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Thisisto certify that the
dissertation entitled
STRUCTURAL ADAPTATIONS OF SUBMERSED VASCULAR PLANTS TO
GAS EXCHANGE AND OXYGEN TRANSPORT

presented by

Jane Lenore Schuette

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MSU Is An Aﬁirmdive Action/Equal Opportunity Institution
chnS-pd

STRUCTURAL ADAPT ATIONS OF SUBMERSED VASCULAR PLANTS

TO GAS EXCHANGE AND OXYGEN TRANSPORT

By

Jane Lenore Schuette

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1990

I

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a

ABSTRACT

STRUCTURAL ADAPTATIONS OF SUBMERSED VASCULAR PLANTS
TO GAS EXCHANGE AND OXYGEN TRANSPORT

By

Jane Lenore Schuette

Submersed vascular plants differ in their ability to transport and release
photosynthetically-derived 02 from their roots. This study examines the structural
features of four morphologically distinct submersed species which may account for
differences in 0, transport known to exist between them. The high transport potential
of Lobelia may be due to a thick cuticle which promotes lacunar storage of 0,, a large
lacunar volume and a short continuous pathway between leaves and roots. Species with
lower transport potentials such as Elodea, Myn’ophyllwn and Potamogeton are
characterized by leaves with thin cuticles, smaller lacunar volumes and long transport
pathways. A hypodermis may protect buried roots from reduced phytotoxins and retard
02 loss.

The lacunar system formed a continuous pathway for gas transport throughout the
length of the stem, although constricted at the nodes by perforated diaphragms. Gas
transport studies showed that diaphragms provide little resistance to diffusion, but
considerable resistance to mass ﬂow. Diffusive resistances measured closely
approximated those predicted by Fick’s first law. As the porosity of the stem increased
from Elodea, Myriophyllum to Potwnogeton, resistance to diffusion decreased, thus

suggesting an increasing ability to transport 0, to the roots among these species. The

Jane Lenore Schuette

results suggest that the 0, transport requirements of the species examined may, during
periods of active photosynthesis, be satisﬁed by diffusion alone. These ﬁndings also
suggest that plants with high internal resistances could not transport enough 02 by
diffusion alone to support a large root biomass.

Measured resistances to mass flow approximated those predicted by Hagen-
Poiseuille equation. Mass flow could occur in these plants under small pressure
differentials, since measured resistances were, with the exception of Elodea, quite low.
The signiﬁcance of mass ﬂow to 0, transport could not be determined.

A model is proposed which describes the distribution of rooted submersed
vascular plants in lakes with increasing sediment 0, demand. It suggests that species
distributed along this gradient may be characterized by a decrease in lacunar
development, relative root production and their dependency on the sediments as a site of

nutrient uptake.

ACKNOWLEDGMENTS

I gratefully acknowledge the encouragement and support from my advisor, Dr.
Karen Klomparens and the technical assistance of everyone at the Center for Electron
Optics. It has also been a privilege to be associated with the Kellogg Biological Station
and I am especially grateful to Dr. Mike Klug for his invitation as well as his inspiration.
I would particularly like to thank Dr. Ken Poff for his advice, encouragement and
continued interest in my research and career. I would also like to acknowledge the
fruitful discussions with committee member Dr. Frank Ewers and with friends and
colleagues, Drs. Fred Payne, Rick Carlton, Mike Kaufman and Rich Losee. Finally, I
would like to thank my family, friends and aerobics for their support throughout this

endeavor.

iv

TABLE OF CONTENTS

LIST OF TABLES ...................................... vi
LIST OF FIGURES ...................................... vii
INTRODUCTION ....................................... 1
MATERIALS AND METHODS .............................. 5

CHAPTER 1. Leaf Anatomy: Structural Resistances to Gas Exchange . . . . 13

CHAPTER II. Anatomy of the Lacunar System: Structural Resistances to Gas

Transport ................................. 36
CHAPTER III. Root Anatomy: Structural Resistances to Gas Exchange ..... 64
CHAPTER IV. Resistance to Gas Transport ...................... 82
FINAL SUMMARY .................................... 117
LIST OF REFERENCES ................................. 125
APPENDIX 1. Standard Curve ............................ 136
APPENDIX 11. Predicted Estimates of Resistance ................. 138
APPENDDI III. Transport Studies in Potamogeton illinoensis ........... 141

Table 1.

Table 2.

Table 3.

Table 4.

Table 5 .
Table 6.

Table 7.

Table 8.

Table 9.

Table 10.
Table 11.
Table AII.1.

Table AII.2.

LIST OF TABLES

Anatomical data for stems of Elodea, Mfyn'ophyllwn and
Potamogeton. ................................. 48

Anatomical data for roots of Elodea, Myﬁophyllum and
Potamogeton. ................................. 49

Porosity values for stems and roots and relative root production of

species examined. .............................. 50
A comparison of the measured diffusive resistance (R, s cm") of

stem sections, with and without nodes, of three species. ....... 96
Statistical analysis of stems of M. spicatum. .............. 97
Diffusive resistances to gas ﬂow ...................... 99
Predicted estimates of resistance of nodes and intemodes to

diffusion. .................................. 101
Evaluation of resistance to diffusion. ................. 102

A comparison of the measured resistance to mass ﬂow (r, kPA s
cm") of stem sections, with and without nodes, of three species. 103

Resistances to mass flow of intemodes and nodes. ......... 104
Evaluation of resistance to mass ﬂow .................. 105
Anatomical characteristics of stems. .................. 139

Morphological and anatomical characteristics of
nodes/diaphrams .............................. 140

vi

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5 .
Figure 6.
Figure 7.
Figure 8.

Figure 9.

Figure 10.
Figure 11.
Figure 12.
Figure 13.
Figure 14.
Figure 15.
Figure 16.
Figure 17.

Figure 18.

LIST OF FIGURES

Cross-section of Elodea leaf. ....................... 23
Surface view of Elodea leaf. ....................... 23
Lower epidermis of Elodea leaf. ..................... 23
Lower epidermis of Elodea leaf. ..................... 23
Upper epidermis of Elodea leaf. ..................... 23
Upper epidermis of Elodea leaf. ..................... 25
Lower epidermis of Elodea leaf. ..................... 25
Lower epidermis of Elodea leaf. ..................... 25
Cross-section of Elodea leaf. ....................... 25
Cross-section of Elodea leaf. ..................... 25
Cross-section of Myriophyllum leaﬂet ................... 27
Cross-section of Myriophyllum rachis. .................. 27
Surface of Myn’ophyllum leaﬂet. ..................... 27
Epidermis of Myn’ophyllwn leaﬂet. ................... 27
Epidemiis of Myriophyllum leaﬂet. ................... 27
Mesophyll of Myn’ophyllwn leaﬂet. ................... 27
Mesophyll of Myn'ophyllum leaﬂet. ................... 27
Cross-section of Potamogeton leaf blade. ................ 29

vii

Figure 19.
Figure 20.
Figure 21.
Figure 22.
Figure 23.
Figure 24.
Figure 25.
Figure 26.
Figure 27.
Figure 28.
Figure 29.
Figure 30.
Figure 31.
Figure 32.
Figure 33.
Figure 34.
Figure 35.
Figure 36.
Figure 37.
Figure 38.
Figure 39.

Figure 40.

Cross-section of Potamogeton leaf at midvein .............. 29
Epidermal cell of Potamogeton leaf. ................... 29
Epidermal cell wall of Poramogeton leaf. ................ 29
Cross-section of Potamogeton leaf. ................... 29
Cross-section of Potamogeton leaf. ................... 29
Cross-section of Lobelia leaf. ....................... 31
Cross-section of Lobelia leaf. ....................... 31
Surface of Lobeh'a leaf. .......................... 31
Surface of Lobelia leaf. .......................... 31
Epidermis of Lobelia leaf. ......................... 31
Cross-section of Lobeh'a leaf. ....................... 31
Mesophyll of Lobelia leaf. ......................... 31
Cross-section of Elodea stem. ....................... 47
Cross-section of Elodea stem. ....................... 47
Cross-section of Elodea root. ....................... 47
Longitudinal section of Elodea stem. .................. 47
Cross-section through node of Elodea stem. .............. 47
Cross-section through node of Elodea stem. .............. 47
Cross-section of Myriophyllwn stem. .................. 52
Cross-section of Myriophyllwn root. ................... 52
Cross-section through node of Myriophyllum stem. .......... 52

Longitudinal section through node of Myn'ophyllwn
stem. ...................................... 52

Figure 41.

Figure 42.

Figure 43.
Figure 44.
Figure 45.
Figure 46.
Figure 47.
Figure 48.
Figure 49.
Figure 50.
Figure 51.
Figure 52.
Figure 53.
Figure 54.
Figure 55.
Figure 56.
Figure 57.
Figure 58.
Figure 59.
Figure 60.
Figure 61.

Figure 62.

Cross-section through node of Myriophyllwn stem. .......... 52
Cross-section through Myn'ophyllum rhizome at the junction of a

root. ...................................... 52
Cross-section of Potamogeton stem. ................... 54
Cross-section of Potamogeton stem. ................... 54
Cross-section of Potamogeton stem. ................... 54
Cross-section of Potamogeton root. ................... 54
Cross-section of Potamogeton stem. ................... 54
Cross-section through node of Potamogeton stem. .......... 56
Cross-section through node of Potamogeton stem. .......... 5 6
Cross-section of Potamogeton stem. ................... 5 6
Cross-section of Potamogeton stem. ................... 56
Cross-section of Potamogeton rhizome. ................. 56
Cross-section of Potamogeton rhizome. ................. 56
Cross-section of LobeIia stern. ...................... 58
Cross-section of LobeIia stem. ...................... 58
Cross-section of LobeIia stern. ...................... 58
Cross-section of Lobelia root. ....................... 58
Cross-section of Elodea root. ....................... 72
Surface of Elodea root ............................ 72
Epidermis of Elodea root. ......................... 72
Epidermis of Elodea root. ......................... 72
Cross-section of Elodea root. ....................... 72

Figure 63.
Figure 64.
Figure 65.
Figure 66.
Figure 67.
Figure 68.
Figure 69.
Figure 70.
Figure 71.
Figure 72.
Figure 73.
Figure 74.
Figure 75.
Figure 76.
Figure 77.
Figure 78.
Figure 79.
Figure 80.
Figure 81.
Figure 82.
Figure 83.

Figure 84.

Cross-section of Elodea root. ....................... 72
Cross-section of Myﬁophyllum root. ................... 74
Cross-section of Myn'ophyllum root. ................... 74
Surface of Myriophyllum root. ...................... 74
Cross-section of Myriophyllum root. ................... 74
Cross-section of Myriophyllum root. ................... 74
Cross-section of Myriophyllwn root. ................... 74
Cross-section of Myriophyllum root. ................... 74
Cross-section of Potamogeton root. ................... 76
Surface of Potamogeton root. ....................... 76
Cross-section of Potamogeton root. ................... 76
Cross-section of Potamogeton root. ................... 76
Cross-section of Potamogeton root. ................... 76
Cross-section of Potamogeton root. ................... 76
Cross-section of Potamogeton root. ................... 76
Cross-section of Lobeh'a root. ....................... 78
Cross-section of LobeIia root. ....................... 78
Surface of LobeIia root. .......................... 78
Cross-section of Lobelia root. ....................... 78
Cross-section of Labelia root. ....................... 78
Cross-section of Lobelia root. ....................... 78

Regression of resistance per cm stem (R, s cm") on NBA (3 cm").

Figure 85.

Figure 86.

Figure AI.l.

Comparison of measured versus predicted diffusive resistance of
stems of four species examined. ....................

Model of distribution of submersed plants within lakes. ......

Standard curve used to estimate resistance to diffusion. ......

121

INTRODUCTION

Submersed vascular plants are exposed to two potential sources of nutrients— those
dissolved in the surrounding water and those in O, deﬁcient sediments. Roots buried in
these sediments require an alternate source of 0,. 0; transport from the shoots to the
roots is necessary not only to satisfy their respiratory demands but also, upon release,
to support an oxidized rhizosphere within the sediments (Sculthorpe, 1967; Hutchinson,
1975; Wetzel, 1975). The lacunar system is a network of intercellular gas spaces found
in leaves, stems and roots of aquatic vascular plants which performs two important
functions in this process. It serves as a reservoir for 0, storage within the plant and
provides a pathway for 0, transport throughout the plant body.

In a diurnal study, Hartman and Brown (1967) showed that 0, produced during
photosynthesis accumulates within the lacunar atmosphere of submersed plants before it
is released to the surrounding water. 02 stored within the lacunar system of the shoots
can be transported to rhizomes and roots and ean also be released to the sediments
(Oremland and Taylor, 1977). Release of 0, from the roots of submersed plants is
known to be much greater in the light than in the dark (Sand-Jensen et al., 1982; Sand-
Jensen and Prahl, 1982; Carpenter et al., 1983; Smith et al., 1984; Kemp and Murray,
1986), thus implying that most of the O, transported to the roots is of direct

photosynthetic origin. It is generally accepted that, on a diurnal basis, respiration in the

2
rhizomes and roots is supported primarily by the photosynthetic activity of the shoots
(Sculthorpe, 1967; Wetzel, 1975; Smith et al., 1984).

Submersed macrophytes have been shown to differ in their lacunar storage and
transport potentials. Among eight species investigated, Sand-Jensen et al. (1982) found
marked differences in the amount of 02 released by the roots relative to that released by
the shoots. In the isoetid species examined, Lobelia dormanna, the amount of 0,
released by the roots as a percentage of total shoot plus root release was high (100%).
This value was low (1-4 96) in the other species investigated where release across the
shoots was favored. These results suggest that submersed vascular plants may possess
different morphological and structural features which inﬂuence the exchange of gases
between the plant and its surroundings.

The exchange of 0, between the lacunar atmosphere and surrounding water can

most simply be described by Fick’s ﬁrst law of diffusion (Armstrong, 1979):

Flux = O, Gradient x W

Resistance

Assuming a constant 0; gradient, the rate of ﬂux across either the shoots or the roots is
thus determined by the surface area and the resistance of the tissue to diffusion. From
a morphological/anatomical standpoint, high release rates of 02 across the roots may be
favored by a combination of the following features:

1) a large surface area of shoots relative to roots

2) a high resistance across leaves

3) a low resistance across roots

4) a lacunar pathway of low resistance.

Release of 02 across the shoots may be facilitated by:

3
1) a large surface area of roots relative to shoots

2) a low resistance across leaves
3) a high resistance across roots

4) a lacunar pathway of high resistance.

Objectives

The purpose of this research was to evaluate differences in the lacunar 02 storage
and transport potentials of submersed vascular plants on the basis of their anatomical
and/or structural features. If a primary function of the lacunar system is to transport 0,
to the roots, one may expect to ﬁnd that differences between submersed species in their
ability to transport 02 may be related to the relative amount of roots that species typically
produces. Four submersed species with known differences in gas exchange
characteristics, as well as contrasting morphologies, root: shoot ratios and distributional
preferences, have been selected for examination to determine the following:

1). What is the general anatomy of the leaves and what features do they
possess that may inﬂuence gas exchange?

2). What is the general anatomy of the lacunar system of the selected species
and what, if any, are the relative differences in the extent of lacunar
development?

3). What is the general anatomy of the roots and what features do they
possess that may inﬂuence gas exchange?

4). What inﬂuence does the structure of the lacunar system have on

mechanisms of gas transport?

4
Each objective is presented in a separate chapter. These chapters are organized
in the sequence of events in which 02 transport occurs, namely: lacunar storage of O,
in leaves (Chapter I); long-distance transport of 0, from the leaves to the roots (Chapter
II); and release ofO, from the roots (Chapter III). In the ﬁnal chapter (Chapter IV), the
inﬂuence of lacunar structure on the ability of submersed plants to transport 0, is
examined. These chapters are then synthesized in a ﬁnal summary which examines the

physiological and ecological signiﬁcance of whole plant strategies to gas exchange.

MATERIALS AND METHODS

Species examined.

The species selected for anatomical and ultrastructural examination include Elodea
canadensis, Myn'ophyllum heterophyllum, Potamogeton praelongus and Lobelia
dortmanna. Hereafter these species are refered to by their generic names, unless it is

necessary to distinguish them from other members of the same genus.

Elodea canadensis

Elodea cwwdensis Rich (Hydrocharitaceae; Monocotyledoneae), a common
aquatic species, is often abundant in small ponds and reservoirs, forming either ﬂoating
mats or dense stands which are sparsely rooted to the sediments (Hutchinson, 1975). It
consists of a segmented stem which is produced by an apical meristcm in a series of
nodes and intemodes. Intemodes are cylindrical in shape, 0.5-2.0 mm in diameter, and
decrease both in length and width towards the shoot apex. Leaves, roots and branches
emanate from the stem as nodal outgrowths (Dale, 1957a). Leaves are linear-lanceolate
in shape, 6-12 mm long, and possess a single midvein. They are sessile, only slightly
constricted at the base and are found most often in whorls of 3. An underground.
rhizome is not produced; roots arise adventitiously from the nodes of the stem and

account for less than 3% of the total plant biomass (Borutskii, 1950 in Westlake, 1965).

6
They are unbranched, often over 20 cm in length and less than 1 mm in diameter. The

mature plant commonly produces both sediment and water roots. Water roots are light
green in color and are devoid of root hairs; those penetrating the sediments are white and

develop an abundant supply of root hairs (Cormack, 1937).

