, is not apparent in the roots. B.

Young primary roots, showing small lacunae

(seen as gaps between cortical cells) . . . 102

FIGURE
A-6

View into the stem lacunae of Myriophyllum
spicatum under scanning electron microscope
(SEM). Septum walls consist of sandbag-shaped
cells, organized into the lacunar arch system.
Many drusae are seen attached to the septae .

View of druse attached to the septum wall of
a lacuna in mature stem of Myriophyllum
spicatum. Photograph made under SEM

Interior view of druse in Myriophyllum
spicatum stem, showing crystal structure
and the cell wall which encloses them.
Notice that the druse wall is continuous
with the walls of the septum cells, arising
from the junction of two septum cells

xi

PAGE

104

106

108

INTRODUCTION

Evidence for physical limitation of vascular hydrophyte
distributions has been established by several authors. Pond
(1905), Pearsall (1920) and Wilson (1935,1937,194l) showed
the dependence of species distributions in lakes upon the
texture, composition and accretion rate of substrata. Sever-
al authors concluded that light levels caused zonations of
species in submersed hydrophyte communities (e.g., Pearsall,
1920; Wilson, 1935,1937,l941; Spence and Chrystal, 1970a,b;
Sheldon and Boylen, 1977; Stross, 1979). The observations
of Payne (1977) suggested that light and temperature both
may have limited submersed hydrophyte distributions in arctic
and subarctic lakes. Hutchinson (1975) has described limi-
tations of hydrophytes by light, substratum and hydrostatic
pressure.

The predictive value of any of these observed relation-
ships is enhanced by knowledge of the mechanisms of limita-
tion. The actions of light and temperature, for example, are
well known from studies on terrestrial as well as aquatic
species (e.g., Bonner and Varner, 1976; Salisbury and Ross,
1978). Although Gessner (1952), Fehrling (1957) and Golubic
(1963) reported that hydrostatic pressure may limit the depth

distribution of vascular hydrophytes in lakes, no mechanisms

2

of limitation have been identified. Vidaver (1972) reported
that hydrostatic pressures greater than 200 atm (20,260 kPa)
altered many metabolic activities in plants. However, the
metabolic changes induced by pressures below 1,000 kPa were
not measurable. Vidaver, (Ibid.) suggested that plants sen-
sitive to pressures of 100 to 200 kPa (cf. Gessner, 1952,
Fehrling, 1957, and Golubic, 1963) must possess a pressure
"transducer"; a mechanism yet undiscovered, which is speci-
fically sensitive to hydrostatic pressure.

Dale and coworkers (Bodkin, et aZ., 1980; Dale, 1981)
found that hydrostatic pressure did not influence growth
of Hippuris vulgaris L. or Myriophyllum spicatum L., in con-
trast to results from Gessner (1952) and Golubic (1963),
respectively. No generalizations can be made from any of the
earlier studies because the experimental growing conditions
almost certainly influenced their results, as will be
shown below. The objectives of this study were to identify
and test potential mechanisms for the limitation of vascular
hydrophyte growth by hydrostatic pressure.

Field studies suggest that the angiosperm hydrophytes
are restricted by hydrostatic pressure to depths less than
10-12 m (Hutchinson, 1975). Observations by Golubic (1963),
Spence (1967), Frantz and Cordone (1967) and Sheldon and Boy-
len (1977) showed that submersed angiosperms do not exceed
the 12 m depth, even in the clearest lakes. Golubic (1963)
specifically tied this limit to hydrostatic pressure in Lake

Vrana, where the light compensation point for shoots of

3

MyriophyZZum spicatum, determined over a 20 h incubation
period, occurred at the 36 m depth. The species was found
to grow only to 7.7 m in the lake, 0.21 times the 20 h light
compensation point.

Hydrostatic pressure does not appear to influence the
depth distribution of non-vascular hydrophytes. These plants
appear to be depth-limited by light, reaching the 1% isophote
which extends to depths as great as 100 m in very clear lakes
(Hutchinson, 1975; Frantz and Cordone, 1967). Stross (1979)
tied the depth limit in NiteZZa flexilis L. to the influence
of spectral composition of light on germination of dissem-
ules. Golubic (1963) found Chara fragilis (C. globularis,
Thuil.) and C. polyacantha (7) reached roughly 0.63 times
their respective 20 h light compensation depths.

The observation which may explain the disparate response
to depth by the vascular and non-vascular hydrophytes is the
presence of lacunar spaces in the tissues of vascular hydro-
phytes. The schizogenous lacunae of leaf and stem tissues
are free gas spaces which develop during normal differentia-
tion (Sculthorpe, 1967). Root and rhizome tissues are
usually found to have lysigenous lacunae (Sculthorpe, Ibid.).
Dale (1957) proposed that schizogenous lacunae are formed
when excess gas pressures achieved by photosynthetic oxygen
production force open small spaces in the meristem region.
The lysigenous lacunae are thought to form by the degenera-
tion of cells and subsequent replacement by gases (Sculthorpe,

1967).

4

Lacunae are generally held to function as conduits for
oxygen supply to buried organs and as reservoirs for the
storage of gases of photosynthetic or respiratory origin
(Hutchinson, 1975; Sculthorpe, 1967). The lacunae seem to
be essential for maintaining internal gas balances, partic-
ularly in light of the relatively slow diffusion of gases in
water. Podostemum ceratophyllum Michx., a non-lacunate vas-
cular hydrophyte, does not support buried organs and is
found only in flowing waters, where plant tissues are contin-
uously bathed in water with gas levels near atmospheric sat-
uration. We are led to suspect that any factor which can
disrupt the normal formation or function of these air spaces
can act to restrict the growth of the lacunate vascular
hydrophytes.

If Dale's (1957) proposed mechanism for the formation
of schizogenous lacunae is correct, gas pressure in these
spaces must exceed the total pressure forcing on the plants'
exterior surfaces; the lacunar gas pressures should be
directly related to depth. Considerations of gas solubility
and diffusion lead to specific predictions which demonstrate
that hydrostatic pressure can be expected to disrupt lacunar
formation and the storage of gases.

Henry's Law defines the solution of gases:
P = K-X (EQN 1)

The vapor pressure of a solute, PA’ is proportional to its

mole fraction, XA, in solution. The constant of

proportionality, K, is the Henry's Law constant. K must be
specified for the solute/solvent system at the temperature
of interest.

For oxygen in an aqueous medium, it is more convenient

to write Henry's Law as:

P02 = K62°I02laq (EQN 1A)

where K6218 expressed as kPa-mg"°l , [02]aq is in

mg02-1'1 , and P02 is expressed in kPa. Approximate values
of K62 can be calculated for temperatures ranging from 5 to
30 C:

K62 = 1.397 + 0.0462-T (EQN 2)

We can use Henry's Law to determine the vapor pressure
of a solute in the atmOSphere overlying a solution, or to
determine the equilibrium concentration of a gas in solution
given its partial pressure in the atmosphere. Henry's Law
shows that an isolated column of pure water will have a uni-
form distribution of any ideal gas in the atmosphere over-
lying the column of water. Since the quantities of gas in
the earth's atmosphere are large, we can view the atmos-
phere as the ultimate source or sink for equilibrating any
consumption or evolution of gases in an aquatic system.
Regardless of depth, lake waters tend toward atmospheric
gas partial pressures.

Because lacunate hydrophytes maintain internal air
spaces, it is important to consider some applications of

Henry's Law. In the absence of surface tension or

6

confinement effects, the total gas pressure at any depth, z,
is the sum of atmospheric (Patm) and hydrostatic (Ph)
pressures:

tot atm h (EQN 3)

and,

Pb

9.80-z (EQN 4)

where Ph is expressed in kPa and z is given in m. Normal
atmospheric pressure is 101 kPa; 10 m of water produces
an additional 98 kPa hydrostatic pressure. The total gas
pressure inside a gas space at the 10 m depth will be 199 kPa.
If the gases dissolved in the water column are in equilibrium
with the overlying atmosphere, Henry's Law points out that
the water surrounding any gas space at the 10 m depth is
undersaturated with respect to a 199 kPa air-water interface.
Gases will exit the space; a gas space at depth can be main-
tained only in conjunction with some source of gas. The rate
of supply from the source must equal the rate of solution
into the surrounding medium to maintain steady-state gas
space volume.

Dale (1957) suggested that oxygen evolved by photo-
synthetic hydrolysis of water is the principle source of gas
for lacunar inflation. Carbon dioxide and methane may also
be present at levels which exceed atmospheric saturation, but
the partial pressures do not approach those of oxygen (Hart-
man and Brown, 1967). The minimum partial pressure of oxygen

can be determined for a gas space at any depth:

7

P02 = Ptot - (PN2 + PCOZ + PCHu + PAr + ...) (EQN 5)

where the sum of gases other than oxygen is approximately
80.1 kPa.

When a submerged gas space is at stable volume, the
concentration of gases in the liquid phase surrounding the
space are determined by Henry's Law. The dissolved oxygen
concentration in the solution surrounding a gas space held
open by excess oxygen pressure can be calculated from Equa-
tions 1A and 5. At 20 C, equilibrium oxygen concentrations
outside a submerged gas space increase linearly from 9.1
mgOz-l"1 at 0 kPa hydrostatic pressure (at the water surface)
to 52.4 mg02-1'1 at 98 kPa hydrostatic pressure (10 m depth).

An oxygen gradient will be established in the water sur-
rounding any gas space with oxygen partial pressures above
normal atmOSpheric levels (6.8-. in excess 0f 21-1 kPa 02)-
For submersed vascular hydrophytes, the production of oxygen
is concentrated in epidermal and mesophyll cells (cf. Grace
and Wetzel, 1976, for Myriophyllum spicatum) which lie
between the lacunae and the surrounding water. There are two
gradients of oxygen diffusion from these source cells: one
inward to the lacunar gas space and a second outward through
the boundary region of still water surrounding the plant sur-
face. The water outside the boundary region in the photic
zone of lakes tends to be near atmospheric equilibrium due
to bulk mixing processes (cf. Hutchinson, 1957).

The flux of oxygen through the gradients is defined

8

by Fick's First Law (Crank, 1956):

A CONCENTRATION - SURFACE AREA
RESISTANCE

FLUX = (EQN 6)
where A CONCENTRATION is the change in oxygen concentration
from the highest to lowest concentrations in the gradient;
SURFACE AREA is the surface area of the plant section from
which the oxygen diffuses; RESISTANCE is the resistance to
oxygen diffusion, a function of the diffusion coefficient
for oxygen in water (for the outward gradient) or air (for
the inward gradient), the shape of the diffusing body (9.8-.
planar, cylindrical or spherical), and the thickness of the
still air or water region adjacent to the diffusing surface
(boundary thickness).

For any plant section, the surface area and diffusion
coefficient for oxygen can be considered as constant for both
gradients. A specification of any two remaining variables
(flux, boundary thickness or A concentration) determines the
third. At steady state, the sum of oxygen fluxes through the
inward and outward gradients equals the net photosynthetic
rate. The rate of oxygen diffusion in the lacunar gas is
4 orders of magnitude faster than in water; thus the oxygen
concentration in the lacunar gas will be relatively homogen-
eous. If the rate of oxygen consumption from the lacunar
space is slow relative to the net photosynthetic rate, the
lacunae act as dead space for gas accumulation. Under this

condition, the concentration of oxygen at a plant's exterior

9

determines the partial pressure of oxygen in the lacunar
space. It can be seen from Equation 6 that the concentra-
tion of oxygen at a plant's exterior surface is a function
of the boundary layer thickness for any specified photosyn-
thetic rate. Also, an increase in net photosynthesis
increases the concentration at a plant's exterior surface
under any specified boundary layer thickness.

Resistance to oxygen movement through the outward grad-
ient can be viewed as back-pressure against which oxygen
levels in the epidermis and mesophyll, and therefore also
the lacunae, can build. Figure 1 provides a graphical rep-
resentation of this viewpoint. In this diffusive gas equi-
librium model, interplay between net photosynthetic rate and
boundary layer thickness for the external oxygen gradient
controls the partial pressure of oxygen in lacunar spaces.
If oxygen partial pressures in lacunar.spaces reflect hydro-
static pressures exerted on a plant's exterior, an increase
in depth requires an increased oxygen concentration at the
plant surface to hold back lacunar oxygen. Previous studies
show that lacunar oxygen partial pressures probably cannot
meet the demand of external hydrostatic pressures at depths
greater than 1 or 2 m.

In studies reported by Hartman and Brown (1967), oxygen
in lacunae of EZodea canadensis Michx. never exceeded 28% of
the volume of gases present; nitrogen levels fluctuated
between 75 and 85%. If lacunar volumes in these plants did

not expand during the photosynthetic period, the highest

Figure 1.

10

The diffusion-controlled gas equilibrium model
for partitioning of oxygen in lacunar spaces,
cell sap, and boundary water for lacunate hydro-
phytes. AMB indicates oxygen activity in water
outside the boundary region; this is the atmos-
pheric equilibrium level. Four conditions are
represented: A. darkness; B. early morning;

C. mid-day with minimum circulation of water
surrounding the plant; D. mid-day with high

circulation of surrounding water.

SURROUNDING WATER

CELL SAP

LACUNAR SPACE

_
IIILIIII
_
---Lnn-u

 

 

 

1.... 1...... Isis—.11... In: .._....i..

 

 

 

B
M
A

AIIII 5585: .3255

2556

 

 

 

12

internal gas pressures could not have exceed 110 kPa, equi-

valent to the total pressure (Pa + Ph) exerted at the 1 m

tm
depth. If the internal gas expanded, lacunar gas pressures
would have been lower than 110 kPa. The shoots could not
have initiated or maintained lacunar spaces at depths greater
than 1 m on the strength of gas pressures alone.

Westlake (1978) determined oxygen content for lacunar

spaces in MyriophyZZum spicatum shoots grown at normal atmos-

2.

pheric pressure under saturating illumination (1,000 uE-m’
sec'1 PAR). The mean of 9 observations was 21.9% 02 with 2
samples at 28%. Again, lacunar gas pressures were too low

to expand against hydrostatic pressure at depths greater than
1 m. Additionally, the low mean oxygen in lacunar gases
probably resulted from stirring of the experimental chambers.
This minimized the thickness of the boundary region, enhancing
outward flux of oxygen as expected from the diffusive gas
equilibrium model (cf. Figures 1C and 1D).

For an oxygen-induced lacunar air space at 20 C, the
equilibrium oxygen concentration outside the plant surface
increases five-fold from the surface to the 10 m depth. But
net photosynthesis, required to maintain boundary layer oxygen
levels, decreases rapidly from the surface downward, even in
very clear lakes (cf. Golubic, 1963, for Myriophyllum spicatum
in Lake Vrana). This concept is developed in Figure 2. In
progressively deeper settings, lacunate hydrophytes should
experience normal lacunar inflation throughout 24 hours, a

zone in which photosynthetic oxygen efflux supports lacunar

Figure 2.

13

Comparative values for equilibrium oxygen concen-
tration outside an oxygen-induced gas space and
net photosynthetic rate, relative to respective
values at the water surface. Henry's Law shows
equilibrium oxygen concentrations for submersed,
oxygen-induced gas spaces increase five-fold from
the water surface to 10 m depth. Decreases in
net photosynthetic rate lower the oxygen concen-
tration in boundary layer water, according to

Fick's First Law.

 

 

 

4.0 --

a
0 0 0.
3 1

means 5 o B< MDA¢> mBH 0a. m>~9<qmm m4m<mm<> zH m02<mo

DEPTH (m)

15

inflation at mid-day (with deflation or collapse for the
balance of the day), and a level below which lacunar infla-
tion cannot be expected at any time. As a result of these
considerations, the principle hypothesis of this research
can be stated as follows:
The partial pressure of oxygen in lacunar spaces of submersed
hydrophytes is determined by the rate of net photosynthesis
and the oxygen concentration in the water outside the plants'
exterior surfaces. The‘diffusive demand on the plants' lacunar
oxygen increases with lacunar oxygen partial pressure. Thus,
net photosynthetic rates and boundary layer thickness determine
a plant's ability to develop excess oxygen partial pressures
which may be necessary to initiate schizogenous lacunae (cf.
Dale, 1957) and to hold lacunar spaces open against the crushing
forces of hydrostatic pressure. Moreover, the ability to store
oxygen in lacunar spaces to meet dark respiration diminishes as
hydrostatic pressure increases, as long as lacunar gas pressures
reflect the prevailing hydrostatic pressures. Consequently,
hydrostatic pressure acts to limit the depth distribution of
lacunate hydrophytes by restricting or eliminating the initiation
and'maintenance of'lacunar gas spaces; a plant's ability to
form and sustain schizogenous lacunar spaces at depth is deter-
mined by the net photosynthetic rate and the development of
elevated oxygen concentrations in the epidermis and’mesophyll

cells and hence in the boundary region surrounding the plant.

16

This hypothesis is attractive because it incorporates
a plausible mechanism for hydrostatic pressure limitation of
submersed vascular hydrophytes. The mechanism itself is
attractive because it eliminates the need to invoke metabolic
changes due to relatively small hydrostatic pressure loads.
It also focuses on the lacunar spaces, the most obvious
difference between plants which appear to be pressure-limited
and those which seem to be unaffected by pressure. Lastly,
the hypothesis entails the resolution of earlier, conflicting
results on hydrostatic pressure limitation (Gessner, 1952;
Fehrling, 1957; Bodkin, et al., 1980; Dale,1981): it is
essential that any experiments performed under hydrostatic
pressure be conducted with atmospheric gas partial pressures
outside the boundary region, just as plants likely experience
in lakes.

Regarding this, Dale (1981) inappropriately utilized
air pressure to develop total pressures in excess of 110 kPa
in experiments on the possible limitation of Myriophyllum
spicatum growth by hydrostatic pressure. Henry's Law clearly
shows that dissolved gas levels surrounding plants in such
an experiment are a function of the depth simulated. Most
importantly, the diffusive demand in such a system would be
similar to that at the water surface in a lake, regardless
of the depth being simulated. There was no opportunity in
Dale's (1981) experiments to exhibit a hydrostatic pressure
influence on lacunar formation or function. Nonetheless, he

concluded that hydrostatic pressure did not influence the

17

growth of Myriophyllum spicatum.

The static pressurization of remaining studies (Gessner,
1952; Fehrling, 1957; Bodkin, et al., 1980) also placed
plants under unrealistic gas balances. If dissolved inor-
ganic carbon levels are sufficient, oxygen in closed, static
systems may reach very high levels, possibly masking hydro-
static pressure effects. Experiments in which mercury man-
ometers have been used to develop excess pressures (Gessner,
1952; Fehrling, 1957) may be further criticized for potential
mercury toxicity to plants. Since mercury concentration in
the water may be influenced by the pressure of mercury at the
mercury/water interface, no adequate control can be estab-
lished for such work.