Myriophyllwn heterophyllwn

Myriophyllwn heterophyllum Michx. (Haloragaceae; Dicotyledoneae), commonly
called water-milfoil, is a perennial aquatic which often exhibits a heterophyllous
condition. This species has recently become abundant in New England where it grows
aggressively in ponds, lakes and streams of low alkalinity. To the west, it typically is
found in waters of high alkalinity (Crow and Helquist, 1983). At maturity, it is
characterized by a rhizotomatous growth habit. New upright stems arise from
overwintering buds which develop either at the base of old stems or on the rhizome.
Stems are long and ﬂexuous and are divided into nodal and intemodal regions.
Intemodes are cylindrical in shape, range from 1-4 mm in diameter, and decrease both
in length and width towards the shoot apex. Leaves are found in groups of 4 at the node.
Submersed leaves are highly dissected and pinnately compound with usually 7-10 pairs
of ﬁne, nearly cylindrical leaﬂets that are arranged ﬂat on the rachis, tapering in length
towards the apex of the leaf. They typically measure less than 1 cm in length and 0.5
mm in diameter. The rhizome is usually short and compact, producing slender and
unbranched adventitious roots at the nodes. Both the roots and rhizome are buried in the
sediments and, in related members of this genus, account for roughly 10% of the total
plant biomass (Nicholson and Best, 1974; Chambers and Kalff, 1985). The roots

cemmonly measure up to 20 cm or more in length and less than 1 mm in diameter.

7
Young buried roots appear silver-grey in color and become dark brown to black at

maturity. Water roots, light green in color, are often produced at nodes of the stem and

are positioned well above the sediment surface.

Potamogeton praelongus

Potamogeton praelongus Wulfen (Potarnogetonacceae; Monocotyledoneae) is a
common deep water annual of temperate lakes (Spence and Crystal, 1970). It consists of
a buried horizontal rhizome system which is segmented and produces long upright leafy
stems and numerous adventitious roots at the nodes. The stem is differentiated into
nodes and intemodes. Intemodes are cylindrical in shape, range from 1.5-4 mm in
diameter and decrease in both length and width towards the shoot apex. Leaves are
produced by an apieal meristcm in alternate arrangement at the nodes. They are oblong-
ovate in shape and commonly measure 10-15 cm long and 2 cm at the widest point. The
venation is characterized by a wide (1-2 cm) central midvein with 5-8 minor midveins
and ﬁbrous ribs on either side. The leaves are sessile and subcordate. Fibrous stipules
are formed in the leaf axils. The root/rhizome system is extensive; in related members
of this genus, it accounts for 12% (Nicholson and Best, 1974) to 40-50% (Ozimek et al.,
1974) of the total plant biomass. Both roots and rhizome are cream in color and mottled
with reddish-orange spots. The roots are slender and unbranched, often measuring 10
cm or more in length and less than 0.5 mm in diameter. Root hairs are usually present

(Shannon, 1953).

Labelia domnanna

Labelia domnanna L. (Lobeliaceae; Dicotyledoneae) is a small isoetid species that
is commonly rooted in shallow sandy areas of soft-water oligou'ophic lakes (Moyle,
1945). It is a relatively slow growing perennial which overwinters as an evergreen,
undergoing only small seasonal ﬂuctuations in total plant biomass (Moeller, 1978).
Leavesarestiffandtubulartostrap—likeinshapeandusuallymeasurelessthanScm
long and 1-3 mm wide. They are produced close together in a basal rosette and are
directly attached to a short (1-3 cm long), compact and unsegmented stem. The stem is
buried in the sediments and produces an abundant root system in close proximity to the
leaves. Usually 2-4 roots are associated with each leaf and are inserted into the stem on
each side of the leaf at its base. Roots are cream in color, unbranched, and typically less
than 10 cm long and 1 mm in diameter. Root hairs are usually not produced
(Senderng and Laegaard, 1977). The roots and stem collectively account for 50-65 %

of the total plant biomass (Sand-Jensen and Sandergaard, 1979).

Collection of plant material for microscopical examination.

Plants of Elodea, growing loosely attached to the sediment, were collected with
a rake from shallow water (1-2 m) in the outdoor experimental ponds at the Limnologieal
Research Laboratory and from ponds at the Waste Water Research Facility of Michigan
State University, Ingham County, Michigan.

Submersed plants of Myriophyllwn and Potamogeton, rooted in calcareous
sediments, were collected either by hand or with a rake in shallow (1-4 m) water at

Lawrence Lake, Barry County, Michigan.

9
Plants of Labelia, growing submersed (1-2 m) in coarse sandy sediments were

collected by hand in the St. Mary’s River, Chippewa County, Michigan. At the time
of collection, plants were gently freed from the sediments and transported to shore in
plastic bags containing lake water. Samples were prepared for microscopical examination
within 1 hour after collection. Despite care taken in handling, much of the loosely
attached periphytic community was dislodged from the plant, this study therefore

examines only the portion securely attached to the plant surface.

3. Tissues selected for microscopical examination.

Samples for anatomical observations were taken from leaves, roots and stems, as
well as the junctions of these organs. For ultrastructral examination, samples were
taken from the midsection of young, fully matured expanded leaf blades and young, fully

mature healthy roots.

Tissue preparation and microscopical examination.

For transmission electron microscopy (TEM), tissues were ﬁxed with cold 4 %
gluteraldehyde in 0.1 M phosphate buffer at pH 7.2 for 2-3 hours, washed twice in same
buffer, post-ﬁxed in 1% osmium tetroxide either for 1.5-2 hours at room temperature or
overnight at 4°C, dehydrated in a graded ethanol series (25%, 50%, 75%, 95%, 100%),
followed by increasing proportions of acetone in absolute ethanol (1:2, 2:1 and absolute)
and embedded in a 1:1 mixture of Spurr’s (1964) and Mollenhauer’s (1969) epoxy resin.
At various steps in the procedure, a vacuum was drawn to remove gases from gas spaces
within the tissue and to facilitate inﬁltration of reagents and resin. Polymerization was

for a minimum of 48 hours at 60°C. Ultrathin sections were cut with glass and diamond

10
knives and stretched with xylene. Gold to silver sections were picked up on copper mesh

grids, stained with uranyl acetate and lead citrate (Reynolds, 1963) and examined in a
Philips 201 Electron Microscope at 60 and 80 kV.

For scanning electron microscopy (SEM), tissues were typically ﬁxed and
dehydrated as above. Post-ﬁxation in osmium was eliminated in some samples. All
samples were dried via liquid C02, mounted on aluminum stubs, coated with gold and
examined in either an 181 Super-III or JEOL 35C Electron Microscope.

For light microscopy, tissues prepared for TEM were sectioned at 1-2 urn, heat
ﬁxed in 5 % acetone to glass microscope slides, and stained with toluidine blue (Peder
and O’Brien, 1968). Samples were also ﬁxed in formalin acetic acid (FAA), embedded
in paraplast, sectioned on a rotary microtome at 10-20 am, and double stained with

safranin and fast green as described by Johansen (1940).

Measurement of Resistance.

Sampler

Resistance estimates were determined using a simple sampler consisting of two
5 ml glass pipets capped with serum stoppers. Stems were inserted into tygon-tubing
collars ﬁtted around the dispensing end of the pipet and were sealed with vaseline to
prevent ﬂooding. The entire sampler was submersed in a water bath. Care was taken
to insure proper ﬁt. Improper ﬁt resulted in either damage to the stem or ﬂooding of the
sampler. Measurements were discarded when these problems were evident. Estimates
of resistance to both diffusion and mass ﬂow were made on the same stem section

without removal from the sampler.

1 1

Diffusion

Resistance to diffusion was determined by constructing a standard curve
(Appendix 1). Sections of glass capillary tubing of known resistance were inserted into
the sampler. Injections of tracer gas (0.1 ml of 10% CH., 90% Ar) were made into one
end to the sampler. After 2 hours, a 0.1 ml gas sample was extracted from the opposite
end. The CH. present in the sample was determined using a Varian gas chromatograph
equipped with a Porapak N column and operated at 50°C. The CPL present was
expressed as a percent relative to control samplers with no added resistance which were
run concurrently. Typically each run consisted of 6 samplers and 4 controls. Regression
analysis was performed on these data and a linear equation derived which describes
percent transported as a function of resistance (Appendix I).

Transport values were determined similarly for samplers containing stem tissues.
Estimates of diffusive resistance (R, s cm") obtained were divided by the length of stem

section and are expressed as resistance per cm stem.

Mass Flow

Mass ﬂow estimates were made prior to diffusion estimates. Gas tight, S-shaped
glass ports were connected to the submersed end of the sampler via 22 gauge needles.
The total volume of the mass ﬂow sampler was 38.5 ml. Injections of 0.1 ml room air
were made through a serum stopper at the emergent end of one port. A Validyne
pressure transducer was connected to a similar port at the opposite end of the pipet
sampler. The change in pressure within the sampler with time after injection was
displayed on a chart recorder. Both the time (t,,) required to reach half the ﬁnal

equilibrated pressure (.SAP) and the pressure at one half AP were determined. The

12
amount of gas that passed through the stem at AP was calculated and the volumetric ﬂow

rate (F, cm3 3") determined by dividing this amount by t 5. Resistance (r, kPA s cm")
was estimated using Hagen-Poiseuille equation (r=A P/F) and represents the mean of 3

individual runs. Estimates are expressed as resistance per cm stem.

Image Analysis

The length and diameter of stem sections were measured with a calipers and the
number of nodes and/or visible diaphragms were recorded.

For image analysis of stem gas space, freehand sections were cut with a razor
blade, mounted on glass slides and video-taped. Images of sections were divided into
quarters, using the center of the vascular cylinder as the midpoint. Images were
analyzed for radius, porosity, gas sapce area, lacunar size and number using a
Microscience Image Analysis System. Each estimate is based on the mean of 3
measurements. For image analysis of nodes and diaphragms, stem sections were ﬁxed
for SEM in 1% gluteraldehyde and processed as above.

Porosity values were also estimated from 35 mm photographs taken from thick
sections of stems and roots embedded for TEM. Gas spaces were delineated on each
photograph. The area occupied by gas space and by epidermal, cortical and vascular
tissues was determined with a planimeter. These areas are expressed as percent cross-

sectional area and represent the mean of 3 measurements.

CHAPTERI

LEAF ANATOMY: STRUCTURAL RESISTANCES TO GAS EXCHANGE

INTRODUCTION

Gases diffuse 2 or 3 orders of magnitude slower in water than in air (Leyton,
1975). In leaves of many submersed species, this physieal constraint has resulted in the
adaptation of several morphological and anatomical features which facilitate the exchange
of gases between the leaf and the surrounding water (Arber, 1920; Sculthorpe, 1967).
Leaves of these plants tend to be long and thin or highly dissected in shape, thus
maximizing the surface area for absorption. Stomates are absent, gas exchange is
thought to occur across the entire leaf surface. The leaves are often only a few cell
layers thick so nearly every cell is in direct contact with the bulk water phase.
Epidermal cells commonly contain chloroplasts and are covered by a thin cuticle. These
features decrease the path length for diffusion of gases as well as the resistance to gas
ﬂow across the leaf surface.

Gas spaces are also present in the leaves of many submersed species and can
facilitate the rapid internal diffusion and storage of gases (I-Iough, 1979; Sandergaard,
1979; Sandergaard and Wetzel, 1980; Sorrell and Dromgoole, 1986). Differences
between species in their ability to store 02 within the lacunae of the leaves are known to
exist (Sand-Jensen et al., 1982), as already discussed. Similarly, differences with

13

14
respect to exchange of CO, have also been demonstrated. In thin leaved species, such

as Elodea, which possess few or no lacunae, release of respired and photorespired CO,
from the leaf is favored. In Labelia and other isoetids that are characterized by large
lacunar volumes, internal 0; storage is promoted (Sandergaard, 1979). Additional
evidence also suggest that plants intermediate in these characteristics show intermediate
lacunar storage capabilities (Sendergaard and Wetzel, 1980). Collectively, these results
suggest that features which promote gas exchange across the leaf surface facilitate both
the uptake of CO, and release of 0,. Those that restrict exchange across the leaf surface
promote the recycling of both of these gases within the lacunar system. It is not
surprising then, to note that in Lobelia and other species of similar growth form, much
of the CO, ﬁxed during photosynthesis is not taken up over the leaf surface but is taken
up over the root surface and is transported to the leaves via the lacunar system (Wium-
Andersen, 1971; Sendergaard and Sand-Jensen, 1979; Wetzel et al., 1985).

The purpose of this investigation was to examine the general anatomy and
ultrastructure of leaves of the selected submersed species and to identify features which
may inﬂuence either the release of gases across the leaf surface or their accumulation
within the lacunar system. Although many studies have examined the ultrastructure of
leaves of different seagrass species (Jagels, 1973; Birch, 1974; Doohan and Newcomb,
1976; Benedict and Scott, 1976; Barnabas et al., 1977; Kou, 1978; Barnabas et al.,
1980; Bamabas, 1982; Cambridge and Kou, 1982), comparatively few freshwater species
have been investigated (Falk and Sitte, 1963; Sitte, 1963; Lunney et al. , 1975; Pendland,
1979; Valanne and Rintamaki, 1982), none of which speciﬁcally examine structural

resistances to gas exchange.

15
RESULTS

Elodea

With the exception of the midvein region, the leaf of Elodea consisted entirely of
two epidermal cell layers and measured 50-75 pm thick (Figure 1). Cells of the upper
epidermis appeared approximately 8 times as large as those of the lower epidermis and
accounted for roughly 2/3 of the total leaf thickness (Figure 1, 2). Cells were
rectangular in shape and arranged in ﬁles along the length of the leaf (Figure 2). Small
triangular to diamond-shaped intercellular gas spaces, measuring 10 to 20 um across,
were regularly found at the junction where the radial walls of the upper epidermal cells
meet the adjacent radial walls of the lower epidermal cells (Figure 1). Due to the
difference in their sizes, each cell of the upper epidermis bordered upon two of these gas
spaces whereas cells of the lower epidermis normally bordered upon only one. The gas
spaces also extend vertically down the leaf blade, and are thought to be continuous
throughout its length (Dale, 1957a). The midvein was multi-layered, cells were densely
packed and relatively devoid of gas spaces (not shown).

The outer epidermal wall was much thicker than either the inner tangential or
radial walls (Figure 1, 3). On the lower epidermis, the inner half of this wall exhibited
a cross-hatched lamellate texture (Figure 4), a feature commonly observed in the walls
of epidermal (Chafe and Wardrop, 1972) and collencyhma cells (Chafe, 1970). In cells
of the upper epidermis, the lamellate structure often appeared loose and disorganized
(Figure 5). The outer half of the epidermal cell wall was homogenous in appearance and
uniformly covered with a thin (30 nm) electron translucent cuticular layer (Figure 6, 7).

Several cone-shaped areas or pores were commonly found in the cuticle of the lower

16
epidermis (Figure 7). They appeared to completely traverse this layer (Figure 8). These

structures were rarely observed in the cuticle of the upper epidermis (Figure 6). The
cuticle was often covered with a thin electron-dense layer to which the periphytic
community is loosely associated (Figure 7).

A distinct feature of the lower epidermis was the presence of several ﬁnger-like
extensions of the cell wall which project from the outer tangential wall into the cell
eavity (Figure 4). The plasma membrane was highly convoluted around these extensions.
Mitochondria with prominent cristae were found concentrated in this region. Cells with
such wall ingrowths are termed ”transfer cells” and, due to the proliferation of the
plasma membrane around these extensions, possess a high surface areazvolume ratio
(Gunning and Pate, 1969).

The striated pattern of the epidermal wall appeared to be continuous around the
entire cell, including the gas spaces (Figure 9, 10). In the comers of the gas spaces, a
slight thickening of the wall was observed. At the junction where gas spaces were not
produced, triangular thickenings in the wall were found (Figure 3). Plasmodesmatal
connections were frequently observed between adjacent cells of the same epidermal layer
(Figure 9), as well as between epidermal layers (Figure 10).

In all sections examined, chloroplasts were found distributed equally along the
walls of lower epidermal cells and randomly throughout cells of the upper epidermis
(Figure 1). They were not found appressed against the outer wall as is commonly
observed in leaves of submersed plants (Sculthorpe, 1967). Chloroplasts were
characterized by well-developed grana, numerous osmiophilic bodies and large starch

grains which often distorted their shape (Figure 3, 9, 10).

l7

Myriophyllwn

The ﬁne cylindrieal leaﬂet of Myriophyllum measured less than 0.5 mm in
diameter and consisted of a single layer of small epidermal cells and an undifferentiated
mesophyll that surrounded a central core of vascular tissue (Figure 11). The mesophyll
was 3 to 5 cells thick. Cells were round to oval in shape and tightly packed together.
Occasionally small triangular to diamond shaped intercellular gas spaces, measuring 2-10
pm across, were visible in the light microscope. They appeared scattered randomly
throughout the mesophyll and were not as large or abundant as those depicted in the
leaﬂet of the related species M. spicatum (Hasman and Inanc, 1957; Grace and Wetzel,
1978).

The rachis showed similarities to both the leaﬂets and the stem (Chapter II) in
general anatomy (Figure 12). The outer mesophyll consisted of a layer of 2-5 closely
packed round cells. Within the inner mesophyll, cells were arranged in a fashion similar
to those of the middle cortex of the stem and formed a single ring of small lacunae that
encircles the vascular cylinder. These gas spaces were round to oval in shape and range
from 10-40 pm in diameter. ChlorOplasts were abundant and distributed equally
throughout both the epidermis and mesophyll.

Epidermal cells were arranged in ﬁles along the length of the leaﬂet (Figure 13).
They contained several chloroplasts that were commonly found lining the relatively thick
outer cell wall (Figure 11, 14). The epidermis was covered by a thin (60 nm), ﬁnely
reticulate cuticular layer (Figure 14, 15).

Triangular thickenings in the cell wall were observed at the point where adjacent

epidermal cells met underlying mesophyll cells (Figure 14). They were also found

18
throughout the mesophyll at the common junction between 3 or 4 adjacent cells (Figure

16). Wherever gas spaces were produced, they were found at the center of these
thickenings (Figure 17).