A pressurization system was developed for this study
which allowed control of gas levels and water flow rates in
the vicinity of the plants. This system allowed realistic
simulation of hydrostatic pressure and eliminated nuisance
covariates.

Three basic questions were established to test the
principle hypothesis:

1. 1s oxygen efflux during the light period influenced by hydro-
static pressure? In metabolic studies by Vidaver(l972), hydro-
static pressure did not depress net photosynthetic rates at the
100-200 kPa level. But Hutchinson (1975) suggested that hydro-
static pressures as low as 50 kPa may depress net photosynthesis

for some vascular hydrophytes.

18

2. Does hydrostatic pressure decrease a plant's ability to
store oxygen in its lacunae? The dissolved gas equilibrium
model shows that hydrostatic pressure can be expected to
promote diffusive losses from oxygen-induced lacunar gas
spaces.

3. What is the normal structure of the lacunar system? Is it
influenced by hydrostatic pressure? As outward diffusion
from plant tissues increases, the ability to form and sus-
tain lacunar spaces decreases. Schizogenous lacunae held
open by gas pressures alone should fail at relatively low

hydrostatic pressures.

Myriophyllum spicatum was selected as the subject for
tests performed during this research. Westlake (1978)
measured lacunar volumes in this species as high as 15% of
plant volume. This indicates that if lacunar volumes were
diminished by the action of hydrostatic pressure, the change
in gas balance may be observable. Lim (1976) measured root
and shoot masses for dense stands of Myriophyllum spicatum.
The mean root/shoot ratio for samples taken in late May was
2.7, suggesting that Myriophyllum spicatum must depend on
lacunae to transport oxygen to its large root mass. Any
inhibition of the lacunar formation or function can be expec—

ted to inhibit plant growth in natural settings.

METHODS AND MATERIALS

Experiments were conducted in a steel pressure chamber
with acrylic windows. This unit is depicted in Figure 3.
The walls and bottom of this tank were 6-mm mild steel
plate, with 6 x 50-mm.steel T-sections welded in place
every 150 mm. All seams were reinforced by 6 x 25-mm steel
angles, welded into the inside of the tank. A window frame
was formed at the top of the tank by 6 x 38-mm steel angles
welded just below the rim of the wall. These were welded
so that a 90° angle was formed between the wall and the un-
derside of the window frame. At 300-mm intervals, 6 x 50-
mm steel T-sections were welded across the top of the window
frame, on top of the angle rim. A flat 6 x 50-mm steel
section was added to the underside of each T-section in
order to make the underside of the window frame smooth and
flat.

Windows were made from 15-mm acrylic plastic. The win-
dow at each end of the tank was permanently sealed in place
with room-temperature vulcanizing rubber (RTV). A rubber
gasket was placed under each of the center window frames,
also sealed with RTV. These open frames served as access
ports to the pressure tank. To close the tank, a light coat

of stopcock grease was applied to the gasket and the windows

19

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22

were pulled into place with suction cups. Hydrostatic
pressure from within the tank enforced the window seals.

A centrifugal pump pressurized the experimental tank,
while providing a constant flow-through of water at 20 C
and atmospheric gas saturation. The water flowed to the
tank through plastic pipe. The outlet stream passed
through fiber (particulate) and activated charcoal (dis-
solved organic carbon) filters on its way to a reservoir.
The reservoir walls were insulated to maintain experimental
temperatures. Continuous aeration by compressed air held
dissolved gases in the reservoir at atmospheric saturation.
A feed line from the reservoir to the pump completed the
circuit.

Pressure in the experimental tank was controlled by
balancing valves on the inlet and outlet streams, creating
a velocity head in the tank. Pressure in the tank was
measured by a standard air/water pressure gauge installed
through the side wall of the tank. The average residence
time for water in the tank was about 60 min. The tank was
designed to be safe at 300 kPa above atmospheric pressure.
An additional margin of safety was achieved by immersing the
tank in a water-filled fiberglass chamber. Temperature
control of the circulating immersion bath was used to main-
tain a 20 C experimental temperature.

Enclosing chambers were constructed to allow determina-
tion of apparent photosynthetic and respiratory rates of

individual shoots of Myriophyllum spicatum. The chambers,

23

shown in Figure 4, were made from acrylic plastic tubing
which was closed at the top by a thin disk of the same
material welded in place by methylene chloride. The dia-
meter of an enclosing chamber was slightly greater than that
of the base unit tube.‘ An enclosure slipped over the base
unit as shown in Figure 4, and was sealed by an "0"-ring
mounted at the top of the base unit tube. The enclosed
volume was 150 ml. Water samples for oxygen analysis were
removed from chambers through a 3—mm Tygon tube located at
the top of the cylinder wall of each enclosure. This tube
led through the wall of the pressure tank to the exterior.
Four small (3-mm) holes, located in the side wall of the
enclosing chamber just above the top of the base unit pro-
vided displacement water. The pressure tank accommodated

8 enclosures, 4 under each of the permanent windows. Water
surrounding the enclosures in the experimental tank was
sampled through a tube drawing water from the center of the
tank. It led through the tank wall in the same fashion as
the sampling tubes from the enclosures.

In order to determine hydrostatic pressure influence
on net photosynthesis in Myriophyllum spicatum, net oxygen
efflux rates were measured using the above enclosures. At
the beginning of a period of measurement, enclosures and
outlet tubes were flushed. Samples from the enclosures
were then collected through the outlet tubes in 300 ml BOD
bottles. A YSI probe and meter were used to confirm that

the initial dissolved oxygen concentration in the enclosures

24

Figure 4. Enclosing chamber and base unit for measurement
of oxygen efflux and influx for shoots of

Myriophyllum spicatum.

SAMPLING

'——

   
  
     
  
 
 

 

ENCLOSING
CHAMBER

 

 

L*—— RELIEF PORT

”o"- RING
SEAL

SILICA SAND

BASE UNIT

26
was at the saturation level for 20 C and atmospheric pres—
sure (near 100 kPa). Outlet tubes were then clamped creat-
ing stagnant conditions in enclosures. Testing showed
that measurable changes in oxygen concentration could be
obtained with these enclosures using one-hour stagnation
periods for shoots 10 cm or more in length. It was also
found that in successive 300-ml samples from enclosures,
the first sample showed deviation from ambient oxygen con-
centration, while the second and subsequent samples did not.
This demonstrated that a 300-ml sample captured the oxygen
produced during closure. The oxygen concentration in the

150 ml closure was then calculated from the concentrations

in the 300 ml sample volume and the ambient sample as

follows:
ENCLOSURE 02 - 2-ENCLOSURE 02 - AMBIENT Oz (EQN 7)
(actual) (sample)

The YSI system used to make these measurements was air-
calibrated and checked against Winkler iodometric deter-
minations (APHA, 1975).

Four pressures and four ranges of irradiance were used
to measure the effect of hydrostatic pressure on net photo-
synthesis. A pressure 10 kPa above atmospheric was obtained
by submersing enclosures at a depth of 0.1 m in an open
circulating tank in the laboratory. Pressures 67, 100 and
152 kPa above atmospheric were established in the pressure
tank to simulate water depths of 6.6, 10 and 15 m, respec-

tively. Ranges of irradiance were obtained by placing

27

fiberglass screens that had been painted black between the
light source and the windows of the pressure tank. Irradi-
ance was mapped over locations occupied by enclosures

using a Li-Cor portable PAR (photosynthetically active
radiation) integrator and underwater sensor. A single light
level of 104 qum‘Z-sec‘1 was used for measurements at 0.1
m depth; 6 Sylvania VHO flourescent bulbs were the light
source. Enclosures in the pressure tank were exposed to
light ranges of 22-29, 47-68 and 115-174 qum‘zssec"l ,
under 2, 1 and 0 screens respectively. Lighting over the
pressure tank was supplied by two 400-W high intensity
discharge (HID) sodium lamps, suspended 1.5 m above the
tank (cf., Appendix I). A 16 h light - 8 h dark cycle was
maintained throughout all experiments.

A stock population of Myriophyllum spicatum has been
maintained in outdoor ponds at the Limnological Research
Laboratory at Michigan State University. This population
developed from transplants from Lake Wingra, Dane County,
Wisconsin, in 1973. Cuttings from outdoor ponds were plant-
ed into plastic trays filled with silica sand enriched with
Ortho plant food pellets. These plants were used as a lab-
oratory stock and were kept at 15 C with PAR at 80 uE-m-Z-
sec‘1 , on a 16 h light - 8 h dark cycle.

Active, low-algae shoot tips were cut from the lab-
oratory stock to 10 cm lengths and planted into enclosure
base units. The lower 2 cm of each shoot was buried in

silica sand enriched with Ortho plant food pellets.

28

Plantings were placed in a recirculating nurturing bath at
20 c with PAR at 104 uE-m‘z-sec-l , 16 h light - 8 h dark.
The length and number of nodes were recorded for each shoot

as it was placed in the nurturing system. Actively growing

plants were used in experiments when they reached a length
above the sand of 10 - 15 cm. This was normally 3 - 5 days

after placement in the nurturing system.

A stock of 20-30 shoots was held in the nurturing sys-
tem continuously. Inactive or algae-contaminated plants,
and those which exceeded 15 cm in length were discarded.
When algal contamination was evident on tank walls and many
plants were affected, the entire shoot population was dis-
carded. The experimental and nurturing systems were then
bleached and the shoot population was re-established from
the outdoor stock pond. Palmella stage Chlamydomonas sp.
was the principle algal contaminant during these experiments.

Four groups of eight plants each were selected from the
nurturing tank for experiments on the influence of light and
pressure on net photosynthesis. Each group was tested at
10 kPa above atmospheric pressure with irradiance at 104
uE-ui“2-sec'1 , then was placed in the pressure tank to con-
tinue a sequence of treatment combinations at pressures 67
to 152 kPa above atmospheric. The sequence of treatments
for each group is given in Table 1. Plants were acclimated
under pressure for 24 h preceeding measurements. Chamber
exit tubes remained open during acclimation to hold ambient

gases in enclosures near atmospheric saturation. Net oxygen

29

QNINN OH mmlmm OH

mNINN ma

mNINN ®.® mNINN ®.m

mmlhv 0H VbHImHH OH

 

 

m<m N m<m N.
m D

 

 

 

 

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mmlmm ma
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mmlhv ®.®
m<m . N
U
EZNSHmmmNm

 

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ma ANV ounce oopsassfim one .usosuaoaxo some mom

 

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>H

HHH

HH

930mm

.H OHQNB

30

efflux measurements were taken during l-h stagnation periods
in all experiments except IV-E which was closed for 2 h.
Chambers were flushed during the intervals between experi-
ments to hold gases at saturation. Times between closures
ranged from 15 to 67 minutes except experiments IV-D and
IV-E for which the interval was 14 h (overnight).

After completion of net oxygen efflux measurements,
each shoot was blotted dry and fresh weights were measured.
The shoots were then extracted in dimethyl sulfoxide at 62
C for 1 h, following the method of Hiscox and Isrealstam
(1979). Chlorophyll-a of the extracts was calculated from
the equations of Arnon (1949). Net oxygen efflux values
(apparent photosynthesis) were recorded as mgOz-h"‘-mg'l
Chl-a.

The influence of hydrostatic pressure on lacunar oxygen
storage was determined by experiments on net oxygen influx
rates (apparent respiration). The diffusion-controlled
gas equilibrium model given in Figure 2 suggests that lacun-
ar storage increases with increasing oxygen concentration
outside the plant surface. Given this, the length of clos-
ure period at constant light would control the concentration
of oxygen in the boundary region and hence the partial pres-
sure of oxygen in the lacunae. By varying closure times we
expected also to vary lacunar oxygen storage.

0n the assumption that oxygen stored in the lacunae
was more readily available to respiring tissues than oxygen

in the surrounding water (cf. Hutchinson, 1975), we

31

expected to observe a reduced apparent respiration rate
during dark periods as long as lacunar oxygen partial
pressures were above those in the surrounding water. Pre-
liminary experiments, reported in Appendix II, showed that
apparent respiration rates were influenced by the ambient
oxygen concentration reached during the preceeding photo-
synthetic period. In order to test the expectation that
hydrostatic pressure would reduce lacunar oxygen storage,
two groups of eight plants each were entered into the exper-
imental closures, one at 67 kPa above atmospheric pressure
and the other at 152 kPa above atmospheric pressure, simu-
lating the 6.6 and 15 m depths, respectively. Six of the
eight enclosures were sealed at times ranging from 3 to 10

h before darkness to achieve a broad range of final oxygen
concentrations in the water surrounding the individual
shoots. The remaining two chambers were left flowing
throughout the light period so gases in the water surround-
ing these shoots would remain at atmospheric saturation. At
the end of the light period the tank was covered with alum-
inum foil and 3-300 ml sample volumes were withdrawn from
each enclosure. Flow through the chambers was then closed.
A single 300 ml sample was withdrawn hourly from each enclo-
sure during the dark period. A sample was drawn from the
ambient tap in the pressure tank at each sampling time. All
samples were analyzed for dissolved oxygen by the Winkler
iodometric method (APHA, 1975) with the titrant strength

reduced to half to improve the precision of the measurements.

32

The oxygen concentration in the first of the three
samples drawn at the end of the light period was used to
estimate the final oxygen concentration in the water sur-
rounding each shoot. The second and third samples were
analyzed to ensure that the chambers had returned to atmos-
pheric oxygen levels prior to the start of respiration
measurements. The preliminary experiments suggested that
only the first of the hourly respiration measurements was
likely to be influenced by lacunar storage. Also, there
was an indication that respiration may decrease near the
end of an 8 h dark period. Given this, basal respiration
was estimated by the mean of hourly rates observed during
the second through sixth hours of darkness. The lacunar
storage (LS) of oxygen was then estimated as the relative
decrease in apparent respiration seen during the first hour
of darkness when compared to the basal rate established
during hours 2 through 6 of darkness. This index value

was calculated as follows:

L8 = (EQN 8)

 

where I1 is the apparent respiration for the first hour of
darkness and I2-5 is the basal rate from hours 2 through 6.
Notice that in the case where the respiration during hour 1
equals the basal rate, the value for LS is 0. If the value
for I; is 0, LS is 1.0. In a case where oxygen continues

to exit the plant during the first hour of darkness, 11

becomes negative and LS is greater than 1.

33

Following sample withdrawl for the eighth hour of dark
respiration, the foil covering was removed from the tank
and hourly sample withdrawl was continued through the light
period. No flushing was allowed between these closure
periods. The results of these tests were used to assess
the stability of oxygen efflux under constant light. The
67 kPa group was blotted dry and weighed then oven-dried
to constant weight at 105 C for fresh/dry weight ratio
estimates and to allow estimation of dark respiration rates
on a.dry weight basis. These estimates were coupled with
earlier net photosynthesis results to allow predictions of
24-h compensation depths.

Plants were grown at 0.3 and 8-m depth simulations to
examine the influence of hydrostatic pressure on the inter-
nal anatomy of Myriophyllum spicatum. Plants at 0.3 m were
held in standard nurturing conditions throughout their
growth period. Eight plants, 5 cm in length, were taken
from the 0.3-m population and planted into the experimental
tank. The tank was then pressurized to the 8-m depth. When
plants reached 15 cm, they were removed after slow decom-
pression. The upper 10 cm of each shoot was removed, rep-
resenting growth which occurred at the 8 m depth. At this
time a group of plants was also removed from the 0.3-m
population. Plant sections were fixed in FAA.

Representative shoot sections were selected from each
experimental group; leaves, stems and roots were separated.

These were dehydrated and embedded in Paraplast following

34
the outline of Sass (1951). Serial sections were made with
an IEC rotary microtome. Apical sections were taken at 10-
um intervals; leaflet, rachis, lower stem and root sections
were made at 20-um intervals. Paraplast ribbons were mounted
on slides with a gelatin/water/formalin adhesive (Sass,
Ibid.). Slides were labelled to maintain serial order of
the sections. The Paraplast was removed with xylene. Saf-
ranin was used as the primary stain with a 30-min exposure.
Fast green provided a counterstain at 30-sec exposure. The
slides were completed by the placement of a cover slip
affixed by Permount.

Sections from the two groups were compared to determine
whether hydrostatic pressure influenced the lacunar structure.
The anatomy of both groups were compared to the Myriophyllum
spicatum sections shown in Hasman and Inanc (1957). Partic-
ular attention was given to lacunar development in the
shoot apices since this information is not available in the
literature.

Mature Myriophyllum spicatum shoots may grow to lengths
in excess of 4 m. The base of these stems are much larger
than any which could be grown in this experimental study.

To examine the anatomy of mature stem sections, plants were
harvested from the 2-m depth in the outdoor stock pond.
Sections from the base of these plants were prepared for
examination under a scanning electron microscope (SEM) by
gluteraldehyde fixation and critical point drying. The

sections were examined under SEM at the Center for Electron

35

Optics at Michigan State University. The results of this
exercise were combined with light microscope examination
of the Paraplast sections to determine the structure and

continuity of the lacunar system for the entire plant.

RESULTS

NET OXYGEN EFFLUX MEASURMENTS

Data regarding the influence of light and pressure on
net oxygen efflux for each experimental treatment used in
this study are given in Appendix III. Figure 5 shows com-

bined results from all treatments. At 10 kPa above atmos-

2 1

pheric pressure with PAR at 104 uE-m' ~sec’ , the mean net
oxygen efflux (NOE) for all shoots was 1.25 mgOZ-h"'mg'1
Chl-a, the standard error of the mean estimate was 0.056
with n 8 31. The results of measurments made at the three
high pressures were combined by pressure level and summar-
ized by logarithmic regression equations. At 67 kPa above

atmospheric pressure,

0.93) (EQN 9)

NOE = -2.50 + 0.82-ln(PAR) (r2

at 101 kPa above atmospheric pressure,

NOE = -2.19 + 0.68-ln(PAR) (r2 0.92) (EQN 10)

and at 152 kPa above atmospheric pressure,
NOE = -2.68 + 0.86-ln(PAR) (r2 = 0.95) (EQN 11)

where NOE values are given in mgOz-h-l-mg'IChl-a and PAR

2 1

values are in uE-m' -sec' The r2 value is the coeffi-

cient of determination for the fit of the logarithmic curve.
The plot of logarithmic equations shown in Figure 6

suggests that the plants grown at 67 and 152 kPa above

atmospheric pressure were similar in their photosynthetic
36

37

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96% «as ADC m2 23 dqv SH gov so gov S as
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maoonm exaeowmm stmemowsmz no“ xsammo sowzxo uoz

.m Opswfim

NET OXYGEN EFFLUX RATE

A .Ioom.~IE.ma v
Am<mv ZOHB<HQ<m M>HBU< >AA<OHBmmezmeHOEQ

 