The mesophyll consisted of uniformly thin-walled cells that, like epidermal cells,
were highly vacuolate and frequently ﬁlled with darkly staining material (Figure 11, 17).
Chloroplasts were long and thin and often contained large starch grains (Figure 14, l6,
l7). Plasmodesmatal connections were not observed between adjacent epidermal cells

(Figure 14), but were frequently found between mesophyll cells (Figure 16).

Potamogeton

The broad leaves of Potamogeton show major differences between the leaf blade
proper and the minor and central midvein regions (Figure 18, 19). The leaf blade
measured roughly 50 pm thick and consisted of two epidermal cell layers and a single
layer of intervening mesophyll cells (Figure 18). Both epidermal layers appeared identical
in gross anatomy and were similar to the mesophyll in both size and shape. Small and
inconspicuous gas spaces were occasionally produced at the junction of the mesophyll and
epidermis. They were not, however, a regular or consistent feature in this region of the
leaf. Around the minor veins, the mesophyll may become 2 or more cell layers thick.
In this region a few small gas spaces were commonly found around the vascular bundles
(not shown). In the thicker midvein region, however, large, round to oval shaped gas
lacunae that measure 100-200 pm in diameter surrounded the vascular bundle (Figure
19). The gas spaces formed continuous canals along the length of the leaf, and were

frequently traversed by diaphragms like those found in the stern (ChapterII).

19
The two epidermal layers appeared symmetrical (Figure 18); no major anatomieal

or ultrastructural differences could be identiﬁed. The outer epidermal wall was several
times as thick as both the inner tangential or radial walls (Figure 20). Microﬁbrils which
form the inner layer of the outer epidermal wall appeared swollen and loosely organized
(Figure 21). It is bound by a thin (50 nm) amorphous electron translucent cuticle, which
was often covered by a layer of ﬂocculent electron dense material (Figure 21).

The internal walls were variable in thickness (Figure 22, 23). Plasmodesmata
were abundant and found in the constricted areas of the walls bordering the mesophyll
(Figure 22, 23).

Chloroplasts were abundant and appeared distributed evenly throughout both the
epidermal and mesophyll layers. Chloroplasts of the epidermis commonly contained
large stacks of grana, osmiophilic bodies and often several small starch grains (Figure
20). Starch grains in the chloroplasts of the mesophyll, however, were usually much
larger (Figure 18, 20, 23). Large starch grains were also found in the mesophyll cells
surrounding the veins (Figure 19). This pattern of starch distribution appears to be

characteristic of the leaf of this species (Rough and Wetzel, 1977).

Lobeh'a

The strap-shaped leaf of LobeIia measured 1-5 mm in diameter and appeared in
cross-sectional view like two hollow tubes of mesophyll cells that were linked together
in the middle by a bridge of mesophyll tissue (Figure 24). This arrangement results in
the formation of two large gas lacunae, measuring roughly 0.5 mm in diameter, that
formed continuous eanals along the length of the leaf. 'Ihe mesophyll consisted of 4 or

more layers of round to oval shaped cells (Figure 25). In addition to the large gas

20
lacunae, intercellular gas spaces of various shapes and sizes, ranging from 1-10 pm

across, were commonly found at the junction between adjacent cells (Figure 25). These
gas spaces were also likely to form continuous, although indirect, gas pathways between
cells of the mesophyll and the central lacunae.

The leaves examined were encrusted with a dense periphytic community
dominated by diatoms (Figure 26). The surface of the leaf underneath this layer was
undulating and irregular in appearance (Figure 27), a pattern also seen in cross-sectional
view (Figure 28). The relatively thick outer epidermal wall appeared uniform in texture
and was covered by a thick ( 1.1 pm) cuticle (Figure 28). The cuticle consisted of a thin
inner reticulate layer and an outer amorphous region. Members of the periphytic
community were conﬁned to the electron dense matrix that surrounds the leaf and did not
appear to disrupt or penetrate the cuticle.

Cells of the epidermis consisted of a large central vacuole ﬁlled with electron
dense material and a thin layer of cytoplasm in which a few organelles were found
(Figure 28, 29). Mesophyll cells had uniformly thin walls and contained numerous
chloroplasts (Figure 25). Chloroplasts were most abundant in cells underlying the
epidermis and decreased in numbers toward the large central gas lacunae. They were
round to oval in shape, and contained several osmiophilic bodies, but relatively little
starch (Figure 29). Plasmodesmata were observed between adjacent epidermal cells and
between epidermal and mesophyll cells, but were most frequently produced between
adjacent mesophyll cells.

Occasionally, plasmolyzed cells containing senescent chloroplasts and vesiculate

debris were found in the mesophyll (Figure 30). These cells were usually isolated and

21
bordered upon normal and unaffected cells. Vesiculate material was also found within

the gas spaces adjacent to these cells.

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

22

Cross-section of Elodea leaf. Light micrograph showing size and
arrangement of epidermal cell layers and the distribution of gas spaces.

Bar=50 um.

Surface view of Elodea leaf. SEM micrograph of A) upper and B) lower
leaf surface. Note differences in cell sizes between cell layers.

Bar=50um.

Lower epidermis of Elodea leaf. TEM micrograph showing structure of

chloroplasts and differences in wall thickness. Bar=50 um.

Lower epidermis of Elodea leaf. Outer wall showing lamellate structure
of wall and transfer cell characteristics. Arrows pointing to wall

lamellations (WL). Bar=l p.111.

Upper epidermis of Elodea leaf. TEM micrograph of outer wall showing

loosened and disorganized inner layer (arrows). Bar=l um.

 

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

24

Upper epidermis of Elodea leaf. TEM micrograph showing electron

translucent cuticle (delineated by arrows). Bar=0.25 um.

Lower epidermis of Elodea leaf. TEM micrograph of cuticle and attached

members of the periphytic community. Bar=0.25 um.

Lower epidermis of Elodea leaf. TEM micrograph of pores (P) traversing

the cuticle. Bar=0.05 um.

Cross-section of Elodea leaf. TEM micrograph illustrating gas space (GS)
between cells of lower epidermis. Wall is striated in appearance, contains
plasmodesmata (PD) and is thickened in the corners of the gas space.

Bar=2.5 um.

Cross-section of Elodea leaf. TEM micrograph of common cell wall
between epidermal layers containing numerous plasmodesmata (PD).

Chloroplasts contain large starch granules. Bar=2.5 um.

25

 

Figure 11.

Figure 12.

Figure 13.

Figure 14.

Figure 15.

Figure 16.

Figure 17.

26

Cross-section of Myriophyllum leaﬂet. Light micrograph showing size and

arrangement of cells. Bar=0.05 mm.

Cross-section of Myriophyllum rachis. Light micrograph showing size and

distribution of gas lacunae (marked by arrows). Bar=0.05 mm.

Surface of Myriophyllum leaﬂet. SEM micrograph. Bar=25 pm.

Epidermis of Myn'ophyllum leaﬂet. TEM micrograph of epidermal cell
showing chloroplast structure, differences in cell wall thickness and

relative thickness of cuticle. Bar=5 pm.

Epidermis of Myn'ophyllum leaﬂet. TEM micrograph of ﬁnely reticulate

cuticle (delineated by arrows). Bar=0.25 um.

Mesophyll of Myn'ophyllum leaﬂet. TEM micrograph showing junction
of 3 mesophyll cells and triangular thickening of common cell wall.

Bar=0.25 um.

Mesophyll of Myriophyllum leaﬂet. TEM micrograph of diamond-shaped
gas space (GS) produced at junction between 4 adjacent mesophyll cells.

Plasmodesmata present between mesophyll cells. Bar=5 pm.

 

 

Figure 18.

Figure 19.

Figure 20.

Figure 21.

Figure 22.

Figure 23.

28

Cross-section of Potamogeton leaf blade. Light micrograph showing size

and arrangement of cells. Bar=50 um.

Cross-section of Potamogeton leaf at midvein. Light micrograph showing
size and arrangement of cells and the distribution of gas lacunae (GL).

Bar=0.2 mm.

Epidermal cell of Potamogeton leaf. TEM micrograph showing relatively
thick outer cell wall and chloroplasts with small starch grains. Underlying

mesophyll (M) contains chloroplasts with large starch grains. Bar=5 pm.

Epidermal cell wall of Potamogeton leaf. TEM micrograph showing
relative thickness of cuticle (delineated by arrows) and loosened

appearance of inner wall (arrows with tails). Bar=0.05 um.

Cross-section of Potamogeton leaf. TEM micrograph at the junction of
epidermis and mesophyll, showing plasmodesmata (PD), variable wall

thickness and lack of gas spaces. Bar=0.05 pm.

Cross-section of Potamogeton leaf. TEM micrograph at the junction of
epidermis (EP) and mesophyll (M) showing small gas space (GS),
plasmodesmata (PD) and differences in chloroplast structure. Bar=0.25

um.

29

18 19

v.-

,4‘ .1: 21,775.?
lose-'2’.

Eran l'-‘ E “are.4.( .« ‘ - _~ .
3:. o.e - v- ‘ . .
.4? | :E'flb d‘ ‘i _ I ‘ n V" cl-
h ;.r a: ‘ “’3‘
“TA-“M

\ -‘A—J-l.‘

. 'r I:
\ ‘ ‘I r
a. _~
‘\I ‘ ‘ , \ Av
.37 ’ ‘
—’ “‘ ,. .———_e_# _>
——

 

Figure 24.

Figure 25.

Figure 26.

Figure 27.

Figure 28.

Figure 29.

Figure 30.

3O

Cross-section of LobeIia leaf. Light micrograph showing large central gas

lacunae (GL). Bar=0.2 mm.

Cross-section of Lobeh'a leaf. Light micrograph showing distribution of
chloroplasts primarily in cells underlying the epidermis. Arrows pointing
to intercellular gas spaces contiguous with gas lacunae (GL). Bar=50

,um.

Surface of Labelia leaf. SEM micrograph of periphytic community

present on leaf surface. Bar=0.l mm.

Surface of LobeIia leaf. SEM micrograph of leaf surface. Bar=0.l mm.

Epidermis of LobeIia leaf. TEM micrograph showing structure and
thickness of cuticle (delineated by arrows) and composition of the

periphytic community. Bar=5 um.

Cross-section of Lobelia leaf. TEM micrograph at junction of epidermis
(EP) and mesophyll (M) showing development of gas space (GS), the lack
of chloroplasts in the epidermis and their abundance in underlying

meSOphyll cells. Bar=10 um.

Mesophyll of LobeIia leaf. TEM micrograph showing chloroplast
degradation, storage of vesiculate material (VM) in cells and gas spaces

(GS), as well as healthy mesophyll cells. Bar=10 pm.

 

32
DISCUSSION

In submersed plants, 02 produced during photosynthesis either accumulates within
the lacunar atmosphere or is released to the surrounding water (Hartman and Brown,
1967). The direction of O2 ﬂow or the partitioning between these two phases, as
described by Fick’s ﬁrst law, is determined by the surface area, the resistance to gas
ﬂow and the 0; gradient (Browse et al., 1977; Payne, 1983). 02 released from the leaf
must diffuse from the chloroplast to the bulk water phase. In this path it encounters
resistances associated with transport from the chloroplast to the epidermis as well as
diffusion through the outer epidermal wall and the surrounding boundary layer.
Accumulation of 02 within the lacunar atmosphere, on the other hand, involves only the
internal resistances of the leaf tissue to gas transport (Sorrell and Dromgoole, 1986).

Diffusion of 0, from the chloroplast to the lacunae occurs in the aqueous phase
and the resistance it encounters is directly related to the length of the diffusion pathway
(Browse, et a1. , 1977). The internal resistance to 02 diffusion in leaves of Elodea would
be expected to be relatively minor since both epidermal layers bordered upon at least one
gas space. Gas spaces, however, were not produced between each cell in leaves of
Potamogeton and Myn'ophyllum. In these leaves the distance between chloroplasts and
lacunae may often be several cells in length. If the resistance to lateral transport over
this distance is greater than the resistance through the epidermis, release of 0, from the
leaf would be expected. In Lobeh'a, chloroplasts were found throughout the mesophyll,
but were not present in the epidermis. Although the mesophyll was several layers thick,

02 transport through this layer is likely to occur through the intercellular gas spaces that

33
appear to be continuous with the central lacunae. This feature would not only facilitate

the transport of 0, but also the transport of CO, from the central lacunae to the outer
mesophyll where chloroplasts were abundant.

Whentheresistance toozreleaseishigh ortlregradientbetween thelacunaeand
the surrounding water is low, accumulation of 0, within the lacunar system is favored.
As O; accumulates within the lacunar atmosphere, the 0, partial pressure increases and
when the 0; gradient overcomes the resistance to outward diffusion, release of 0, will
occur. The steepness of this gradient will be determined by the lacunar volume and
pressure, the photosynthetic rate and the rate at which 02 is removed from the leaf as it
is transported down the stem and into the roots (Chapter II), (Sand-Jensen and Prahl,
1982; Sorrell and Dromgoole, 1986).

The diffusion of gases through the boundary layer provides the major resistance
to CO, uptake in leaves of submersed plants and can limit the rate of photosynthesis,
even under well-stirred conditions (Browse et al. , 1979; Smith and Walker, 1980; Black
et al., 1981). Since 02 diffuses along the same pathway (Armstrong, 1979), it is also
likely to provide a signiﬁcant resistance to 02 release. The increase in the rate of 02
release observed with increasing current velocities, and hence decreasing boundary layer
thickness, supports this conclusion (Westlake, 1967; Madsen and Sendergaard, 1983;
Sorrell and Dromgoole, 1987).

The cuticle of submersed vascular plants is typically very thin and is thought to
provide little or no resistance to the diffusion of gases or solutes (Sculthorpe, 1967 ;
Sharpe and Denny, 1976; Denny, 1980). Although the resistance of the cuticle of

submersed plants to the diffusion of gases has not been examined directly, the cuticle of

34

Potamogeton lucens was found to provide some resistance to the diffusion of water,
although 1,000 times less than that of terrestrial species (Schonherr, 1976).

The electron translucent cuticles of Elodea, Myriophyllwn, and Potamogeton
appeared to ultrastructurally similar to cuticles of other freshwater submersed plant
species (Sharpe and Denny, 1976; Halloway, 1982). They were all of similar thicknesses
(0.03-0.06 um). The thick (1.1 um) cuticle of Labelia, on the other hand, resembles
the cuticle of many terrestrial species, both in size and structure (Halloway, 1982). If
similar resistances can also be expected, the cuticle should offer a substantial resistance
to gas exchange since the cuticle of terrestrial plants provides an effective barrier which
minimizes passive water loss across the leaf (Ting, 1982).

The lower epidermis of Elodea was associated with the presence of several small,
electron dense areas or pores which traverse the cuticle. Structures of this nature have
not been reported elsewhere for submersed plants.

The periphytic community was embedded in a matrix of electron dense material
that did not appear to attach directly to the cuticle. Cellulolytic bacteria have been
isolated from members of this community (Robb et al. , 1979) and may be associated with
cuticular peeling and rupture of the epidermal cell wall (Howard-Williams, et a1. , 1978).
Bacterial colonization of the leaf surface has also been correlated with a progressive
internal disorganization of the epidermis, a swelling and loosening of the cell wall,
followed by invasion into the epidermis and mesophyll and ultimately cell death (Rogers
and Breen, 1981). The inner portion of the outer epidermal wall appears swollen and
loosely organized in leaves of both Elodea and Potamogeton, and may be undergoing the

initial stages of senescence as characterized above (Rogers and Breen, 1981). In none

35
of the leaves examined by TEM was there evidence of direct bacterial damage to the

cuticle or epidermis. Unfortunately surfaces of these leaves were not viewed by SEM
at magniﬁcations necessary to examine the extent of this activity. Rogers and Breen
(1981), however, note that swelling and disorganization of the epidermal wall ean result
without extensive damage to the leaf surface.

The effects of these bacteria on the leaf surface can be seen within 6 weeks after
emergence and within 14 weeks can result in leaf senescence (Howard-Williams et a1. ,
1978). In Elodea, Potamogeton and Myriophyllwn, where growth rates are relatively
high, a signiﬁcant proportion of the leaf biomass may die before the seasonal maximum
is attained (Rich et al., 1971; Adams and McCraken, 1974). The evergreen leaves of
Lobelia examined in this study may be close to one year old (Moeller, 1978). The
degradation of chloroplasts and the storage of vesiculate material within a few cells of
the mesophyll may be interpreted as symptoms of an age related process (Mahlberg,
1972). It is therefore surprising that the well-developed periphytic community present
on the leaves of LobeIia had no apparent effects of the cuticle surface or on the
ultrastructure of the epidermis. In this slow growing annual, the thick cuticle may
provide a substantial resistance not only to gas exchange, but also to the pathogenic

effects of the periphytic community.

CHAPTERII

ANATOMY OF THE LACUNAR SYSTEM: STRUCTURAL RESISTANCES TO GAS
TRANSPORT
INTRODUCTION

Although gas spaces are found throughout the plant kingdom, they reach their
greatest development, in terms of both size and proportion, within tissues of aquatic
plants (Sifton, 1945; 1957).

Gas spaces develop by either schizogeny or lysigeny. Schizogenous gas spaces
arise by the splitting apart of the common wall between adjacent cells and include the
small intercellular gas spaces as well as the large gas lacunae characteristically found in
leaves and stems of submersed plants. In Elodea, stem lacunae are initiated at the shoot
apex as small intercellular spaces and attain their characteristic size and shape by the
regular growth and development of bordering cortical cells (Dale, 1957a). These lacunar
initials are thought to form due to increasing 0, pressures which develop in the stern
during photosynthesis (Dale, 1957b). Lysigenous gas spaces result from cell death and
disintegration. They are common not only in roots of aquatic plants, but also develop
in the roots of many terrestrial species that are able to survive waterlogging conditions

(Sifton, 1945; 1957; Kawase, 1981).

36

37
It is commonly stated that the lacunar system of submersed macrophytes occupies

a signiﬁeant, yet variable, proportion of the total plant body and that it serves as both
a reservoir for the storage of 0, and as a pathway for its transport (Sculthorpe, 1967 ;
Hutchinson, 1975; Wetzel, 1975). Little, however, is actually known about the variation
that exists between species and the functional signiﬁcance of these differences with
respect to gas transport.

A number of recent studies have established a correlation between the ability of
submersed plants to transport and release 0, from their roots and the degree of their
lacunar development . As already discussed, the results of Sand-Jensen et a1. (1982)
show that the isoetid species, with their large lacunar volumes, are better adapted to 0,
transport than species characterized by small lacunar volumes. Penhale and Wetzel
(1983) noted an increase in the lacunar development of roots of seagrasses distributed
along a gradient of increasing sediment O, demand and suggested that plants with the
more developed lacunar system would facilitate higher 0, ﬂux rates. Smith et a1. (1984)
observed higher rates of 0, release from roots of mature seagrasses; young individuals
were characterized by poorly developed lacunar systems. In Potamogeton perfoliatus,
Kemp and Murray (1986) found that the amount of 0, released from the roots was
inversely related to the overall length and mass (speciﬁc gravity) per unit length of the
stern. Their results suggest that both a decrease in the length of the pathway and an
increase in percentage gas space or porosity, as reﬂected by low mass per unit stem
length, increases the rate of transport from the shoots to the roots.

Armstrong (1979) examined the inter-relationship of porosity and path length on

the transport of 02 down the roots by measuring its release from the roots. Increases in

38
porosity were found to have more of an overall effect on reducing resistance than

decreases in root length. This is due, at least in part, to proportionate decreases in the
amount of respiring tissue (Williams and Barber, 1961; Armstrong, 1972; 1979; Penhale
and Wetzel, 1983). length of stems and roots are known to vary with both age and
environmental conditions (Sculthorpe, 1967). Lacunae, on the other hand, can form
relatively consistent patterns within tissues of submersed plants (Arber, 1920; Sculthorpe,
1967). Porosity should therefore be a reliable feature on which to evaluate the efﬁciency
of lacunar transport, provided that no additional barriers to transport exist.

The purpose of this investigation was twofold. First, to examine the anatomy and
continuity of gas spaces throughout the plant body and to identify features which may
restrict or impede gas transport. And secondly, to evaluate the functional signiﬁcance
of the lacunar system in gas transport by examining differences in porosity that may exist

between the selected species.

39
RESULTS

Elodea

The gas space system of the mature Elodea stem was characterized by a double
ring of large gas lacunae that encircled the stem throughout the middle cortex (Figure
31). The lacunae were round to oval in shape and were formed by the arrangement of
cells into single rows or ﬁles which interconnect in honeycomb pattern. The lacunae also
extended vertically down the stem to form gas canals that were continuous throughout
the length of the intemode (Figure 32). In the stems examined, the lacunae ranged from
25-200 um in diameter and occupied over 30% of the cross-sectional area of the cortex
or roughly 26% of the volume of the intemode (Table 1).

Throughout the inner and outer cortical regions, small intercellular gas spaces,
measuring 1 or 2 pm in diameter, were also produced (Figure 31). They were not
common, however, at the junction between epidermal and underlying cortical cells. The
vascular tissues were densely packed and also devoid of obvious gas spaces.

The lacunar system of the mature sediment roots of Elodea examined consisted
entirely of small intercellular gas spaces of schizogenous origin (Figure 33). Gas spaces
were found throughout the cortex and were regularly produced at the junction between
4 or more adjacent cells. They ranged from 4- to 10-sided polygons in shape and varied
from 10-50 pm in diameter. Gas spaces occupied approximately 33% of the cortical area
or roughly 22% of the volume of the root (Table 2, 3).

The root was bound by a single layer of large epidermal cells (Figure 33).
(Chapter III). Gas spaces were not common at this junction nor were they prominent

between cells of the vascular cylinder.

4O
Vasculartracestoboththeleavesandtherootscrossthecortexofthestematthe

nodes (Dale, 1957a) (not shown). In regions unoccupied by these tissues, small plates
of cells, called diaphragms, were found traversing the large gas eanals (Figure 34, 35).
Diaphragms were usually I, sometimes 2 cell layers thick and were made up of
hexagonally shaped cells (Figure 36). Small triangular gas spaces or pores, measuring
roughly 2-5 run across, were present at the comers where 3 adjacent cells of the
diaphragm met. The effect of diaphragms on gas transport is examined in Chapter IV.

Leaves were inserted into the node through a slight constriction at their bases.
The smaller cells of the lower epidermis enlarge at this junction (not shown) and
although this enlargement should also result in the formation of larger gas spaces, their
continuity with the stem could not be veriﬁed. The junction of the mature root to the

stem was not examined.

Myriophyllum

The most striking feature of the stem of Myn'ophyllum was the highly lacunate
structure of the middle cortex (Figure 37). Cells in this region were rectangular in shape
and arranged end-to-end in single ﬁle. Columns of these cells bridge the outer and inner
cortical regions and radiate around the stem much like the spokes of a wheel. A single
oval-shaped lacuna was formed between the columns. The lacunae also extended
longitudinally down the stem and formed gas canals that were continuous throughout the
length of the intemode. In the stems examined, the lacunae measured roughly 100 um
wide and 400 um long and accounted for more than 50% of the area of the cortex or

roughly 48% of the stem’s volume (Table 1, 3).

41
Between cells of the inner and outer cortex, small intercellular gas spaces,

measuring 1-2 pm in diameter were produced (not shown). The cortex was tightly bound
by a single layer of small epidermal cells (Figure 37). Gas spaces were absent at this
junction and within tissues of the central vascular cylinder.

Both lysigenous and schizogenous gas spaces were produced in the mature root
of Myﬁophyllwn (Figure 38). Lysigenous gas spaces extended primarily from the outer
to the mid-cortieal region and were formed by the collapse of radially aligned groups of
cells. The walls of these cells produced thin partitions which separate adjacent lacunae.
Small diamond-shaped schizogenous gas spaces were found between the apparently
healthy cells that scattered throughout the cortex. Collectively, these gas spaces occupied
around 46% of the cortical area or roughly 32 % of the volume of the roots examined
(Table 2, 3).

The outermost protective layer of the root consisted of 3 or more layers of
densely packed, thick walled cells that were irregular in shape (Figure 38) (Chapter III).
Gas spaces were not found between cells of this layer. They were also absent within the
tissues of the vascular cylinder.

Leaves are usually arranged in whorls about the node and are directly inserted
into the stem by the rachis. The lacunae of the rachis appeared uninterrupted up to their
insertion into the stem (Figure 39). It could not be determined whether the gas spaces
of the rachis formed a continuous pathway through these layers to the lacunae of the
stem.

The gas canals of the stem, although continuous throughout the length of the

intemode, were interrupted at the node by diaphragms and tissues associated with leaf

42
traces (Figure 39, 40). The leaf trace consisted of a core of vascular tissue surrounded

by a compact layer of parenchyma cells embedded within a multi-layered sheath of small
globose cells (Figure 41). Intercellular gas spaces, roughly 10 um across, were present
at the rounded comers of these cells. The inﬂuence of diaphragms on gas transport is
examined in Chapter IV.

Rhizomes were similar to the stem in general anatomy (Figure 39, 42). The
vascular traces to the numerous adventitious roots could be seen traversing the cortex of
the rhizome (Figure 42). The gas spaces of the root appeared to be continuous from the
external root into the tissues of the root trace. The root trace, like the leaf trace, was
also surrounded by several layers of small globose parenchyma cells. The pathway

across this tissue could not be determined.

Potamogeton

In the stems on Potamogeton, cells of the cortex were arranged into single rows
or ﬁles of cells which interconnect at right angles and form a honeycomb or net-like
pattern (Figure 43). This arrangement resulted in the formation of 3-5 rings of large,
round to oval-shaped gas lacunae that encircle the stem. The lacunae typically ranged
from 50-350 pm in diameter and decreased in size toward the center of the stem. They
occupied nearly 70% of the cortical area or roughly 63 % of the volume of the intemodes
examined (Table l, 3).

At the outer edge, the stem was bound by a layer of small square-shaped
epidermal cells and a single layer of underlying cortical cells (Figure 44). Gas spaces
were not abundant at this junction. The vascular cylinder was surrounded by layer of

3 to 5 cortical cells (Figure 45). Small intercellular gas spaces, measuring 1-2 pm in

43
diameter, were produced between these cells. Gas spaces were not obvious between cells

of the vascular tissues.

The mature root of Potamogeton contained both lysigenous and schizogenous gas
spaces (Figure 46). Collectively, these gas spaces accounted for 61% of the total cross-
sectional area of the cortex or roughly 46% of the volume of the roots examined (Table
2, 3). Lysigenous gas spaces developed only in the outer third of the cortex and formed
a concentric ring around the root. The gas spaces appeared to be continuous along the
length of the root, but were separated radially by thin partitions formed by remains of
collapsed cell walls. Cells of the middle and inner cortex appeared round, turgid and
apparently in healthy condition. Small diamond-shaped, schizogenous gas spaces,
measuring roughly 10 pm across, were formed between these cells.

The cortex was bound at the outer edge by a single layer of large, turgid cortical
cells and a layer of small thick walled, irregularly shaped hypodermal cells (Figure 46).
Gas spaces were not found between cells of these two layers (Chapter 111) nor are they
present between tissues of the vascular cylinder.

The gas canals of the stem were frequently interrupted throughout the length of
the intemode (Figure 47) as well as at the node (Figure 48) by the presence of horizontal
diaphragm plates. Diaphragms consisted of a single layer of small, hexagonally shaped
cells which produced small triangular pores at their comers (Figure 49). The effect of
these diaphragms on gas transport is examined in Chapter IV.

The leaf attaches to the stem through a constriction at its base. At this junction,

the gas spaces surrounding the veins of the leaf (Chapter II) enlarge considerably and

44
appeartomerge into the tissues ofthe stem (FigureSO, 51). It could notbedetermined

if these gas spaces are directly continuous with those of the stem.

Rhizomes were similar to stems in general anatomy (Figure 52, 53). The gas
canals of the rhizome were also traversed by diaphragms as well as by vascular traces
tothe manyrootsproducedatthenode. Thegaslacunaeoftherooteanbeseen entering
the cortex of the rhizome within the tissues of the root trace (Figure 52, 53). The traces
were, however, surrounded by a few layers of small compact cells. It could not be
determined whether the gas spaces of the root were continuous with the gas canals of the

rhizome across this layer.

LobeIia

The stem of Lobeh'a consisted of a discrete ring of vascular tissues, a few cell
layers thick, which delineated an inner pith and outer cortical region (Figure 54). Small
intercellular gas spaces were interspersed throughout the large cells of the pith as well
as the small globose parenchyma of the cortex. In neither of these tissues, however,
were cells organized into the highly lacunate structure found in the stems of the other
species examined.

The stem was not differentiated into nodes and intemodes. Vascular traces to
both the leaves and the roots were commonly seen traversing the outer cortical layer
(Figure 55, 56). leaves attached directly to the stem and were associated with 2 or 4
roots that were also inserted into the stem on each side of the leaf. The lacunae of both
the leaves and the roots extended well into the cortical tissues of the stem (Figure 55 ,

56). Although it was not evident from a single cross-section, examination of a series of

45
sections show that the lacunar system of the leaf is continuous with the lacunae of the

roots (Figure 5456).

The cortex of the mature LobeIia root was occupied almost entirely by very large
and irregularly shaped lysigenous gas lacunae (Figure 57). The wall remains of
collapsed cortical cells formed thin partitions that traversed the cortex and, in many
instances, provided the only structural connection between the inner and outer cortical
regions. Small schizogenous gas spaces were also found between the remaining inner
cortical cells that surrounded the vascular bundle. Approximately 75 % of the cortex or
roughly 58% of the root’s volume was occupied by gas space (Table 2, 3).

The lacunae were bound at the epidermis by a single layer of large irregularly
shaped cortical cells and a layer of small thick walled hypodermal cells (Figure 57)
(Chapter III). Gas spaces were not produced between the cells of these different layers

nor between cells of the vascular tissues.

'lw‘

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Figure 31 .

Figure 32.

Figure 33.

Figure 34.

Figure 35.

Figure 36.

46

Cross-section of Elodea stern. Light micrograph showing size and

arrangement of gas lacunae (GL). Bar=0.2 mm.

Cross-section of Elodea stem. SEM micrograph illustrating continuity of

gas canals along length of intemode. Bar=0.2 mm.

Cross-section of Elodea root. Light micrograph showing size and

distribution of schizogenous gas spaces (arrows). Bar=70 um.

Longitudinal section of Elodea stem. SEM micrograph through node
showing vertical distribution of gas canals and presence of diaphragms.

Bar=0.2 mm.

Cross-section through node of Elodea stem. SEM micrograph showing

placement of diaphragms throughout cortex. Bar=0.4 mm.

Cross-section through node of Elodea stem. SEM micrograph of
diaphragm showing size and distribution of small triangular pores.

Bar=50 um.

 

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Figure 37 .

Figure 38.

Figure 39.

Figure 40.

Figure 41.

Figure 42.

51

Cross-section of Myn‘ophyllum stem. Light micrograph showing size and

arrangement of gas lacunae. Bar=0.5 mm.

Cross-section of Myn’ophyllum root. Light micrograph showing

development of lysigenous gas spaces (GS). Bar=0.1 mm.

Cross-section through node of Myriophyllum stem. SEM micrograph
showing leaf trace and part of associated sheath. Arrows denote gas

pathway from leaf into stem. Bar=0.5 mm.

Longitudinal section through node of Myn’ophyllwn stem. SEM
micrograph showing insertion of diaphragm through gas canals (arrows).

Bar=0.5 mm.

Cross-section through node of Myﬁophyllwn stem. SEM micrograph of

diaphragm showing size and distribution of pores (arrows). Bar=0.5 mm.

Cross-section through Myn’ophyllum rhizome at the junction of a root.-
SEM micrograph showing gas spaces of root extending into cortex.

Arrows denote gas pathway from rhizome to root. Bar=0.5 mm.

52

 

Figure 43.

Figure 44.

Figure 45 .

Figure 46.

Figure 47.

53

Cross-section of Potamogeton stem. Light micrograph showing size and

arrangement of gas lacunae (GL). Bar=0.5 mm.

Cross-section of Potamogeton stem. Light micrograph of epidermis.

Bar=50 pm.

Cross-section of Potamogeton stem. Light micrograph of vascular tissue.

Note lack of obvious gas spaces. Bar-=50 um.

Cross-section of Potamogeton root. Light micrograph showing
development of lysigenous gas spaces (GS) in outer cortex only. Barr-0.1

mm.

Cross-section of Potamogeton stem. SEM micrograph showing gas canals

interrupted by diaphragms. Bar=0.1 mm.

1
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Figure 48.

Figure 49.

Figure 50.

Figure 51.

Figure 52.

Figure 53.

55

Cross-section through node of Potamogeton stem. SEM micrograph

showing presence of diaphragms in gas canals. Bar=0.25 mm.

Cross-section through node of Potamogeton stem. SEM micrograph

showing size and distribution of pores. Bar=50 um.

Cross-section of Potamogeton stem. Light micrograph of junction of leaf.

Arrows denote gas pathway from leaf to stem. Bar=0.25 mm.

Cross-section of Potamogeton stem. SEM micrograph of leaf/ stem

junction. Arrows denote gas pathway from leaf to stem. Bar=0.25 mm.

Cross-section of Potamogeton rhizome. Light micrograph through node
showing root traces. Arrows denote gas pathway from rhizome to root.

Bar=0.5 mm.

Cross-section of Potamogeton rhizome. SEM micrograph through node.
Gas spaces of root appear to enter rhizome. Arrows denote pathway from

rhizome to root. Bar=0.25 mm.

 

Figure 54.

Figure 55.

Figure 56.

Figure 57.

57

Cross-section of LobeIia stem. Light micrograph showing insertion of

leaf. Arrows denote two large leaf lacunae. Bar=0.5 mm.

Cross-section of Lobeh'a stem. Light micrograph showing the close
proximity of leaf and root at their insertion into the stem. Arrows denote
gas pathway from leaf (2 arrows) through stem to root (1 arrow).

Bar=0.5 mm.

Cross-section of LobeIia stem. Light micrograph showing insertion of leaf

(1 arrow) and root (2 arrows) into stem. Bar=0.5 mm.

Cross-section of LobeIia root. Light micrograph showing lysigenous gas

spaces (GS). Bar=0.l mm.

 

59
DISCUSSION

In the rosette growth form of Labelia, leaves and roots are produced close
together on a short compact stem. 02 that is stored within the lacunae of the leaves
(Chapter I) can be transported to the roots via an uninterrupted continuation of the
lacunae throughout the stem. The short distance between leaves and roots as well as the
large, continuous and uninterrupted pathway should facilitate the transport of both 0,
(Sand-Jensen et al., 1982; Sand-Jensen and Prahl, 1982) and C02 (Wium-Andersen,
1971, Boston et al., 1987) throughout the plant body. The segmented stems of Elodea,
Myriophyllwn and Potamogeton, on the other hand, are long and ﬂexuous, producing
leaves and roots at the nodes. This design increases not only the transport distance but
also the complexity of the transport pathway. In these species, 0, must be transported
down the leaf into the stem lacunae, down the stem across several nodes to the rhizome
and from the rhizome into and down the root.