 

oom emu OOH om
P V b I D E P P I h P b .- b h b Pl.
‘ A“ I IGI — d d d. d u N in d d _ d 1
Q
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11 q 0 w 0
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9m
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.. 0 Am 0
m 0
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OD
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(D_Iq31_sw't_q°z03m)
HLVH XHTJJH NEDAXO LEN

39

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zaasofiuonpsszposa mo :OHuosSH a we maoonw Esseommm Es~Nmedomsmz

as xsfimmo somhxo am: so“ mo>u=o scammoswos OaEnpfismwoq

.m whsmfim

com

A AuoomJuEé: v

Am<mv zo~e<~n<m m>HEU< >JJ<UHBm=Bz>mOPO=Q

omH OOH on

.H

( o-{q3t_3w.t_q.203w )

NHOAXO LEN

HLVH XQTJJH

41

.masonm Hayseedsoaxo was and

use so on» new O>A=O OfisnufisawOH one mOpaOfioefi mesa OHHOm one

. QI>H.OI>H.mI>H use m|H~.QIHH.ouHH . mIH.QIH.OIH moososcom as
eo>aomoo Ohms mesouusa ssfifisam .mufiswa oosoefinsoo fiom Ouaofiosfi when
”HO>OH anmfia same one as scape-a memos escapees» one esosm mo=Hs>

. QIH.OIH.mIH ooeoscom usosusoau one madame ~O>OH pan- :H momssno

msflBOHHOm xsammo somuxo Hos museum mcfinoxos AOm mxa was» one

.5 oasmflh

A .Ioom.~ls.m: v

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b -

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I-
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( o-{q3,_8m.t_q.203w )

HLVH XOTHJH NHDAXO LEN

43

response to light. Plants grown at 101 kPa above atmos-
pheric pressure had a lower light response. From these
data, hydrostatic pressure could not be shown to influence
net oxygen efflux in shoots of Myriophyllum spicatum. Al—
though the results from 101 kPa above atmospheric pressure
were lower than the other three pressures, hydrostatic
pressure was expected to act in a continuous manner, dimin-
ishing the photosynthetic response at 152 kPa above atmos-
pheric pressure at least to the extent that the 101 kPa
plants were influenced.

Two factors other than hydrostatic pressure may have
contributed to the lowered net oxygen efflux estimates in
the shoots grown at 101 kPa above atmospheric pressure. The
mean chlorophyll content per gram fresh weight (FW) was
lower for experimental Group IV than for any of the other
plant groups (cf. Appendix III). There also appears to
have been a long lag-time for re-establishing steady net
oxygen efflux rates after changes in light level during ex-
periments. An example of this is shown in Figure 7, where
treatment I-D (101 kPa) may have inadvertently been influ-
enced by the earlier NOE rate established at a lower light
level. This equilibration pattern was anticipated in the
diffusive gas equilibrium model shown earlier in Figure 2.
However, the minimum 15 min flushing time was originally
thought to be adequate to return the lacunar compartment to
atmospheric equilibrium.

Points from the logarithmic prediction equations were

44

entered into the Lineweaver-Burke double-reciprocal plot
to estimate kinetic coefficients at 20 C. Since the curves
did not pass through the origin, the value 1/(PAR - PARC)
was substituted for l/(PAR), where PAR was the actual light
(i.e. substrate) level and PARC was the light compensation
point for net oxygen efflux, estimated by the X-intercept
from the logarithmic plots. This procedure shifts the Y-
axis in the Michaelis-Menton kinetics plot to the compensa-
tion point. The resulting kinetic constants were as follows
for combined data from 67 and 152 kPa above atmospheric
pressure:

vmax for NOE . 2.13 mg02°h'1-mg'1 Chl-a

Km for light a 75 uE-m’z-sec'1

Light compensation for NOE a 21 uE-m‘zosec'1
For data from 101 kPa above atmospheric pressure:

vmax for NOE = 1.79 mgOz-h‘l-mg‘IChl-a

Km for light 8 92 1.1E-m"2-sec'1

1

Light compensation for NOE 25 uE0m'2'sec'

The net oxygen efflux (apparent photosynthesis) from

shoots of Myriophyllum spicatum can be modelled as follows:

PAR - PAR
( C) (EQN 12)

 

NOE = V -
max Km + (PAR - PARC)

Equation 12 follows the general form of the Michaelis-
Menton enzyme kinetic equation, with the Y-axis shifted to

the light compensation point for net oxygen efflux.

45

LACUNAR OXYGEN STORAGE ESTIMATES

Lacunar storage of oxygen (LS) was estimated for plants
at 67 and 152 kPa above atmospheric pressure by depression
of net oxygen influx (apparent respiration) as described in
Equation 8, above. The results of these experiments are
shown in Figure 8, and are tabulated in Appendix III. Stor-
age at 67 kPa above atmospheric ranged from 0 to 1.0 with

ambient oxygen concentrations ranging from 8.1 to 31.7

mgOz-l‘1 . A logarithmic curve was fitted to these data as
follows:
LS 3 -0.73 + 0.49-ln(02) (r2 = 0.75) (EQN 13)

As expected, these results demonstrated the existence of a
relation between oxygen storage in the lacunae and oxygen
concentrations in the surrounding water.

Storage at 152 kPa above atmospheric pressure ranged
from -0.22 to 1.0 with external oxygen concentrations from
8.1 to 20.4 mgOZ-l’1 . A logarithmic curve was established
for these data:

LS - -1.35 + 0.68-1n(02) (r2 = 0.38) (EQN 14)
The comparison of these two groups of data does not support
the hypothesis that hydrostatic pressure decreases lacunar
oxygen storage by enhancing outward diffusion.

Fresh and dry weights (DW) were determined for the
group of plants tested at 67 kPa above atmOSpheric pressure.
Respiration and photosynthesis values for these experiments
were normalized by gDW and are tabulated in Appendix III.

The mean respiration rate for the second through sixth

46

.ousmmoaa OfisosamOEps
952..» max Adv «mu use on to as Esaoommx SsNmemoH-smz
m0 mucosa meiossosssm heads on» a“ sofiuaaasoosoo sommxo

oo>HommHO HO sofiaossu a ma ommpOpm somzxo panacea m0 movesaumm

.m ossmum

ov

A

1788. C onSmeszzoo zmosxo 9583 4

on em

Ca

o.A

(ST) HHTVA XHONI HOVHOLS HVNOOVT

48

hours of darkness was 0.872 mgOz-h'l-g'lDW; the standard
[error for the mean estimate was 0.038 with n = 40. FW/DW
and Chl-a/FW ratios were calculated from shoots involved in
lacunar storage and net oxygen efflux experiments, respec-
tively. These calculations showed approximately 2.005
mgChl-a/gDW . Based on this conversion factor, the net
oxygen efflux rates during the photosynthetic period Of the
lacunar storage experiment were similar to those of the

67 and 152 kPa results reported in Figures 6 and 7.

LACUNAR STRUCTURE

Paraplast thin sections were made from shoots grown at
pressures equivalent to 0.3 and 8 m depths. No differences
in internal anatomy could be determined from sections of
these two experimental groups. All leaflet, rachis, stem
and root sections had normal morphological organization as
shown by comparison with the anatomy of Myriophyllum spicatum
given by Hasman and Inanc (1957). Representative sections
were photographed under the light microscope and are shown
in Appendix IV.

Lacunar development was assesed from apical sections,
sections taken 10 cm below the shoot apex, and sections
taken 2 m below the apex. Experimental plants were used for
apical and lO-cm sections. Specimens for the 2-m sections
were collected from outdoor stock ponds, and sections were
made at the position just above the sediment surface.

Figure 9 depicts the lacunar development in the apical

Figure 9 .

49

Development of lacunar spaces in the meristem
region of Myriophyllum spicatum. A. Apical
cells, 0.04 mm below shoot apex; B. Transverse
stem cross-section, 0.4 mm below shoot apex;

C. Transverse stem cross-section, 0.7 mm below
shoot apex; D. Detail Of C, showing septum
cells.

 

0.4 mm

 

0.2 mm

0.1mm

0.4 mm

 

 

51

region. Tissues were poorly defined 40 um below the shoot
apex (Figure 9a). Lacunae first began to open at 200 um
below the apex; the 15 lacunae seen in mature stems were
all at least partially open 400 um below the apex (Figure
9b). The lacunar architecture seen in mature stem sections
was clearly evident 700 um below the apex. This stage is
shown in Figures 9c and 9d.

Stem sections taken 10 cm below the apex were about
1 mm in diameter, as shown in Figure 10. Structure of the
cortex in this region of the stem is depicted in Figure 11.
Each lacuna has a distinct arch of cells on the inner and
outer radii. Lacunae are bounded on the sides by septae
which have a load collector cell at the end joining two
outer arches, and a load distributor cell on the inside
joining two inner arches. Given a uniform radial load
applied externally (eg., hydrostatic pressure) the lacunar
arch system dissipates a portion of the load in the outer
and inner arches and redirects the remaining load safely
inward to avoid collapsing the lacunar air space. In all
regions of the cortex, the external load is directed such
that it tends to push cells toward each other rather than
pulling them apart. In a fashion analogous to the lacunar
arch system, the strength of arches in ancient Roman bridges
was determined by the strength of the bricks rather than
the mortar.

Sections from 2 m below the shoot apex were taken from

the outdoor stock pond and examined under a scanning electron

52

Figure 10. Transverse cross-section through stem
of Myriophyllum spciatum. Section taken

10 cm below shoot apex.