The most efﬁcient transport pathway from the leaves into the stem would be by
a direct continuation between the leaf lacunae and the stem lacunae. In Potamogeton and
Myriophyllwn, the large gas canals surrounding the major veins of the leaf merge directly
into tissues of the stem and appear to be continuous with tissues of the leaf trace. It is
not clear, however, whether the gas spaces within the leaf trace are continuous with the
stern lacunae. In Elodea, the pathway from the small gas spaces of the leaf into the stem
could not be determined. If gas spaces are not continuous across this junction, 0,
transport would have to occur in an aqueous phase and the resistance associated with

transport into the stem would be directly related to the length of the pathway (Sorrell and

Dromgoole, 1986).

60
0, transport down the stem is facilitated by the formation of large gas eanals that

are continuous throughout the length of the intemode. They are interrupted at the node,
however, by tissues associated with traces to the leaves and roots as well as by perforated
plates of cells called diaphragms. Porous diaphragms were found traversing the gas
canals at the nodes of stems of Elodea and Myriaphyllum and, in Potamogeton, were also
found throughout the internode of the stem as well. The inﬂuence of diaphragms on gas
transport is examined in Chapter IV. Vascular traces to the leaves and roots also
traversed the stem cortex. It was not determined, however, whether these tissues
completely interrupted the continuity of the intervening gas canals. If the resistance
across these tissues were sufﬁciently high, lateral transport into adjacent gas canals might
be expected. The walls bordering the lacunae were usually one cell thick and did not
appear to contain pores. If lateral transport through the stem were necessary, it would
likely be via aqueous diffusion through the common cell between adjacent lacunae.

In Potamogeton and Myn’aphyllwn, the gas canals of the stem appeared to be
continuous with those of the rhizome. Plants of Elodea do not produce a rhizome, roots
arise adventitiously from the nodes of the stem. This junction was not examined. In
Myn’ophyllwn and Potamogeton, however, the root/rhizome junction is analogous to the
leaf/ stem junction. The gas spaces of the root appeared to be continuous within the root
trace, but were bound by a few layers of cells which may provide some resistance to 0;
transport into the root. Coult (1964) described the gas pathway across the root/rhizome
junction of the emergent species, Menyanthes, as discontinuous and, since gas spaces in
this plant are as abundant within the vascular tissues as they are in the cortex, suggested

that much of the 0, transported into the root is supplied via the vascular tissues. It is

61
unlikely that this pathway is of any signiﬁeance to 0, transport in the species examined,

since the vascular tissues of both the stems and roots are relatively devoid of gas spaces.
In other emergent species, 0, transport is thought to be continuous across the
shoot/rhizome junction, although the pathway may be constricted and transport through
these tissues may occur at reduced rates (Armstrong, 1979; Justin and Armstrong, 1983).

The lacunar system of Lobeh‘a is continuous and provides a short and
uninterrupted pathway for gas transport (Sand-Jensen and Prahl, 1982). The lacunar
system has also been examined in the seagrass, Halophila (Roberts et al., 1984) and in
Egeria densa (Sorrell and Dromgoole, 1986), and is thought to form a continuous
pathway for transport form the shoots to the roots, In the stems of Elodea,
Myriophyllwn and Potamogeton examined in this study, the lacunar system also appeared
to form a continuous pathway for 02 transport. If the transport pathway from the leaves
to the roots is not continuous in these species, and if aqueous diffusion is imposed, it is
likely to occur at the junction of these organs to the stem.

In the stems of Elodea, Myn‘ophyllwn and Potamogeton, the lacunae varied widely
in size, shape, numbers and distribution. Porosity values also differed signiﬁcantly
between these species and increased from Elodea (26%), Myn’ophyllum (48 96) to
Potamogeton (63 96). The development and distribution of gas spaces in the roots also
varied widely. In the roots of Elodea, lysigenous gas spaces were not produced. In
Potamogeton, lysigenous gas spaces were conﬁned to the outer cortieal region, while in
Myriophyllwn and LobeIia, the lacunae traversed the entire cortex. Porosity values
differed signiﬁcantly between species and increased from Elodea (22 %), Myriophyllum

(32 96), Potamogeton (46 96) to Labelia (58 96).

62

Increasing porosity is known to decrease resistance and increase 0, transport
(Armstrong, 1979; Kemp and Murray, 1986). The increasing porosity of stems and roots
observed in this study suggests that the ability to transport 02 to the roots should also be
expected to increase from Elodea, Myﬁophyllwn, Potamogeton to Lobeh'a (Chapter IV).
The amount of roots these species produce also increases respectively. These results
suggest that the ability to transport 0, to the roots is directly correlated with the amount
of roots a species typically produces. In emergent plants, the ability to transport and
release 0, from the roots appears to be directly related to their distribution by
determining the O, demand of the sediments which it can tolerate (Armstrong, 1964,
1979, Yamaski, 1987). This relationship, however, does not appear to apply to
submersed plants, since LobeIia and related species of isoetid growth form are best able
to transport and release 02 from their roots (Sand-Jensen et al., 1982; Sand-Jensen and
Prahl, 1982), yet are characteristically found in sandy sediments with low 0, demands
(Moyle, 1945; Seddon, 1972). This would suggest that submersed plants distributed in
sediments with higher 02 demands are characterized by features which decrease the
amount of 02 released from the roots. Increasing the O, demand of the sediments
increases the 02 gradient between the lacunar system and the water surrounding the roots.
According to Fick’s ﬁrst law, decreasing the amount of 02 released from the roots under
conditions of increasing 0, demand, could only occur by either decreasing the relative
surface area of the roots or by increasing the resistance to diffusion across the roots
(Chapter III). Decreasing the surface area across the roots ean be achieved by simply
decreasing the amount of roots produced. This would suggest that the distribution of

submersed plants in sediments with increasing 0, demands may be inversely related to

63
the amount of roots produced and, according to the results presented, a decreasing ability

to transport 02 to the roots.

CHAPTERIII

ROOT ANATOMY: STRUCTURAL RESISTANCES TO GAS EXCHANGE
INTRODUCTION

Roots of submersed vascular plants are, in comparison with most terrestrial and
emergent aquatic plant species, reduced both in size and structure (Arber, 1920;
Sculthorpe, 1967). Although these features were at one time also interpreted as a
reduction in function, a considerable amount of evidence now indicates that the roots of
submersed vascular plants play a signiﬁcant role in nutrient uptake (Denny, 1972;
Bristow, 1975; Barko and Smart, 1981).

Aquatic sediments are often completely anaerobic a few millimeters below the
sediment-water interface. The lack of 02, however, is only one potential problem for
plants rooted in these sediments. Anaerobic processes of microbes in sediments can
result in the production of reduced soluble ions and volatile fatty acids which are
potentially toxic to plants (Ponnamperuma, 1984; Drew and Lynch, 1980). Although
some plants are temporarily able to metabolically adapt to anoxic conditions (Crawford,
1978), most aquatic plants try to avoid anoxia by transporting O, to the roots. 0,
transport is necessary not only to support aerobic respiratory processes but can also be
utilized in the oxidation and detoxiﬁcation of reduced compounds within the sediments

(Armstrong, 1979, 1982; Drew and Lynch, 1981). 0, released across the root surface

64

65

can form a protective oxidized layer immediately surrounding the root. This layer can
be visualized when the rust-colored precipitates of ferric hydroxides which form upon
oxidation of ferrous iron, are deposited on or around the roots (Bartlett, 1961; Green and
Etherington, 1977; Chen et al., 1980; Armstrong, 1982; Taylor et al., 1984). The
dimensions of this layer, and hence it oxidative ability to protect the root, will depend
largely on the redox potential of the surrounding sediment and the amount of 0, released
from the root.

0, release from the root follows Fick’s ﬁrst law and is therefore a function of the
surface area of the root and the resistance of the root wall to radial diffusion. The
concentration difference across the root surface provides the driving force for O2
diffusion and will determine the amount of 02 released by a given root. In emergent
species, the release of O2 is not uniform along the length of the root but has been found
to occur primarily across the root tip (Armstrong, 1964; Armstrong and Armstrong,
1988). A decrease in the sensitivity of roots of wetland plants to soil anoxia and
phytotoxins has been observed upon suberization of the root wall (Sanderson and
Armstrong, 1978). Suberin deposits in the wall of hypodermal cells are known to
decrease the permeability of the root to both water and ions (Ferguson and Clarkson,
1976; Robards et al., 1979) and may also inﬂuence the diffusion of gases across its
surface (Armstrong and Armstrong, 1988). The purpose of this investigation was to
examine the general anatomy of the outer wall of roots of the selected species and to
identify features which may inﬂuence the release of 02 across this layer. Although the

general anatomy of roots of several submersed species has been examined (Arber, 1920;

66
Sculthorpe, 1967 ; Tomlinson, 1969), little information exists concerning the

characteristics of the outer layers of the root.

67
RESULTS

Elodea

Roots of Elodea were bound by a single layer of large epidermal cells that often
appeared irregular in outline (Figure 58). These cells were highly vacuolate, contained
few recognizable organelles and possessed relatively thick outer cell walls. In surface
view, this wall showed evidence of epidermal pitting and peeling, presumably due to the
activities of the numerous bacteria present (Figure 59). At higher magniﬁeations, the
extent of bacterial degradation became more evident. It was particularly advanced within
the outer layer of this wall (Figure 60, 61) where the remaining wall material appeared
as a matrix of loose ﬁbrils of varying electron density (Figure 60). Within the inner
portion of the wall electron translucent lamellae were often found in a loose transverse
arrangement (Figure 60). These lamellae were similar in structure to suberin deposits
found in the walls of hypodermal cells (Peterson et al., 1978). Suberin deposits,
however, are not commonly observed in epidermal cells (Peterson et al., 1978).

Bacteria were not conﬁned to the surface, but were also found throughout the wall
of the epidermis. They were particularly common along the inner tangential wall
between adjacent epidermal cells where the more advance stages of wall degradation
usually occurred (Figure 61). The epidermis was covered by a thin, rather persistent
cuticular layer which appeared to be resistant to bacterial decay (Figure 60, 61).

A layer of closely appressed cortical cells lined the epidermis (Figure 58). Gas
spaces were not produced at this junction (Figure 62). A few electron-translucent

lamellate structures similar to those found in the outer epidermal wall could also be found

68
along the edges of the internal walls of these cortical cells. Walls of cortical cells

internal to this layer, however, were normal in appearance (Figure 63).

Myn’ophyllwn

Roots of Myn'ophyllwn were bound by a layer of small epidermal cells, two layers
of hypodermal cells and a single layer of large cortical cells (Figure 64). Epidermal cells
appeared collapsed in outline and were eroded away from much of the roots surface.
When present, they were characterized by very thin and partially dissolved cell walls and
contained electron dense granular material (Figure 66, 67). Cells of the underlining
hypodermis were thick-walled and usually consisted of 2 distinct cell types. Cells of the
outer hypodermal layer also contained electron dense granular material and showed signs
of dissolution along the side facing the epidermis (Figure 66, 67, 68). The internal walls
of this layer, however, appeared to be more electron dense than those of the bordering
inner hypodermal layer (Figure 68). An examination of this common wall showed that
the two hypodermal layers were separated by a distinct electron translucent band which
was composed of several ﬁne parallel lamellae (Figure 69). This pattern is characteristic
of hypodermal cells walls which contain suberin deposits (Peterson et al. , 1978; Robards
et al., 1979). A similar layer was also found along the inner tangential and radial walls
of the hypodermal cells bordering the cortex (Figure 70). Individual lamellae, however,
could not be resolved within this electron translucent band. Cells of the inner
hypodermal layer also contained electron dense material which formed hemispherieal
bodies that lined the periphery of the cell (Figure 68). Neither gas spaces nor

plasmodesmata were observed between cells of these layers. Bacteria were also not

69
observedwithinthewallsofthesecells. Theyappearedtobeconﬁnedtotheouter

epidermal region.

Numerous organelles were observed within the underlying layer of cortical cells
(Figure 66). They were not found within the hypodermal or epidermal cells (Figure 67,
68). The internal walls of these cells were also examined and did not appear to contain

suberin deposits.

Potamogeton

Roots of Potamogeton were bound by a single layer of large cortieal cells, a
layer of thick-walled hypodermal cells and an oceasional epidermal cell (Figure 71).
Although root hairs were present, most had been sloughed from the root’s surface
(Figure 71, 72). The outer hypodermal wall was typically covered by a thin layer of
electron dense material of unknown origin (Figure 73, 74). Many suberin lamellae were
found along the outer edge of this wall (Figure 75). They also appeared to continue
down the inner tangential wall and along the inner radial wall which borders underlying
cortical cells (Figure 76). The suberin layer divided the wall between these two cells
into a lighter hypodermal and darker cortical side (Figure 76). The walls were also
impregnated with a substance which produced small electron dense inclusions upon
staining (Figure 75, 76). The distribution of these inclusions appeared to be greater
within the walls surrounding the cortical cells than in the walls of the hypodermis (Figure
76, 77).

Suberin deposits were not found in the internal walls of the cortieal cells.

Neither gas spaces nor plasmodesmata were observed between cells of the hypodermis

70

and cortex. Although numerous bacteria were present of the root surface (Figure 72),

they were not found within the walls of these cells.

Lobelia

The root of Labelia was bound by a compact outer layer composed of epidermal,
hypodermal and cortical cells (Figure 78, 79). Epidermal cells were small, possessed
thin cell walls and were usually collapsed in outline (Figure 79, 80). The underlying
hypodermal cells were irregular in shape and possessed a relatively thick outer cell wall
(Figure 79). A rather wide electron translucent lamina was found in the middle of this
wall, which appeared to completely encircle the cell (Figure 81, 82). This layer was
similar to the layer found within the inner hypodermal wall of Myriophyllum roots
(Figure 70) and may also be composed of suberin. Suberin deposits were not found
within the internal walls of the cortical cells. Some of these cells were, however, lined
with the wall remains of adjacent cortical cells which collapsed during the formation of
lysigenous gas spaces (Figure 83). Cellular organelles were not observed within cells
of either the hypodermal or epidermal layers, but were frequently found within the
underlying cortical cells (Figure 79, 83). Neither gas spaces nor plasmodesmata were
observed between cells of these layers. Bacteria also appeared to be conﬁned to the

outer epidermal region of the root (Figure 79, 81).

Figure 58.

Figure 59.

Figure 60.

Figure 61.

Figure 62.

Figure 63.

71

Cross-section of Elodea root. Light micrograph showing size and

arrangement of epidermal and cortieal cells. Bar=0.l mm.

Surface of Elodea root. SEM micrograph showing epidermal peeling and

presence of bacteria. Bar=50 pm.

Epidermis of Elodea root. TEM micrograph illustrating outer and inner
regions of the outer epidermal wall. Note outer cuticular layer (arrows)
and electron translucent lamellae present within the inner region. Bar=1

p.111.

Epidermis of Elodea root. TEM micrograph showing degradation of outer

and anticlinal regions of the epidermal cell (EP) wall. Bar=1 pm.

Cross-section of Elodea root. TEM micrograph of junction between
epidermis (EP) and underlying cortical (C) cell. Walls are lined with a

few electron translucent lamellae (arrows). Bar=1 pm.

Cross-section of Elodea root. TEM micrograph showing walls of adjacent

cortical cells forming a gas space. Bar=2.5 pm.

 

 

 

Figure 64.

Figure 65 .

Figure 66.

Figure 67.

Figure 68.

Figure 69.

Figure 70.

73

Cross-section of Myriophyllum root. Light micrograph showing size and

arrangement of outer layer of cells. Bar=0.1 mm.

Cross-section of Myriophyllum root. TEM micrograph through epidermal

(EP), hypodermal (HY) and cortical (C) cells. Bar=5 um.

Surface of Myriophyllwn root. SEM micrograph showing epidermal

peeling. Bar=20 p.111.

Cross-section of Myn’ophyllum root. TEM micrograph showing
degrading walls of epidermal (EP) and hypodermal (HY) cells. Bar=2.5

um.

Cross-section of Myn’ophyllum root. TEM micrograph at junction

between hypodermal (HY) and cortical (C) cells. Bar=2.5 um.

Cross-section of Myn‘ophyllum root. TEM micrograph of electron
translucent suberin lamellae (arrows) in wall between hypodermal cells.

Bar=0.1 um.

Cross-section of Myriophyllum root. TEM micrograph showing band of
suberin (arrows) in wall between hypodermal and cortical cells. Bar=2.5

pm.

 

 

Figure 71 .

Figure 72.

Figure 73.

Figure 74.

Figure 75 .

Figure 76.

Figure 77.

75

Cross-section of Paramogeton root. Light micrograph showing the size

and arrangement of outer cell layers. Bar=0.1 mm.

Surface of Potamogeton root. SEM micrograph showing root hairs,

presence of bacteria and epidermal peeling. Bar=50 um.

Cross-section of Potamogeton root. TEM micrograph through outer layer

of root showing hypodermal (HY) and cortical (C) cells. Bar=10 um.

Cross-section of Potamogeton root. TEM micrograph of outer wall of

hypodermal cell. Bar=1 pm.

Cross-section of Potamogeton root. TEM micrograph of anticlinal wall
of hypodermal cell showing suberin lamellae and electron dense

inclusions. Bar=0.5 um.

Cross-section of Potamogeton root. TEM micrograph at common wall
between hypodermal (EP) and cortical (HY) cell delineated by electron
translucent suberin lamellae. (Note abbreviations are mislabeled).

Bar=0.5 um.

Cross-section of Potamogeton root. TEM micrograph of cortical (C) cell

bordering hypodermis (HY) and lysigenous gas space (GS). Bar=25 pm.

 

 

Nil:

HY

76

 

Figure 78.

Figure 79.

Figure 80.

Figure 81.

Figure 82.

Figure 83.

77

Cross-section of Lobeh'a root. Light micrograph showing size and

arrangement of outer layer of cells. Bar=0.1 mm.

Cross-section of LobeIia root. TEM micrograph through epidermal (EP),

hypodermal (HY) and cortical (C) cells. Bar=5 um.

Surface of LobeIia root. SEM micrograph showing collapsed appearance

of epidermal cells. Bar=25 um.

Cross-section of LobeIia root. TEM micrograph showing epidermal (EP)

cells and underlying hypodermal cells. Bar=1 pm.

Cross-section of Lobelia root. TEM micrograph at junction of
hypodermal and cortical cell layers showing thick translucent suberin

layers (arrows). Bar=5 um.

Cross-section of LobeIia root. TEM micrograph of inner cortical (C) cell
bordering lysigenous gas space (GS) which is lined with remains of

collapsed cell walls. Bar=2.5 pm.

 

(EP)

78

 

79
DISCUSSION

Hypoderrml cells containing suberin lamellae were found in the roots of
Myn‘ophyllwn, Potamogeton and Lobelia. Plasmodesmata were not observed between
cells of the hypodermis and the epidermal and cortical cells adjacent to this layer. If
plasmodesmata are not present in these walls, it is likely that water and ions transported
through these cells would have to cross the suberin layer. Suberin is known to form a
relatively impermeable barrier which can drastically reduce the rate of transport across
the cell wall (Robards et al., 1973; Ferguson and Clarkson, 1977; Robards et al., 1979).
Although direct evidence does not exist, it is likely that suberin also restricts the
transport of dissolved gases. In the roots of the sand sedge, Carex arenaia, the suberized
walls of the hypodermal cells are thought to form an effective barrier to radial diffusion
of 0, into the root (Robards et al., 1979). In Lobeh'a, transport of 0, through the root
wall was found to provide substantial resistance to 0, release (Sand-Jensen and Prahl,
1982). These observations support the interpretation that suberized walls may restrict
gas transport across the root. The presence of suberin in the wall does not, however,
always indicate a low permeability of the cell to the transport of water or ions (Clarkson
et al., 1978; Clarkson et al., 1987). It is not known how the presence or absence of
plasmodesmata through the suberin layer of hypodermal cells affects root permeability.

Hypodermal cells were not produced in the roots of Elodea. Although electron
translucent lamellae were found in the walls of some epidermal and underlying cortical
cells, these structures differed in both their appearance and location from the suberin

layers observed in hypodermal cells of the other species examined. Since they were

80
typieally found in walls undergoing dissolution, they may represent the degradation

product of some wall component.

Suberization of the hypodermal cell wall may not only retard transport across the
root, but may also restrict the rate of bacterial invasion within the walls of the internal
cells. Roots of Elodea do not appear to produce a hypodermal layer and may therefore
be more susceptible to the activities of soil bacteria. In these plants, roots are produced
adventitiously from the nodes of an upright stem and are often loosely anchored in the
sediments. The need for an outer protective layer may not be as great in this species.
Roots of Myn’ophyllum, Potamogeton and Lobeh'a, however, are produced on an
underground stem or rhizome and buried in the sediments and may have an increased
need for protection. Although suberization of the hypodermis may increase the
resistance of this wall, it must eventually break down, since bacteria and fungi can be
found in hypodermal cells and in cortical cells internal to this layer (Kuo et al., 1981).

Roots of Potamogeton are oﬁen rust-colored and probably contain iron. The
unusual speckled staining of inclusions observed in these roots suggests that iron may
have precipitated within the cell walls. Large crystals of iron have been identiﬁed within
the wall of cortical cells of rice roots exposed to anaerobic conditions (Green and
Etherington, 1977). In Sparrina, iron deposits are conﬁned to the wall of epidermal cells
(Mendelssohn and Postek, 1984). Iron is soluble in water in its reduced state and is
highly mobile within sediments (Ponnamperuma, 1984). The presence of iron within
walls of these roots would suggest that at some point in time release of 0, from the root
was reduced to a level which led to a reduction in the thickness of the oxidized layer and

the penetration of reduced iron into the root (Armstrong, 1979). The extent to which

81
iron penetrates the root should therefore reﬂect both the permeability of the root wall to

iron transport and the point at which oxidation and immobilization of iron occurred
(Taylor et al., 1984).

The amount of 02 released by the root is a function of its surface area, its
resistance to O; diffusion and the difference in 0, concentration between the lacunar
atmosphere of the root and that of the surrounding sediments (Armstrong, 1979). The
development of a suberized hypodermal layer in roots may confer two important adaptive
advantages. First, it may provide a substantial resistance to the radial loss of 0, from
the roots and thus promote long distance transport of O; to the root tip where it is
especially needed for growth and development. Many studies which examine gas
exchange across the roots assume permeability is uniform along the length of the root
(Sand-Jensen and Prahl, 1982; Boston et al., 1987a, 1987b). If the mature root wall is
relatively impermeable to gases, then calculations based on total surface area may not
accurately measure ﬂux of gases from the roots. Secondly, the hypodermis may also act
as a barrier to the entry of phytotoxic compounds. The resistance of this layer may
become increasingly signiﬁcant when, during periods of reduced 0, availability, a
reduction in the oxidative powers to protect the root from the reduced nature of the

sediments occurs.

CHAPTER IV

RESISTANCE TO GAS TRANSPORT

INTRODUCTION

1 0, transport to and release from the roots of submersed plants increases
dramatically during the light (Sand-Jensen et al., 1982; Carpenter et al., 1983; Smith et
al. , 1984; Thursby, 1984; Kemp and Murray, 1986). 0, produced during photosynthesis
is therefore thought to provide the major source of 02 for transport to roots buried in O2
deﬁcient sediments (Wetzel, 1975; Smith et al., 1988). Submersed plants have been
shown to differ in their 0, transport abilities (Sand-Jensen et al., 1982). These
differences may be of both physiologieal (Penhale and Wetzel, 1983; Smith et al., 1988)
and ecological (Carpenter et al., 1983; Smith et al., 1984; Thursby, 1984; Kemp and
Murray, 1986) signiﬁcance.

Gradients of decreasing 02 concentrations from the shoots to the roots are known
to exist within the lacunar atmosphere of aquatic macrophytes (Barber, 1961; Teal and
Kanwischer, 1966; Dacey, 1981; Brix, 1988). 0; transport has been shown to occur
down this gradient at rates consistent with gas phase diffusion (Barber et al. , 1962;
Armstrong, 1964; Sorrell and Dromgoole, 1987). Thus, the lacunar atmosphere of
aquatic plants has traditionally been thought of as a static system where gas transport
occurs purely by molecular diffusion along established concentration gradients.

82

83
Mass ﬂow of gases through the lacunar system, however, has recently been

demonstrated in a number of emergent species. Twa basic mechanisms have been
proposed. One is a ﬂow-through system as described by Dacey (1980, 1981) for
waterlilies, which has also been shown to operate in Phragmites (Armstrong, 1989).
According to this mechanism, pressures develop in young leaves as they heat up during
the day. This pressurization generates a mass ﬂow of gases down the petiole, through
the rhizome and out older leaves of the plant. The other mechanism proposed by Raskin
and Kende (1983, 1985) is based upon a pressure deﬁcit that is generated by the
solubilization of respired CO, into the water surrounding the plant.

In Phragmites, mass ﬂow of O, to the roots and rhizomes during the day
transports roughly 30 times more 0, than is capable by diffusion alone (Armstrong and
Armstrong, 1988). During the night, when pressure gradients no longer are formed,
diffusion becomes signiﬁcant as steep O, gradients develop between the shoots and the
roots (Armstrong and Armstrong, 1988; Brix, 1988; Konealova, 1988).

0, produced during photosynthesis preferentially partitions into a gas phase
(Sorrell and Dromgoole, 1986). This increases not only the 0; concentration within the
lacunar atmosphere of submersed plants (Hartman and Brown, 1967 ; Oremland and
Taylor, 1977), but also the pressure within the lacunar system (Sorrell and Dromgoole,
1988). Thus the potential for mass ﬂow exists in submersed plants. Little, however, is
actually known about the relative signiﬁcance of mass ﬂow and diffusion with respect to
0, transport in these plants. Sorrell and Dromgoole (1987) concluded that the rates of

0, transport they observed from the roots of Egeria could be satisﬁed by diffusion alone.

84
Smith et a1. (1984), on the other hand, report that the 0, transport rates they observed

for seagrasses were greater than that possible by diffusion alone.

The purpose of this study was twofold. First to examine the relationship between
lacunar structure and gas transport by estimating the resistances associated with both
diffusion and mass ﬂow. And secondly, to evaluate the physiologieal/ecological
signiﬁcance of the lacunar system by comparing the transport abilities of selected species.

Diffusion of 02 down a simple stem can most easily be described by Fick’s ﬁrst

law (Armstrong, 1979; Sorrell and Dromgoole, 1987) as:

1.31%). (Equation 1)

where J =ﬂux density (cm3 cm‘2 s“) and AC = concentration gradient (cm3 cm“) and
R=resistance of the tissue to diffusion (s cm").

The ﬂux of 0, down a simple stem by mass ﬂow can be deﬁned by the Hagen-
Poiseuille equation (Nobel, 1983; Sorrell and Dromgoole, 1988) as:

LAT? (Equation 2)

where AP=pressure gradient (kPa cm") and R=resistance per cm to mass ﬂow (kPa s
cm"). Note that in diffusion, the concentration gradient is the driving force, whereas in
mass ﬂow, the pressure gradient is the driving force.

Stems of submersed plants are typically divided into nodes and intemodes.
lacunae form large gas canals that are continuous throughout the length of the intemode,
but are interrupted at the node by perforated plates of cells called diaphragms (Chapter

11). Resistance (R,, s cm") of the intemode to diffusion is determined by:

85

[tr-é; (Equation 3)

where L=length of the stem segment (cm), D =diffusion coefﬁcient of gas in question
(cm2 s") and A=cross-sectional area through which gas is diffusing (cmz’.
Resistance (ri, kPa s cm") of the intemode to mass ﬂow is deﬁned as:

81.1.
Nrtr4

 

where n=viscosity of lacunar gas (kPa s), N =number of gas lacunae and r=mean
lacunar radius (cm). Note that in Equation 3, RI is a function of the diffusion coefﬁcient
of a speciﬁc gas, whereas in Equation 4, ri is a function of n, the viscosity of the lacunar
gases. This comparison emphasizes the fact the diffusion is the movement of a speciﬁc
gas, whereas mass ﬂow is the movement of the bulk gases present.

According to these equations, resistance to transport through the intemode can be
inﬂuenced morphologically by the length of the path and anatomically by the extent of
lacunar development. lacunar structure, however, affects resistances to diffusion and
mass ﬂow differently. Diffusive resistance is a function of the total gas space area and
is not inﬂuenced by the actual number and size of the lacunae (assuming r>than free
mean path of gas, Nobel, 1983). Resistance to mass ﬂow, on the other hand, is a
function of both the number of lacunae and the 4th power of the radius of the lacunae.

Equations which characterize resistances across the nodal diaphragms are
essentially analogous to Equations 3 and 4. Resistance (RN, 3 cm") of the node to

diffusion is determined by:

86

l+2r
..__L (Equation 5)
N 0nd,

where l is the depth of the pore, r, is the radius of the pore, n is the number of pores
across the node and a, is the average area per pore.
Resistance (r_, kPa s cm") of the node to mass ﬂow is analogous to that of the

internode and is determined by:

will (Equation 6)
"I"

where l is the depth of the pore and n is the number of pores across the node. Note
again that resistance to diffusion is a linear function of the total gas space area across the

node, while resistance to mass ﬂow is an exponential function of the radius of the pore.

87
RESULTS

Estimates of resistance to both diffusion and mass ﬂow of gases were determined
for stems of Elodea, Myn’ophyllwn heterophyllum, M. spicamm and Potamogeton.
Resistance values are expressed as resistance per cm stem length and are denoted as R
(s cm”) for diffusion and r (kPa s cm") for mass ﬂow. Morphological and anatomical
data were also collected on the stems and nodes/diaphragms examined. Predicted
resistances were ealculated using this information and are compared with measured
values. Estimates of resistance were used to examine the inﬂuence of lacunar structure
on diffusion and mass ﬂow and to evaluate the potential signiﬁcance of these mechanisms
to gas transport. The results are organized into two sections in this chapter, namely

diffusion and mass ﬂow.

Diffusion

Resistance through nodes.

The inﬂuence of nodal tissue on diffusion of gases through the stem was examined
by comparing the resistances of stem sections with nodes to sections without nodes.
Resistances were measured in stems of three different species, all of a given stem
diameter, and in M. spicatum, across a range of stem diameters (.12-.24 cm). No
signiﬁcant differences in R were detected between stem types (1 node) of the three
species examined (Table 4). Nodes also appeared to have no measurable effect on R in
stems of M. spicatum over the range of diameters examined (Analysis of Covariance,

P < .8222; Table 5).

88
It was assumed that nodes offered little resistance to diffusion of gases through

the stems examined and that the data for both stem types could be pooled for further

analysis.

Resistance and gas space.

In M. spicatwn, porosity values increased with increasing stem diameter (Table
5). Increasing porosity resulted in an increase in the cross-sectional gas space area (A)
and a decrease in R (Table 5). R is deﬁned by Fick’s ﬁrst law as IJDA (Equation 3),
where D is a constant (ie.- the diffusion coefﬁcient of a given gas). Estimates of R
obtained for M. spicatwn were plotted against llDA (Figure 84). R increased as A
decreased (r2= .58). Although the regression of this relationship was highly signiﬁcant
(P<.0001), the slope differed signiﬁcantly from 1 (P<.05), thus suggesting that the
observed relationship roughly approximates Fick’s ﬁrst law.

Stems of M. spicatum are anatomically similar to stems of M. heterophyllum
(Chapter II). When stems of equal diameter (d=.20 cm) were compared between these
two species, signiﬁcant differences in both porosity and A were found (P< .05, Table
6). lacunae occupied a larger proportion of the cross—sectional area of stems of M.
spicatum. This increase in A was associated with a signiﬁcantly lower estimate of R as
compared to stems of M. heterophyllum (P< .05, Table 6). Estimates of R were not
signiﬁcantly different (P > .05), however, when stems of different diameters but equal
A were compared (Table 6). Differences in both lacunar development and A appeared

to account for the observed differences in R between these two species.

89

Estimates of mean porosity, A, and R of Potamogeton and Elodea were
determined (Table 6). Both porosity and A decreased in order from Potamogeton,
Myn’ophyllwn to EIodea. R also increased across species as A decreased. Thus the
relationship of R to 1/DA also appears to be consistent across species of different

anatomies (Chapter II) as well as within a given species over a range of stem diameters.

Predicted values of R.

Estimates of intemodal resistance (R,) were derived from porosity data according
to Equation 3 and calculated for each observation. Measured values of R were plotted
against these predicted values (Figure 85). The regression of this relationship was highly
signiﬁeant (P< .0001), thus conﬁrming a correlation between measured and predicted
values (r’= .73, n= 170). The slope of this line, however, differed signiﬁeantly from
1 (P< .001). This result questions the accuracy of this method of measurement. The
ratio of mean measured R to mean predicted R is listed for each species in Table 6. The
mean measured R for all observations combined was 590 which is 1.5 times greater than
the mean predicted R of 391. Measured R was over 2.5 times the predicted R for stems
of high resistance (Elodea), but agreed reasonably well with predicted values in stems
of low resistance (Table 6, Figure 85).

Estimates of nodal resistance (RN) were predicted for each species according to
Equation 5 (Table 7). The anatomieal data from which these estimates were derived are
presented in Appendix II. Estimates of RN ranged from 13-72 s cm" among the species

examined and tended to increase with increasing values of Rl (Table 7).

90
The signiﬁeance of RN to diffusive gas transport was examined by constructing

hypothetical 1 cm stem segments which contain a single node. The relative values for RN
and R, for this stem were calculated (Table 7). Among the species examined, nodal
diaphragms were estimated to account for only 3-15 96 of the total resistance to diffusion.
These ﬁndings agree well with the observations of measured resistances in that nodes
appeared to have no signiﬁcant effect on the diffusion of gases through the stem (Table

4).

Resistance and 0; transport.

With these estimates of resistance, one can, through manipulations of Fick’s ﬁrst
law, determine the 0, gradient necessary to drive a given rate of 0, transport from the
shoots to the roots by diffusion alone. Two estimates of rates of 0; transport (F) were
obtained from the literature. Sorrell and Dromgoole (1987) measured a transport rate of
6.28 pl 02 h" (1.74x10‘ cm3 O2 s") for individual stems of Egeria. This value
corresponded to the rate of O2 consumption and release by a 5 cm root segment subjected
to Oz-depleted water. Sand-Jensen et al. (1982) measured a rate of .50 pg 02 mg’1 plant
dry wt h“ for plants of Potamogeton under similar conditions. This value was converted
into a mean transport rate of 85.5 ul 0; h" (2.375x10" cm3 02 s“) for a 50 cm stem
using 228 mg dry wt per 50 cm shoot as a conversion estimate (Kemp and Murray,
1986). The two rates of 0; transport are termed LOW and HIGH, respectively.