 

mm

 

54

.msoum Esueowmm ssNmemomsmz HO xOpHOO on» ma Sesame rose sersoeN one

.HH msswfim

moeaemema 3.0..

55.5%

mzzmmoim

(2304‘

flimuooozm. IO”?

0 o O o o O
u ..a . .
. ........

 

 

muzz.

 

 

 

 

 

 

Iom< mmhao

”5.53.50 9‘04

56

microscope (SEM). These sections were approximately 2 mm
in diameter; one is shown in Figure 12. In this region of
the stem, the septae are long and thin and the outer arches
are expanded, diminishing the load-distributing character
of the lacunar arch system relative to that seen in the
younger stems. Hasman and Inanc (1957) show the presence
of storied cork in mature stems of Myriophyllum spicatum.
The presence of storied cork cannot be confirmed for the
mature sections in this study since SEMjprocedures do not
distinguish secondary tissues. If present, however, storied
cork would enhance the strength of the stem against crush-
ing forces.

The continuity of the lacunar system was determined
from the Paraplast sections shown in Appendix IV. No con-
nection was found between lacunae in the leaflets, rachis
and stem sections. Along the stem, the lacunar system was
continuous except for partial interruptions by vascular leaf
traces at the nodes. In stem sections where adventitious
roots had developed, blockage of lacunae by roots was sub-
stantial. It is important to note, however, that adventi-
tious roots are usually only present in shoots beginning to

fragment from the parent plant.

57

.huama0>fisa madam
sawazofia as mofipao cosaOOAm you sowsoo one as oaoomosofis
:OAHOOHO msfisssow some: mosaedxm .xons one Beams E N

sons» assesses stNSxmoesmz mo :Oapoomummono Scum

.NH mesmHm

 

DISCUSSION

The principle hypothesis in this work held that hydro-
static pressure should limit the depth distribution of sub-
'mersed vascular hydrophytes by preventing the formation and
function of lacunar spaces. The hypothesis was not support-
ed by the results of this research; hydrostatic pressure
could not be shown to influence Myriophyllum spicatum shoots
in any of the observations made during this study. Three
issues must be pursued based on the observations given above;
(1) gas balances behaved as predicted by the diffusive gas
equilibrium model, (2) the Observation that lacunar storage
was not diminished by hydrostatic pressure suggests that
lacunar gas pressures were not controlled by hydrostatic
pressure, and (3) the influence of hydrostatic pressure on
mature shoots may not be reflected in the performance of
young shoots like those used in this study.

Two groups of observations supported the diffusive gas
equilibrium model. During net oxygen efflux experiments a
time-lag for equilibration of oxygen efflux occurred follow-
ing changes in light level. This was shown in Figure 7.

The diffusive gas equilibrium model showed that a back-pres-
sure for oxygen movement must be established as oxygen exits

plant surfaces, allowing accumulation of oxygen in lacunar

59

60

air spaces. Any change in light level caused a like change
in the net photosynthetic rate and a concomitant change in
back-pressure for oxygen accumulation. Before stable oxygen
efflux was achieved, the boundary region and lacunar air
spaces must have reached equilibrium oxygen levels. In the
case of decreasing light between experiments, this entailed
unloading of the lacunae, cell sap and boundary region oxygen
to the new, lower oxygen equilibrium levels. Oxygen efflux
during unloading was higher than the net photosynthetic rate.
Following an increase in light, oxygen efflux was lower than
the net photosynthetic rate until the system was loaded to
its equilibrium level. This followed from Equation 6; stable
efflux was not achieved until the A CONCENTRATION value
reached its equilibrium level following filling of the
gradient with oxygen.

Westlake (1978) observed similar equilibration lags in
Myriophyllum spicatum and Vallisneria americana Michx.,
both lacunate hydrophytes. Net oxygen efflux values were
initially depressed following the start of a light period.
After a mean lag of 9.5 min, stable oxygen efflux was ob-
served. Equilibration lags were also observed following
rapid flushing with water at atmospheric saturation. Break-
down of the still boundary region forced unloading of the
lacunae, cell sap and boundary region oxygen. After flush-'
ing was complete, oxygen efflux lagged while oxygen levels
were re-established in the three compartments.

The relation between ambient oxygen concentration and

61

lacunar storage indices developed in this study also follow-
ed from the diffusive gas equilibrium model. By allowing
oxygen levels in the boundary region to build during extend-
ed stagnation, back-pressure for lacunar oxygen storage
increased. These results confirm the notion that lacunar
oxygen storage should be enhanced in stagnant waters where
boundary layer thickness is greater than in flowing water.
For example, lacunar storage must be enhanced in the massive
mats of Myriophyllum spicatum which appear near the water
surface in summer.

Given the diffusive gas equilibrium model, it was
expected that hydrostatic pressure would act to diminish or
eliminate lacunar gas storage. This prediction was based
on the notion that gases would exit the lacunar spaces
whenever internal gas partial pressures exceeded partial
pressures at a plant's exterior surface. If internal gas
pressures were determined by hydrostatic pressure as suggest-
ed in the literature (of. Dale, 1957; Sculthorpe, 1967),
outward diffusion would have depleted lacunar oxygen levels,
except possibly at the highest net photosynthetic rates.
Failure of this prediction forced consideration of the
possibility that lacunar gas pressures were independent of
prevailing hydrostatic pressures.

Dale (1957) showed that rates of lacunar formation in
the apical region of Elodea canadensis were directly related
to illumination. Dale (Ibid.) and Sculthorpe (1967) con-

cluded that oxygen evolved by the photosynthetic hydrolysis

62

of water pressurized lacunae and forced their expansion. TO
force expansion at depth, lacunar gas pressures must exceed

total pressures (Pa + Ph) forcing inward on plant surfaces.

tm
But it is also possible that growth of cells in the apical
region causes lacunar formation. If cells in the apex were
appropriately positioned and shaped, expansion of the cells
by turgor pressure during normal growth processes would form
and expand lacunar spaces. Dale (1957) and Sculthorpe (1967)
did not give cross-sectional anatomy for developing Elodea
canadensis apices. However, shoot apices of Myriophyllum
spicatum examined during this study showed a distinctive
cellular arrangement, the lacunar arch system. The lacunar
arch structure may force expansion of lacunar spaces without
gas pressurization.

Septae in stem sections of Myriophyllum spicatum enlarg-
ed primarily by cell expansion and to a lesser extent by cell
division. At 0.7 mm below the shoOt apex (Figures 9C and
9D) the septum was composed of 3-4 cells averaging 3 um in
length along the stem radius. Septae from 10 cm below the
apex were composed Of 4-5 cells, 25 um in length (Figure 10).
Sections from outdoor stock ponds taken 2 m below the shoot
apex had septae composed of 9-10 cells averaging 35 um in
length along the stem radius (Figure 12). Cell expansion
is driven by turgor pressurization combined with relaxation
of cell wall fibers (Salisbury and Ross, 1978). Sculthorpe
(1967) pointed out that cell turgor pressures are generally

high in young hydrophyte tissues, increasing as metabolic

63
activities elevate sugar and ion levels. Turgor-driven cell
expansion can progress independently from gas pressurization
of lacunar spaces. Increased metabolic activity associated
with increased illumination may explain the correlation
between illumination and lacunar expansion rates found in
Elodea canadensis by Dale (1957).

The lacunar arch system in Myriophyllum spicatum
allows the expansions and divisions of lacunar development
to proceed against substantial external loads. In this
study, normal lacunar development was observed in shoots
grown under 83 kPa hydrostatic pressure. As long as the
lacunar arch system dissipates hydrostatic pressure loads,
stems of Myriophyllum spicatum should be able to maintain
lacunar gas pressures near atmospheric levels (101 kPa),
regardless of their depth Of immersion.

Lacunae in leaf tissues of Myriophyllum spicatum were
not as well developed as stem lacunae. But oxygen efflux
measurments made during this study provide evidence that
leaf lacunar gas pressures also could not have matched pre-
vailing hydrostatic pressures. Equation 6 showed that mag-
nitude of the oxygen gradient at steady state, A CONCENTRA-
TION (AC), is determined by efflux of oxygen from a specified
surface area and resistance to oxygen movement, R, through
the boundary region. Having made net oxygen efflux measure-
ments for shoots of Myriophyllum spicatum, it is only
necessary to specify values for the remaining variables in

Equation 6 to estimate values for AC.

64

According to Campbell (1977), resistance to gas move-
ment, R, is a function of thickness of the boundary region,
shape of the diffusing body and the diffusion coefficient
for oxygen in water. For a leaflet of Myriophyllum spicatum

(a cylindrical source),

-2. 2.
R - D 1na (EQN 15)

where a is the radius of the leaflet cross-section, D is the
diffusion coefficient for oxygen in water (0.002 mmZ-sec"1
at 20 C) and b is the sum of the leaflet radius, a, and the

boundary layer thickness. The surface area, S, for a leaflet

section of length l is given by:
S = ZN-a-l (EQN 16)

Rearranging Equation 6,
AC (mgoz.mm—3) 3 FLUX (mgOz-sec'1)- R (sec-mm'l) (EQN 17)
S (mmz)
For Myriophyllum spicatum plants growing at PAR levels
between 150 and 170 uE-m'z-sec'1 , the leaflet radius aver-
aged 0.2 mm. 0n the assumption that all photosynthesis
occurred in leaflet tissues, the oxygen efflux for a leaflet
section 1 mm long was approximately 8.4 x 10"9 mgOz-sec-1.

Substituting for FLUX, a and l, and converting from mm3 to

liters,
AC (mg02-1‘1) = 0.667-ln(UE§) (EQN 18)

Values for b, the sum Of leaflet radius, a, and boundary

65

layer thickness, can be specified to develop values for AC.
Browse, et al., (1979) and Smith and Walker (1980) pointed
out that boundary-thickness for macroscopic objects in water
must be at least 10 um in highly stirred systems and no more
than 500 um in "still" water. Equation 18 was plotted in
Figure 13 for boundary thickness ranging from 0 to 1,000 um.
These results indicate that even if boundary layer thickness
reached 1 mm, leaflets of Myriophyllum spicatum cannot
develop oxygen partial pressures at the outer plant surface

greater than 2 kPa above atmospheric saturation, when light

levels are below 170 uE-m'z-sec'l.

Westlake (1978) found that shoots of Myriophyllum spi-

2 1

catum were light-saturated at 1,000 uE-m‘ ~sec' ; oxygen
efflux at light saturation was 1.85 times greater than rates
measured at 150-170 ilE-m'z-sec"1 during this research. Sub-
stituting the light-saturated estimate for FLUX into Equa-

tion 17,
AC (mgOz-l-l) = 1.23-1n(59§) (EQN 19)

Equation 19 was also plotted in Figure 13, showing AC values
at the highest reported oxygen efflux for the species.

This exercise shows that even when oxygen efflux reaches
light-saturated levels and there is minimal water movement,
Myriophyllum spicatum leaflets cannot develop oxygen partial
pressures at the exterior surface greater than 5 kPa above
atmospheric saturation. As light levels (hence oxygen efflux)

decrease, oxygen partial pressures at a plant's surface

Figure 13.

66

Magnitude of the oxygen gradient outside leaflets
of Myriophyllum spicatum, as a function of boun—

dary layer thickness. A. Values calculated from

Equation 18, using oxygen efflux values at 150 -

170 nE-m"2-sec-1 PAR. B. Values calculated from

Equation 19 using estimated oxygen efflux at

1,000 uE°m-2-sec'1.

 

v _

0

1..
A.-H.~0msc one<eezmonO <

 

1.000

500

BOUNDARY LAYER THICKNESS (um)

68

also decrease. The cylindrical shape of Myriophyllum
spicatum leaflets allows rapid dispersion of photosynthetic
oxygen production, minimizing back-pressure for lacunar
oxygen accumulation. Like the stem lacunae, leaflet lacunae
in this species must be formed and held open by cell struc-
ture.

The continuity of stem lacunae in Myriophyllum spicatum,
shown in Figure A-4, provides the final evidence that gases
in the internal atmosphere of these plants are stored at

pressures less than the total pressures (Pa + Ph) forcing

tm
inward against plants' exterior surfaces. A plant rooted

at 4 m depth which reaches the water surface experiences

39 kPa hydrostatic pressure at its base and 0 kPa hydrostatic
pressure at its apex. Since the stem lacunar system is con-
tinuous, gas pressures must be the same throughout. In a
lacunar system held open entirely by gas pressurization, the
apical region would be required t0~h01d 39 kPa excess pres-
sure. Alternatively, if the lacunar arch system held the

gas spaces open, it would be required to hold against 39

kPa hydrostatic pressure just above the sediment surface.
Partial excess gas pressurization resulting from photosyn-
thetic oxygen production would lead to slight over-pressur-
ization at the apex and reduced under-pressurization at the
stem base. Oxygen diffusion considerations given above show
that it is unlikely these plants can develop excess oxygen

pressures as high as 39 kPa. Considering this, significant

under-pressurization must occur in the basal portions of

69

stems greater than 2 m in length.

The strength of the lacunar arch system is lower in
basal portions of long stems than in younger sections. The
load-bearing character of the lacunar arch system is deter-
mined partly by the ratio of length to width for the septae.
As length for a column increases relative to its width, its
load-bearing ability decreases (Baumeister, 1958). This
ratio increased from 1.0 in apical sections of Myriophyllum
spicatum (Figure 9D) to 11.0 in sections taken 2 m below
the shoot apex (Figure 12). Consequently, mature stem
sections near the sediment surface appear more likely to
collapse under hydrostatic pressure loads than younger,
apical sections. This research and Dale's (1981) incor-
porated young, apical shoot sections Of Myriophyllum spi-
catum, and both studies failed to link hydrostatic pressure
to depth limits for this species in lakes.

Mature root sections (Figure A-5A) also had lacunar
structures which were not suited to bear external crushing
forces. These lysigenous lacunae had thin septae but lacked
arch-like organization. Younger roots (A-5B) had small
lacunae which may stand greater external pressures than
mature sections, but expansion of these gas spaces by addi-
tional lysing weakens the strength of the cross-section.
Dale (1981) found that root growth in Myriophyllum spicatum
was significantly decreased at hydrostatic pressures greater
than 135 kPa, yet he concluded that hydrostatic pressure

probably had not limited depth distribution of this species

70

in lakes. Grace and Wetzel (1976) concluded that nutrient
uptake from the root system in this species was an important
aspect of its competitive ability. The depth limitation of
Myriophyllum spicatum by the action of hydrostatic pressure
on the plants' root system cannot be ruled out by existing
data.

Evidence from field studies (Golubic, 1963; Frantz and
Cordone, 1967; Spence, 1967; Sheldon and Boylen, 1977)
pointed toward a disparity between depth distributions of
lacunate and non-lacunate hydrophyte species. The principle
hypothesis of this study held that hydrostatic pressure acted
against the formation and function of lacunar spaces in the
lacunate hydrophytes, restricting their depth distributions.
The promotion of outward gas diffusion was proposed as the
mechanism for lacunar disruption by hydrostatic pressure.
The results of this research have shown that hydrostatic
pressure did not promote outward gas diffusion because gases
were stored at pressures which were independent of hydro-
static pressure. This was made possible by the lacunar
arch system, which bore the load of hydrostatic pressure,
avoiding the compression of lacunar gases within the range
of pressures simulated. These results could not be general-
ized to mature plants in natural settings. The lacunar
arch system in mature stem sections was weaker against
external crushing forces than in apical shoot material used
for this research. The root system may also be susceptible
to crushing by hydrostatic pressure; data given by Dale (1981)

support this contention.

LITERATURE CITED

LITERATURE CITED

American Public Health Association. 1975. Standard Methods
for the Examination gf‘Water and WasteWater. 14th ed.
Washington, D.C., American Public Health Association.
1193 pp.

 

 

Arnon, D.I. 1949. Copper enzymes in isolated chloroplasts.
Polyphenoloxidases in Beta vulgaris. Plant Physiol.
24:1-15.

Baumeister, T. 1958. Mark's Mechanical Engineers' Handbook.
6th ed. New York, McGraw-Hill. 2278 pp.

Bodkin, P.C., U. PosluSzny, and H.M. Dale. 1980. Light and
pressure in two freshwater lakes and their influence
on the growth, morphology and depth limits of Hippuris
vulgaris. Freshwater Biol. 10:545-552.

Bonner, J., and J.E. Varner. 1976. Plant Biochemistry. New
York, Academic Press. 925 pp.

Browse, J.A., F.I. Dromgoole, and J.M.A. Brown. 1979. Photo-
synthesis in the aquatic macrophyte Egeria densa. III.
Gas exchange studies. Aust. J. Plant Physiol. 6:499-
512.

Campbell, G.S. 1977. Ag Introduction £2 Environmental Bio-
physics. New York, Springer-Verlag. 159 pp.

Crank, J. 1956. The Mathematics 23 Diffusion. Oxford, Clar-
endon Press. 341 pp.

 

Dale, H.M. 1957. Developmental studies of Elodea canadensis
Michx. I. Morphological development at the shoot apex.
Can. J. Bot. 35:13-24.

1981. Hydrostatic pressure as the controlling fac-
tor in the depth distribution of eurasian watermilfoil,
Myriophyzzum spicatum L, Hydrobiologia 79:239-244.

 

Fehrling, E. 1957. Die Wirkungen des Erhoten hydrostatischen
Druckes auf Wachstum und Differenzierung submerser
Blutenpflanzen. Planta 49:235-270.

72

73

Frantz, T.C., and A.J. Cordone. 1967. Observations on deep-
water plants in Lake Tahoe, California and Nevada.
Ecology, 48:709-714.

Gessner, F. 1952. Der Druck in seiner Bedeutung fur das Wach-
stum submerser Wasserpflanzen. Planta, 40:391-397.

Golubic, S. 1963. Hydrostatischer Druck, Licht, und submerse
Vegetation im Vrana-See. Int. Rev. gesamten Hydrobiol.
48:1-7.

Grace, J.B., and R.G. Wetzel. 1976. The production biology of
eurasian watermilfoil (Myriophyllum spicatum L.): a
review. J. Aquat. Plant Manag. 16:1-11.

Hartman, R.T., and D.L. Brown. 1967. Changes in internal
atmosphere Of submerged vascular hydrophytes in rela-
tion to photosynthesis. Ecology, 48:252-258.

Hasman, M., and N. Inanc. 1957. Investigations on the anatom-
ical structure of certain submerged, floating and am-
phibious hydrophytes. Istanb. Univ. Fen Fak. Mecm.,
ser. B, 22:137-153.

Hiscox, J.D., and G.F. Israelstam. 1979. A method for the
extraction of chlorophyll from leaf tissue without
maceration. Can. J. Bot., 57:1332-1334.