Under steady state conditions, the rate of 0, transport from the base of the shoot
expressed per unit area is termed the ﬂux density (J, cm3 02 cm‘2 s") and is determined
by the transport rate (F, cm3 02 3") divided by the cross-sectional area of the stem (cm’)

(Sorrell and Dromgoole, 1987). According to Fick’s ﬁrst law, I is also equal to the O;

91

concentration gradient divided by the resistance (Equation 1). Thus the concentration
gradient required to meet a speciﬁed ﬂux can be determined if the resistance to transport
is known. The 0, concentration of the lacunar atmosphere increases during
photosynthesis and can range from 30% (Hartman and Brown, 1967; Oremland and
Taylor, 1977; Roberts and Moriarty, 1987) up to 60% Oz (Sorrell and Dromgoole,
1987). An upper limit of 40% O, in the shoots during photosynthesis was arbitrarily
chosen and the lower limit assumed to be 0% O2 surrounding the roots. These conditions
set the maximum gradient at 40% 0,. Therefore plants whose estimates of AC (AC =JR)
exceed 40% 0, cannot transport enough 0, to the roots by diffusion alone to meet the
O, demand imposed (LOW or HIGH transport rates). The concentration gradients
required to meet both the LOW and HIGH 0, transport rates (F) were determined for
50 cm stem sections of each species using this relationship and values of R measured and
R predicted (Table 6). The results indicate that the LOW rate of 0, transport could be
supported by diffusion alone in all species examined when calculations are based on R
predicted (Table 8). Elodea is the only exception when AC is based on R measured,
since the 0, required to drive diffusion (64%) exceeds the 40% 02 upper limit. The
results also show that only two of these species could supply enough 0, by diffusion
alone to support the HIGH rate of 0; transport. They are Potamogeton, the genus from
which the transport rate was determined, and the larger stemmed individuals of M.
spicatum. Thus resistances encountered along the lacunar pathway are too large in stems
of Elodea, the smaller stems of M. spicatum, and the less porous stems of M.

heterophyllum to support the HIGH rate of 0; transport.

92
Mass Flow

Resistance through nodes.

The inﬂuence of nodes on resistance to mass ﬂow was examined by comparing
stem sections with nodes to sections without nodes (Table 9). The presence of a node in
the stem section signiﬁcantly (P<.05) increased r in all 3 species examined. In M.
spicatum, an increase in r was also detected within the different size classes examined
(P < .05). These values of I were not direct estimates of nodal resistance, since the
resistance of the node was expressed on a per cm stem basis. The data were further
analyzed to determine an estimate of nodal resistance by subtraction of the intemodal
resistance from the total resistance measured.

Intemodal resistance (r,, kPa s cm"), the resistance to longitudinal transport
through the stem lacunae, was examined in M. spicatum and M. heterophyllum (Table
10). No signiﬁcant differences between size classes of M. spicatum stems or between
the 2 species were detected (P > .05). Estimates of ri could not be measured in Elodea
and Potamogeton. Intemodes of Elodea were too short and delicate to handle without
damaging. Intemodes of Potamogeton are interrupted by diaphragms throughout their
length (Chapter 11). Thus measurements of ri for this species also included the resistance
associated with these structures.

Values of ri were predicted for each species using Equation 4 (Table 10). The
anatomical data from which these estimates are derived is given in Appendix II.
Predicted values were very low (.02-. 13) in both Myriophyllwn species. They were,

however, from 1 to 2 orders of magnitude lower than the measured values for these

93
stems. The differences between measured and predicted values may represent problems

associated with detecting a relatively low resistance. The large conﬁdence interval
associated with these measurements supports this interpretation. Stems of Elodea were
characterized by the smallest lacunar radius (Appendix II) and had a relatively large
predicted r, (3.14).

Estimates of nodal resistance (r,, kPa s cm") were obtained by subtracting r, from
r, total resistance measured. For Potamogeton and Elodea, predicted values of r, were
multiplied by 10 to adjust for measurement discrepancies. An estimate of intemodal
diaphragm resistance (rd) for Potamogeton was ﬁrst determined by subtracting predicted
r, from the r of stem sections without nodes and determining a mean r,I by dividing this
value by the number of diaphragms present. The mean r,I ealculated was then multiplied
by the number of diaphragms present in the nodal section. This value represented the
total resistance attributed by the diaphragms. rll was then estimated by subtracting this
value from r.

Measured estimates of r. ranged from 4 to 203 kPa s cm'3 among the 4 species
examined (Table 10). r, was lowest in M. heterophyllum and increased from M.
spicatum, Potamogeton to Elodea. In M. spicatwn, a nearly 5-fold difference in r, was
detected between the smallest (66 kPa s cm'3) and largest (14 kPa s cm”) nodes examined
(P < .05).

Values of r, were predicted for each species using Equation 6 (Table 10). The
anatomical data from which these estimates were derived is presented in Appendix II.
Predicted estimates were typically within an order of magnitude of the measured values

but ranged from 10% to nearly 300%.

94
The relative resistance of nodes and intemodes to mass ﬂow was evaluated by

constructing hypothetical 1 cm stem sections containing a single node. Of the species
examined, nodes accounted for 87-97 % of the measured resistance (Table 10). Resistance
through the intemodes was relatively insigniﬁeant (3-8%) in all species except in Elodea
(13 %).

The total resistance to mass ﬂow was estimated for 50 cm sections of stems for
each of the 4 species examined using measured estimates of r,I and predicted estimates
of r, (Table 10). Since nodes and intemodes are constructed in series, their resistances
are directly additive. Total resistance (r,, kPa s cm") was calculated as the sum of r.
times the number of nodes estimated for a 50 cm stem and ri expressed over the 50 cm
length of the stem (Table 11). Estimates of total resistance for a 50 cm stem ranged
from 300 to 13,770 among the species examined. The lowest r, was determined for
Poramogeton and the highest for Elodea. In stems of M. spicanan, a relatively small
decrease in stem diameter resulted in a nearly 5 fold increase in the total resistance of
the pathway. In Potamogeton, 1'. was determined to be roughly 30 times greater than r,
(Table 10). In the stems examined, there were 8 j: 1 (n=60) diaphragms visible per
intemode. Intemodal diaphragms accounted for only 17% of the total resistance to mass
ﬂow, the remaining 83% attributable to the node (Table 11) since resistance through the

lacunae themselves (without diaphragms) was predicted to be insigniﬁcant (Table 10).

Mass Flow and 0, transport.
The rate of transport (F, cm3 3") through a stem by mass ﬂow is deﬁned by the

Hagen-Poiseuille equation (Equation 2) and is equal to the pressure gradient along a

95
given length of stem (kPa) divided by R (kPa s cm"), the total resistance encountered in

the pathway. Given a speciﬁc 0; transport rate (F), one can, using this equation,
determine the pressure gradient required to support that ﬂow rate. Estimates of LOW
and HIGH O2 transport rates used in the analysis of diffusion (Table 8) were adjusted to
account for mass ﬂow of bulk gases. The lacunar atmosphere was again assumed to be
40% 02, thus rates were multiplied by 100/40 = 2.5 to account for transport of bulk
gases and not just 0,. The AP required to support both LOW (4.35x10" cm3 gas s“) and
HIGH (5.94x10-5 cm3 gas s-l) rates of 0; transport are given in Table 11. Estimates of
AP are very low for Potamogeton, M. heterophyllum and the larger stems of M. spicatwn
and relatively very large in Elodea. Stems of EIodea would require a pressure gradient

5 -60 times larger than the other species examined in order to acheive the same ﬂux rate.

96

 

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106
DISCUSSION

Nodal and intemodal resistance.

Gases were transported down the stem through large gas canals that were
continuous throughout the length of the intemode (Chapter II). These canals were
interrupted at the node, and in Potamogeton throughout the intemode as well, by ﬁnely
perforated diaphragms through which gases must pass. Although the gas phase is
continuous across the pores of the diaphragm, the pores examined in this study were
relatively small (2-5 um in diameter) and occupied only minor fraction of the total cross-
sectional area (.015-.061) of the diaphragm. Studies were therefore to examine the
effect of pores on the resistance to gas transport.

Diaphragms had a signiﬁcant impact on the rate at which gases can be transported
through the stem. In hypothetical 1 cm stem sections containing a single node, the node
accounts for roughly 3-10% of the total resistance to diffusion and 87-97 96 of the total
resistance to mass flow. Although the porosity of the stem decreased dramatically at the
node, diffusion did not appear to be severely affected. This was likely due to the
relatively short pathlength through the diaphragm (1-10 pm, estimated). Resistance to
mass ﬂow, however, is an inverse function of the radius of the pore to the fourth power.
Thus as the radius of the pore decreases, the resistance increases exponentially.

Since nodes had relatively little effect on diffusion, the resistance to transport
becomes largely a function of the porosity of the stem and the length of the diffusive
path. In mass ﬂow, where most of the resistance was determined by the nodes, the

overall resistance of the stem becomes largely a function of the numbers of nodes

107
distributed throughout the length of the stem. These two mechanisms are discussed

separately in later sections.

Review of methods.

The mean measured resistance (R) to diffusion was 1.5 times greater than the
mean predicted R. In stems of relatively low resistance, values of measured R were in
close agreement with predicted values. In stems of high resistance, as in Elodea, the
measured values of R were nearly 4 times greater than the predicted R. Although the
resistance of nodes of Elodea were not examined directly, they were predicted to provide
little resistance. It is not clear why measured values were larger than predicted for this
species.

Stems of aquatic plants were easily susceptible to damage. It is likely that cutting
of the stem segment results in rupture of cells and the blockage of gas canals. Since
diffusion of gases occurs 10,000 times slower in water than air (Leyton, 1975), ﬂooding
of gas lacunae would result in less gas transported and an overestimation of the
resistance. Gas transport can be completely blocked in ﬂooded stems of Potamogeton
infested with chironomid larvae (Appendix III). Such damage to the plant may have a
signiﬁcant impact on gas transport throughout the plant body.

In Phragmites, Armstrong and Armstrong (1988) occasionally found sections of
rhizome with unusually low transport capabilities. This was apparently due to the
production of callus-like wound tissue or 'tylosoids" , which drastically increased

resistance across the node. Occasionally stem sections were also found in this study with

108
uncharacteristically high resistances. These stems were not examined. It is not known

whether a similar type of wound tissue is also produced in stems of submersed plants.
Predicted esitmates of intemodal resistance to both diffusion and mass ﬂow were
based on anatomical observations of fresh tissue. Measured estimates were of the same
order of magnitude as predicted estimates. Predicted estimates of nodal resistance were
based on anatomical observations from SEM photographs taken of nodes that were ﬁxed,
dehydrated and critical point dried. Although this process shrank the stem tissue
(Appendix 11), measured estimates of resistance were also within an order of magnitude

of predicted estimates corrected for shrinkage.

Porosity and diffusion.

The results presented in Chapter 11 support the claim that the lacunar system
occupies a signiﬁcant, yet variable, proportion of the total plant body (Sculthorpe, 1967;
Wetzel, 1975). Porosity values were found to increase for both stems and roots from
Elodea, Myriophyllum to Potamogeton. The purpose of this section was to establish the
relationship between these differences in lacunar development and their resistance to gas
transport.

As the gas space area of the stems examined increased, resistance to diffusion
decreased. The observed relationship closely approximated Fick’s ﬁrst law (Equation 3)
and was valid across species as well as across stem diameters. Resistances varied widely
among species and increased from Potamogeton, M. spicatum, M. heterophyllum to

Elodea. Thus according to Fick’s ﬁrst law, the ability of these species to transport 02

109
by diffusion should decrease in this order. The signiﬁeance of these differences in

resistance with respect to 0, transport to the roots are evaluated in the following section.

Diffusive resistance and 0, transport.

Estimates of RN (6 s cm‘) and RI (249 s cm”) reported by Sorrell and Dromgoole
(1987) for Egeria dcnsa are of the same magnitude as the species examined here. The
LOW 0, transport rate used in the preparation of Table 5 was taken from their work and
represents the rate of 0, released and respired by a 5 cm section of Egeria root exposed
to O; depleted water. The authors calculated a concentration gradient equivalent to 2.7 %
0, over a 50 cm stem in order to meet this rate. Since lacunar 02 concentrations are
often over 50% during photosynthesis and the concentration gradient necessary to satisfy
this rate was much less than that (2.7%), they concluded that the rate of 0, transport to
the roots could be supported by diffusion alone.

Stems of Egeria and the 4 species examined here are typieally at least 1 m long.
Most of the young healthy leaves are concentrated at the shoot apex, leaves decline in
vigor towards the base of the shoot. In addition light is also attenuated down through
the water column, thus concentrating photosynthesis at the shoot apex. A 50 cm segment
of stem tissue was approximated to represent the base of the stem where 0, contributions
to the lacunar atmosphere would be low. Calculations presented in Tables 5 and 7 are
based on this length, thus AC occurs across this distance.

Elodea is very similar to Egeria in growth form. Both produce segmented stems
with whorls of small-lanceolate leaves and adventitious roots at the nodes. The biggest

difference between the Egeria examined by Sorrell and Dromgoole (1987, 1988) and the

1 10
Elodea examined here is stem diameter, .30 cm and .12 cm respectively. Porosity values

for these species were essentially equal, .23 and .25, respectively. The predicted RI for
Elodea (1931 3 cm3), however, is 6 times greater than that for Egeria (311 s cm").
Since AC is proportional to resistance, Elodea would require an 0; gradient 6 times
greater than Egeria to satisfy the same rate of 0; transport into the roots.

Setting the upper limit to the 0; gradient within the lacunar atmosphere at 40% ,
all species examined, including Elodea, would be able by diffusion alone to satisfy the
LOW transport rate. The LOW 0, transport rate was determined for Egen'a (Sorrell and
Dromgoole, 1987) and also may apply to the small rooting biomass of Elodea. The
other species examined, however, have much larger rooting systems and are likely to
have higher 0, transport requirements. The HIGH 0, transport rate used in preparation
of Table 5 is an average for 5 Potamogeton species examined by Sand-Jensen et al.
(1982). Of all species examined, only Potamogeton and the large stemmed M. spicarwn
would be able to transport enough 02 to the roots to satisfy this rate. Thus Elodea, the
small stemmed M. spicatum and M. heterophyllum would not be able to support this rate
by diffusion alone or would require steeper (higher) 0, gradients than the upper limit of
40% 0,. Based on similar calculations, the Egeria examined by Sorrell and Dromgoole
(1987) would require a 37 96 02 gradient, which approaches this upper limit. The
40% upper limit was chosen as an estimate of the average 02 concentration within the
lacunar atmosphere during active photosynthesis. Reported values range from 30%
(Hartman and Brown, 1967; Oremland and Taylor, 1977; Roberts and Moriarty, 1987)
to 60% O, (Sorrell and Dromgoole, 1988). The lacunar 0, concentration is dynamic,

however, and reﬂects the photosynthetic rate of the plant and therefore undergoes large

1 1 1
diurnal ﬂuctuations. Hartman and Brown (1967) report a lacunar concentration of 10%

for Elodea during the night; Oremland and Taylor (1977) report a value of 8% for
seagrasses. If the upper limit to the 0, gradient is set at 10%, all species except Elodea,
would be able to transport 0, to the roots by diffusion alone at the LOW 0, transport
rate. Elodea, the species examined for which this rate may apply would not (17.9%
required). None of the species examined would be able to satisfy the HIGH rate of 0,
transport given a 10% upper limit to the 0, gradient. Even at atmospheric equilibrium
(21% 0,) only Potamogeton would be able to transport enough 0, to the roots by
diffusion alone to sustain this rate.

The HIGH 0, transport rate includes 0, released from the roots and O, utilized
in root respiration. Root respiration alone accounts for roughly 68 % of this rate (Sand-
Jensen et al. , 1982). Reducing the transport rate by this amount (multiply AC in Table
5 by .68) indicates that only Potamogeton could support the respiratory rate of the roots,
given an upper limit of 21% 0,. It could not meet this rate at a lacunar concentration
of 10% 0,, which is an estimate of the lacunar 0, concentration during the night. These
calculations suggest that during low or non-photosynthetic periods, plants with high
internal resistances and long diffusion pathways would not be able to transport enough
0, to the roots to support high rates of respiration and/or 0, release.

Smith et al. (1988) found that in seagrasses, 0, transport to the roots was
sufﬁcient to sustain aerobic respiration during high rates of photosynthesis. The roots
undergo anoxia/hypoxia, however, throughout the night or during periods of low
photosynthesis. Not only do roots undergo anaerobic metabolism during this period

(Smith et al., 1988; Penhale and Wetzel, 1983), but there is also a decline in the amount

112
of 0, released from the roots (Oremland and Taylor, 1977; Smith et al., 1984). This

decline leads to a decrease in the thickness of the oxidized rhizosphere surrounding the
roots and an increase in the exposure to potentially toxic compounds in the sediments
(Chapter III).

The results presented here are consistent with Fick’s ﬁrst law of diffusion
(Armstrong, 1972; 1979). As the gas space area of the stem increases, resistance to
diffusion m. Since diaphragms at the nodes are relatively insigniﬁcant to the
overall resistance of the transport pathway, the ability of the plant to transport gases
throughout the stem becomes largely a function of the porosity of the stem and the
pathlength for diffusion. The signiﬁcance of this relationship becomes apparent when
one compares the relative transport abilities of plants with different porosities. For
example, small stems of M. spicatum would not be able to satisfy the same 0, demand
by the roots as the larger stemmed individuals. This pattern is also observed when a
comparison is made between M. spicaaon and M. heterophyllwn with stems of equal
diameter. The lacunar system of M. spicatum is able to transport more 0, to the roots
than the less porous lacunar system of M. heterophyllum. The 0, transport rates from
which these comparisons were made are within physiologieal limits for submersed plants.
Thus the small differences in porosity and resistance observed between the plants
examined may be of considerable physiological as well as ecological signiﬁcance. These

ﬁndings are further discussed within the ﬁnal summary.

1 13
Mass Flow and 0, Transport

Estimates of r, (.42 kPa s cm") and r. (3.882 kPa s cm") reported by Sorrell and
Dromgoole (1988) for Egeria are of similar magnitude as those measured/predicted in
this study. The resistance summed over a 50 cm stem with an intemodal distance of 0.8
cm is equal to 265 kPa s cm". This value is less than similar values determined for the
species examinedinthis study. ItisSOtimeslessthanthatmeasuredand 18 times less
than that predicted for Elodea.