Hutchinson, G.E. 1957. A Treatise 92 Limnology. I. Geography,
Physics, and Chemistry. New York, John Wiley and Sons.
1015 pp. '

 

. 1975. A Treatise 9g Limnology. III. Limno-
logical Botany. New York, john Wiley and Sons. 660 pp.

 

 

Lim, P.G. 1976. A field experiment with granular 2,4-D for
control of eurasian watermilfoil, 1976. Studies on
aquatic macrophytes, Pt. 10. British Columbia, Water
Investigations Branch, Report No. 2613. 105 pp.

Payne, F.C. 1977. Floristics of Harding Lake, Alaska. M.S.
Thesis. Michigan State University. 99 pp.

Pearsall, W.H. 1920. The aquatic vegetation of the English
Lakes. J. Ecol., 8:163-199.

Pond, R.H. 1905. The relation of aquatic plants to the sub-
stratum. Rep. U.S. Fish. Comm., 21:483-526.

Salisbury, F.B., and C.W. Ross. 1978. Plant Physiology. Bel-
mont, California, Wadsworth Pub. Co. 436 pp.

74

Sass, J.E. 1951. Botanical Microtechnique. Ames, Iowa, The
Iowa State Univ. Press. 228.

 

Sculthorpe, C.D. 1967. The Biology 9; Aquatic Vascular Plants.
London, Edward Arnold, Ltd. 610 pp.

 

 

Sheldon, R.B. and C.W. Boylen. 1977. Maximum depth inhabited
by aquatic vascular plants. Amer. Midl. Nat., 97:248-
254.

Smith, F.A., and N.A. Walker. 1980. Photosynthesis by aquatic
plants: effects of unstirred layers in relation to
assimilation of C02 and HCOa‘ and to carbon isotopic
discrimination. New Phytol. 86:245-259.

Spence, D.H.N. 1967. Factors controlling the distribution of
freshwater macrophytes with particular reference to the
lochs of Scotland. J. Ecol., 55:147—170.

Spence, D.H.N. and J. Chrystal. 1970a. Photosynthesis and
zonation of freshwater macrophytes. 1. Depth distri-
butions and shade tolerance. New Phytol., 69:205-215.

1970b. Photosynthesis and

zonation of freshwater macrophytes. II. Adaptability
of species of deep and shallow waters. New Phytol.,

69:217—227.

 

Stross, R.G. 1979. Density and boundary regulations of the
Nitella meadow in Lake George, New York. Aquat. Bot.,
6:285-300.

Vidaver, W. 1972. Effects of pressure on the metabolic pro-
cesses of plants. In: M.A. Sleigh and A.G. MacDonald,
eds., The effects of pressure on organisms. Proc.
Symp. Soc. Exp. Biol., 26:159-174.

Westlake, D.F. 1978. Rapid exchange of oxygen between plant
and water. Verh. Int. Ver. Limnol., 20:2363-2367.

Wilson, L.R. 1935. Lake development and plant succession in
Vilas County, Wisconsin. Pt. I. The medium hard water
lakes. Ecol. Monogr., 5:207-247.

1937. A quantitative and ecological study of the
larger aquatic plants of Sweeny Lake, Oneida County,
Wisconsin. Bull. Torrey Bot. Club, 64:199-208.

 

1941. The larger aquatic vegetation of Trout
Lake, Vilas County, Wisconsin. Trans. Wisconsin Acad.
Sci., Arts, Lett., 33:135-146.

 

APPEND I CE 8

APPENDIX I

SPECTRAL COMPOSITION OF HIGH INTENSITY DISCHARGE
SODIUM LAMPS AND FOUR NATURAL LIGHT ENVIRONMENTS

75

Figure A1.

76

Spectral characterization of photosynthetically
active radiation (PAR) for four sources: 1. Clear-sky
terrestrial solar curve at East Lansing, Michigan,

at noon on the summer solstice (‘7); 2. Lake George,
New York, at 10 m depth under clear sky (0); 3. Lake

Michigan off Grand Haven at 10 m depth under high

overcast (t3); 4. Lawrence Lake, Barry County, Michigan,

at 10 m depth during July, under clear sky ((3).

Each curve is adjusted to a total PAR of 1,000

l

uE-m‘Z-sec- for comparison.

 

 

15 7-

 

A ~IE:._IOOm.~uE.m—: V
wfimmzma xDAm :DBZ¢DG

WAVELENGTH (nm)

78

Figure A2. Spectral characterization of photosynthetically
active radiation (PAR) emitted by high intensity

discharge (HID) sodium lamps. The curve is

adjusted for total PAR of 1,000 uE-m"2-sec‘1

154-

db

db

+

‘I

 

 

 

10 —r-

A _IE:.

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700

600

500

400

WAVELENGTH (nm)

APPENDIX II

RESULTS OF PRELIMINARY EXPERIMENTS ON THE INFLUENCE

OF EXTERNAL OXYGEN CONCENTRATION ON LACUNAR OXYGEN

STORAGE IN MYRIOPHYLLUM SPICATUM.

80

81

Table A1. Results of preliminary experiments on the
influence of external oxygen concentration on
lacunar oxygen storage. Experiment 1: all
chambers were closed for oxygen accumulation
during the final three hours of the light period,

then were flushed to ambient oxygen level.

NET OXYGEN INFLUX
mgOz-h'I-g'lDW

 

 

CHAMBER" 'HOUR’l” "HOUR'2' HOUR 12
1 0.273 0.545 0.455
2 0.200 0.400 0.440
3 0.232 0.588 0.510
4 0.000 0.434 0.528
5 0.310 0.310 0.000
6 +0.276+ 0.423 0.331
7 0.000 0.527 0.344
8 0.000 0.483 0.420
1

Positive influx value indicates oxygen was
released during the first hour of darkness.

Table A2.

82

Results of preliminary experiments on the
influence Of external oxygen concentration on
lacunar oxygen storage. Experiment 2: chambers
2, 3, 6 and 7 were closed for the final 8 hours
of the light period, then were flushed to ambient
oxygen concentration. The remaining 4 chambers
flowed at 150 ml-min‘1 throughout the light period.

NET OXYGEN INFLUX
mgOz 'h-l -g-1DW

 

CHAMBER HOUR 1 HOUR 2
1 0.364 0.636
2 +0.267+ 0.200
3 +0.232f 0.387
4 0.283 0.660
5 0.517 0.000
6 +0.092'r 0.276
7 0.229 0.573
8 0.839 1.049

 

+Positive influx value indicates oxygen
was released during the first hour of
darkness.

Table A3.

83

 

Results of preliminary experiments on the
influence of external oxygen concentration on
lacunar oxygen storage. Experiment 3: Chambers
2, 3, 6 and 7 were closed for the final 8 hours
of the light period, then were flushed to ambient
oxygen concentration. Chambers 1, 4 and 8 flowed
at 150 ml-min'1 throughout the light period;
chamber 5 flowed at = 5 ml-min"1 throughout the
light period.
NET OXYGEN INFLUX
mgOz-h‘log'lDW
CHAMBER ‘ "HOUR’1“‘ "HOUR’2‘ 'HOUR 3

1 0.545 0.727 0.727

2 0.000 0.533 0.400

3 0.000 0.464 0.619

4 0.943 0.943 0.943

5 0.414 0.207 0.000

6 0.000 0.368 0.368

7 0.458 0.229 0.229

8 1.049 1.049 1.049

 

APPENDIX III
RESULTS OF EXPERIMENTS ON THE INFLUENCES OF HYDRO-

STATIC PRESSURE AND LIGHT ON NET OXYGEN EFFLUX

AND LACUNAR OXYGEN STORAGE IN MYRIOPHYLLUM SPICATUM.

84

85

 

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Table A8. Estimates of fresh weight (FW) and dry weight
(DW) for shoots involved in lacunar storage

experiments at 67 kPa above atmospheric pressure.

 

 

PLANT. . , FW (g) . DW (g)
1 0.697 0.184
2 0.501 0.113
3 0.621 0.148
4 0.491 0.096
5 0.502 0.091
6 0.517 0.099
7 0.555 0.104
8 0.560 0.129

 

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APPENDIX IV

RESULTS OF ANATOMICAL STUDIES OF MYRIOPHYLLUM

SPICATUM.

97

98

Figure A3. Typical leaf structure in Myriophyllum spicatum.
A. Transverse cross-section through rachis; lacunae
are seen as clear areas in the mesophyll.
B. Transverse cross-section through leaflet; lacunae
are seen as small gaps between mesophyll cells.

 

0.5 mm

 

T

   

0.5mm

Figure A4.

100

Typical configuration of a node in the stem of
Myriophyllum spicatum, demonstrating the maximum
constriction of the stem lacunar system. A. Trans-
verse cross-section through node showing leaf
traces; notice that rachis lacunae are also visible,
but do not join stem lacunae. Leaf traces decrease
lacunar cross-section by 40%. B. Transverse cross-
section above node at the point where adventitious
roots exit the stem. Lacunar cross-sectional area
is reduced 60-70% in this section. No lacunae are
visible in the root. C. Longitudinal cross-section
through node, showing leaf traces crossing through
lacunae.

 

mm

Figure A5.

102

Transverse cross-sections through Myriophyllum
spicatum roots. A. Mature primary root, showing
large, lysigenous 1acunar_spaces. Notice that

the lacunar arch structure visible in stem sections
and, to a lesser extent in the rachis secitons, is
not apparent in the roots. B. Young primary root,
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cells).

 

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