The gradient required to meet the LOW 0, transport rate by mass ﬂow alone can
be calculated by the Hagen-Poiseuille equation, AP = FR (Equation 2). Both LOW and
HIGH transport rates (F) were adjusted (see results) to account for mass ﬂow of bulk
gases. The pressure gradient (AP) required to meet this rate is equivalent to .001 kPa
over a 50 cm section of stem. Actual gradients measured after the onset of photosynthesis
were .9 kPa m’1 or .5 kPa per 50 cm stem (Sorrell and Dromgoole, 1988). Since
measured gradient (.5 kPa) is greater than that ealculated (.001 kPa) as necessary, mass
ﬂow of 0, is likely to occur during this phase. At the AP observed for Egeria (.5 kPa,
Sorrell and Dromgoole, 1988) all 4 species examined would be eapable of supporting the
LOW 0, transport rate. All species, except Elodea, would also be eapable of supporting
the HIGH 0, transport rate by mass ﬂow.

Pressure gradients generated by Egeria after the onset of photosynthesis
equilibrate rapidly (rate = 0.02 m s", Sorrell and Dromgoole, 1988). Therefore even
though mass ﬂow may occur during the equilibration period, its duration will be
relatively short. Changes in the environment, such as water velocity, light intensity and

temperature, may temporarily induce mass ﬂow by generating pressure gradients along

114
the length of the stem. The pressure gradient established, however, is likely to

equilibrate rapidly, thus reducing the time and potential signiﬁcance of mass ﬂow to 0,
transport.

Equilibration of pressures is not a mechanism for sustained mass ﬂow. For mass
ﬂow to operate continuously, a pressure differential must be maintained. Since the
pressure gradients required to induce mass ﬂow are relatively small, it may be possible
that a decline in the photosynthetic rate along the length of the stem would be sufﬁcient
to generate and sustain a pressure gradient. 1 would suggest that this may have more
signiﬁcance in stems with higher resistances, since a pressure gradient would not
equilibrate as rapidly. This hypothesis would be very difﬁcult to examine. One would
have to discriminate between mass ﬂow and diffusion, and therefore would need to
measure AP as well as AC. The total volume of gases in stems of submersed plants is
relatively small, on the order of 1-4 ml. Thus sampling even 0.1 ml of the lacunar gas
would dramatically alter the pressure. Additional problems may also be associated with
detecting the rather small pressure differentials required to generate mass ﬂow.

Pressure differentials could also be induced in aquatic plants by the solubilization
of respired CO, from the roots according to the mechanism described by Raskin and
Kende (1983, 1985). Since this mechanism does not require thermal inputs, mass ﬂow
could occur during both the day and night. Recent doubt has been shed on the
signiﬁeance of this mechanism for plants growing in aquatic sediments. Aquatic
sediments are typically rich in CO, and reverse gradients into the roots are commonly
observed. This ean result in net CO, diffusion into the plant, thus preventing mass ﬂow

(Raskin and Kende, 1985; Konealova, 1988). Another concern is that as CO, is released

115
from the root, concentration gradients develop around the root. In this situation, mass

ﬂow then becomes regulated by the diffusion of CO, away from the root (Beckett et al.,
1988). If this mechanism occurs in submersed plants, it is likely to be of little
signiﬁcance to 0, transport since roots of these plants typically undergo anoxia during
the night (Smith et al., 1988), when this mechanism should still be operable.

In the waterlily Nuphar, pressure differentials are created between young and old
leaves (Dacey, 1980, 1981). Pressures are produced within young leaves across small
pores in the mesophyll and are generated due to gradients in temperature and water vapor
(Dacey, 1980, 1981; Schréder et al., 1988). Porosity of the leaf increases with age, thus
decreasing the leaf’s ability to pressurize. The increasing pressure in the leaf is
transmitted down the petiole through the rhizome and out the old leaves which serve as
vents. This ﬂow-through mechanism also appears to apply to the emergent species,
Phragmites (Armstrong and Armstrong, 1989). In this rhizotomatous plant, pressures
are generated in young culms and are vented out old culms. Although it has not been
examined, it is difﬁcult to imagine how a mechanism that is dependent on thermal inputs,
such as this one, could operate in submersed plants given the high thermal conductivity
of water.

Since pressurization occurs within these plants during photosynthesis (Sorrell and
Dromgoole, 1988), a mechanism for mass ﬂow exists. However, if mass ﬂow is to be
of any duration, a pressure gradient must be sustained for an extended period of time.
Potamogeton illinoensis is a rhizotomatous submersed plant that produces a small spike
of ﬂowers which emerges up through the water column. These structures may provide

a means of venting pressures which build up in the plant (Appendix III). Thus a pressure

l 16
differential may develop between young pressurized stems and the stems with emergent

ﬂowers in a manner analogous to the ﬂow-through system described for waterlilies

(Dacey, 1980, 1981).

FINAL SUMMARY

Although roots of submersed vascular plants usually comprise around 10% of the
total plant biomass, much variation exists (Westlake, 1965). Among submersed species,
Lobelia and Elodea probably represent the high and low extremes of the rooted
condition. Since submersed plants are capable of taking up nutrients from both the
leaves and the roots (Denny, 1972), the amount of roots a given species produces may
reﬂect the degree to which it depends on the sediments as a source of nutrients
(Hutchinson, 1975). This would suggest then that in Lobeh'a, most of the nutrients are
supplied by the sediments and are taken up by the roots, while in Elodea, most of the
nutrients are supplied from the water column and are taken up across the leaves.
Potamogeton and Myn’ophyllum are intermediate in their root production and may utilize
both the sediments and the surrounding water as sources of nutrients (Barko, 1983).

The availability of nutrients in the sediments increases as the organic content of
the sediments increases (Wetzel, 1975; Ponnarnperuma, 1984). Increasing the organic
content of the sediments also increases the O, demand of the sediments. This results in
an increase in the 0, gradient across the roots buried in these sediments and consequently
an increase in the amount of 0, released by the roots (Armstrong, 1964; 1979; Yamasaki
and Saeki, 1979; Brix, 1989; Weisner and Graneli, 1989). Increasing the organic

content of the sediments has been shown to reduce the growth and vigor of submersed

117

 

 

 

 

 

 

118
plants (Barko and Smart, 1983; Carpenter et al., 1983). It is thought that a high 0,

demand in the sediments may inhibit growth by exceeding the plants ability to transport
sufﬁcient O, to meet this increase in demand (Barko and Smart, 1983; Carpenter et al.,
1983).

The amount of 0, transported to and released from the roots of submersed plants
increases dramatically in the light (Sand-Jensen et al., 1982; Sand-Jensen and Prahl,
1982; Carpenter et al., 1983; Smith et al., 1984; Kemp and Murray, 1986; Sorrell and
Dromgoole, 1986; 1987; 1988). It is therefore thought that photosynthesis provides the
major source of O, for transport in these plants (Sculthorpe, 1967; Wetzel, 1975; Smith
et al., 1984; Smith et al., 1988). In seagrasses, enough 0, is transported to the
roots/rhizomes during periods of active photosynthesis to sustain aerobic metabolism.
During the night or periods of reduced photosynthesis, the 0, concentration within the
lacunar atmosphere decreases dramatically, thus reducing the amount of 0, available for
transport (Oremland and Taylor, 1977; Sand-Jensen et al., 1982; Smith et al., 1984;
Smith et al., 1988). Uptake of 0, from the water is often difﬁcult. Not only is the 0,
concentration frequently low in the water surrounding the plant, but it also diffuses at a
rate 10,000 times slower than in it does air (Sculthorpe, 1967; Leyton, 1975). These
restrictions signiﬁcantly retard 0, uptake, especially under stagnant conditions when thick
boundary layers develop (Sculthorpe, 1967; Westlake, 1967). Therefore, 0, availability
for submersed plants is likely, at least on a diurnal basis, to be in limited supply (Smith
et al., 1984; Smith et al., 1988). It seems reasonable then, to expect that submersed
plants have adapted to reduced sediments in a manner which is conservative with respect

to 0,. One possible adaptation would be to increase 0, availability by increasing the

1 19
porosity and hence lacunar storage eapacity of the plant body. This does not appear to

be an adaptation found among submersed plants, however, since the results of this study
show that, on a cross-sectional basis, species which typically root in sediments with high
0, demands are characterized by tissues of low porosity.

Porosity values differed signiﬁcantly among the species examined. Decreasing
porosity was found to be directly related to an increase in resistance to diffusion and a
decrease in the plants ability to transport 0, to the roots. The differences in porosity
observed between species were evaluated and considered to be of physiological
importance. Under the same conditions, species with low diffusive resistances should
be able to transport more 0, to the roots than species with high diffusive resistances. If
0, transport occurs primarily by diffusion, these results also suggest that species with
higher porosity or low diffusive resistance should be able to support a larger rooting
biomass than species with lower porosity values. This trend was observed among the
species examined. Decreasing porosity was found to be correlated with an increase in
the resistance to diffusion and a decrease in relative root production.

A decrease in the amount of roots a plant produces should, due to a decrease in
the volume of tissue, reduce the total amount of 0, required for respiration (Williams and
Barber, 1967) and should also, due to decrease in the surface area, reduce the amount
of0,released fromtheroot. 'I‘hesefeatureswouldleadtoareductioninthetotal
amount of 0, required for transport. Suberin deposits in the cell wall of hypodermal
cells may restrict the amount of 0, released across the roots. This would restrict release
of 0, from the roots and promote diffusion of 0, down the root to the root tip where it

is especially needed for growth and development.

120
This discussion leads to the following model for the distribution of submersed

plants within lakes. In this model, relative root production and porosity are plotted
against sediment Q demand (Figure 86). Typically within lakes, release of nutrients
from the sediments to the overlying water increases as the Q demand of the sediment
increases (Wetzel, 1975). As the concentration of nutrients in the water increases, the
productivity of phytoplankton also increases which can result in a decrease in light
penetration (Wetzel, 1975). Therefore along a gradient of increasing Q demand, one
would expect to ﬁnd an increasing concentration of nutrients in the water and an increase
in the extinction coefﬁcient for light penetration. The results of this study suggest that
species distributed along this gradient will be characterized by a decrease in the relative
amount of roots produced as well as a decrease in the porosity of the plant body.
LobeIia is typically found in nutrient poor sediments with low Q demands
(Moyle, 1945 ; Seddon, 1972). It produces an abundant root supply by which it obtains
nutrients (Moeller, 1978). In these sediments, Lobeh'a releases enough Q from its roots
to maintain a fully oxidized rhizosphere (Wium-Andersen and Andersen, 1972). It is
characterized by a short rosette growth form, yet may receive enough light for
photosynthesis due to the high penetration of light in these oligotrophic environments.
The high Q storage potential of this species may be due to a large lacunar volume and
a thick cuticle across the leaves which restricts the release of Q. The lacunar system
provides a short and continuous pathway and therefore should also facilitate the transport
of Q from the leaves to the roots. Lobeh’a is unable, however, to transport enough Q
to the roots to exist in more organic sediments characterized by higher Q demands

(Moyle, 1945 ; Farmer and Spence, 1986). Features which promote 0, storage may also

121

A A Labella

 

 

 

 

>.
'65 ‘3 Potamogeton
O o
‘- r:
o
a. 32 Myriaphyllum
Elodea
)
Sediment oxygen demand
)
Nutrients In water
)

 

Extinction coefficient

Figure 86. Model of distribution of submersed plants within lakes.

122
restrict O, uptake during the dark and thus limit the amount of 0, available for transport

to that stored within the lacunar system during the day. This supply may be inadequate
when the O, demand around the roots increases. In this species, both nutrient uptake and
gas exchange would be expected to occur primarily across the roots and very little across
the leaves.

Elodea, on the other hand, is commonly found in eutrophic lakes, where it often
forms large ﬂoating mats (Sculthorpe, 1967). This growth form is likely to be adaptive
for photosynthesis, since light is rapidly attenuated throughout the water column in these
lakes. Eutrophic lakes are nutrient rich systems. The sediment 02 demand is very high
and nutrients are abundant in both the sediment and in the overlying water (Wetzel,
1975). Since this species produces very few roots, nutrient uptake across the leaves
would be expected to predominate. Decreasing the amount of roots buried in these
sediments reduces the amount of 0, required for transport. An efﬁcient transport system,
one characterized by low resistance to diffusion for example, may not be required to
support this small rooting biomass. Features such as thin leaves, thin cuticles and small
lacunar volumes may enhance the release of 01 across the leaves. These features should
facilitate not only the uptake of 02 and CO, from the water column but also the uptake
of nutrients across the leaves. Thus in Elodea, most of the gas exchange and nutrient
uptake would be expected to occur across the leaves and very little across the roots.

Species such as Potamogeton and Myﬁophyllum are intermediate between Lobelia
and Elodea with respect to these characteristics. Stems of Potamogeton were more
porous than stems of the Myn'ophyllwn species examined. These results suggest that, due

to a lower diffusive resistance, Potamogeton would be able to support a larger rooting

123
biomass than Myriophyllwn. Similarly differences in porosity between the Myriophyllum

species would suggest that M. spicatum would be able to support a larger rooting system
than M. heterophyllum. These results, however, could also be interpreted that, at a given
root biomass, M. spicatum would be expected to transport more 02 to the roots than M.
heterophyllum and therefore should be able to root in sediments with higher 0, demands.
The physiological/ ecological signiﬁeance of these ﬁndings should be examined further.

The ability of emergent aquatic plants to transport and release 02 from their roots
appears to be directly related to their distribution; plants with high rates of 0, release are
able to root in sediments with high 02 demands (Armstrong, 1964; 2979; Yamasaki,
19876). This relationship does not appear to directly apply to submersed plants. The
results of this study suggest the inverse. Plants with high diffusive transport abilities are
characterized by large rooting systems which are buried in sediments of low 0, demand.
Plants with low 0, diffusive transport abilities are characterized by small rooting systems
and are typical of lakes with high 02 sediment demands.

The 02 concentration within the lacunar system of submersed plants varies
throughout the day and is highly dependent upon the photosynthetic rate of the plant.
The results of this study showed that a steep 0, gradient between the shoots and the
roots, as is typically found during active photosynthesis, may be sufﬁcient, by diffusion
alone, to satisfy the O, demands of the roots. During the night or during periods of low
photosynthesis, the 0, gradient is likely to be too small to support this rate of O2
transport. This suggests that environmental factors, such as changes in light intensity,
will also affect the ability of submersed plants to transport 02 to the roots. Therefore,

the photosynthetic rate of the plant may determine not only the availability of

124
carbohydrates for root growth, but also the availability of 02 for root respiration and

release to the sediments.

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APPENDICES

APPENDIX I

Standard Curve

A standard curve of 1/ %transported versus resistance was constructed (Materials
and Methods) and used to measure the resistance of stem tissue to the diffusion of CH,
(Figure AI.1). The resistances of capillary tubing inserts were determined using Fick’s
ﬁrst law, R=L/DA (Chapter IV), where L is the length and A is the cross-sectional gas
space area of the tubing. D, the diffusion coefﬁcient of CH, in air, was estimated from
data reported by Boynton and Brattain (1929)(D=.20 cm2 s“, estimated). Resistance
values employed ranged from 130-16800 s cm". The regression was highly signiﬁcant
(P< .0001, r’=.92, n=135).

136

137

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APPENDIX II

Predicted Estimates of Resistance

Estimates of intemodal resistance to both diffusion and mass ﬂow were based on
anatomical observations of porosity, lacunar area and lacunar number from fresh tissue.
Lacunar diameter was estimated from the mean lacunar area. These estimates and
predicted estimates of resistance are presented in Table AII.1.

Estimates of nodal resistance to both diffusion and mass ﬂow were based on
anatomical observations of pore size and frequency from SEM photographs taken of
nodes that were ﬁxed, dehydrated and critical point-dried. A study was conducted to
examine the effects of this procedure on the dimensions of these features. Cross-sections
of fresh stem tissue of Potamogeton were compared with processed tissues taken from
the same stem sections and were examined for porosity, mean pore area and number of
pores per node. Pore diameter was determined from the mean pore area. Processing of
stems resulted in a 26% :t 6% (95% conﬁdence interval, n=9) shrinkage of the tissue.
The data were adjusted to account for shrinkage (Table AII.2). Predicted estimates of

nodal resistance were calculated using these adjusted values (Table AII.2).

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APPENDIX III

Transport Studies in Potamogeton illinoensis

In the ﬂow-through system of waterlilies, a pressure differential is established
between young pressurized leaves and old leaves which serve as vents (Dacey, 1980;
1981). Potamogeton illinoensis was chosen for study to demonstrate the potential for
mass ﬂow in submersed plants. This rhizotomatous plant produces a small spike of
emergent ﬂowers. It was hypothesized that these ﬂowers may provide a means of
venting pressures which may build up in the plant during photosynthesis. If a pressure
differential develops between submersed stems and stems with emergent ﬂowers, then
one would expect a mass ﬂow of gases down the submersed stem, through the rhizome
and out stems with emergent ﬂowers.

As gases ﬂow through the rhizome of waterlilies they become enriched in CH, which
diffuses into the rhizome from the sediments (Dacey and Klug, 1978). Flowers of P.
illinoensis were bagged and sampled for CIL over a 24 hour period (Dacey and Klug,
197 8). The CIL concentration increased considerably during the photosynthetic period
in some bags, while in others CPL levels remained relatively low. The results were
confusing at the time until stems were examined carefully. Chironomid larvae bury into

these stems and eventually ﬂood the lacunar system. Flooding blocks the pathway to

141

142
both diffusion (n=11) and mass ﬂow (n=-11). It may be possible that stems with ﬂowers

that did not release CH4 were infected with these larvae. If the lacunar system were
ﬂooded, CH, could not be transported up from the rhizome and released from the ﬂower.
If this explanation is correct, then release of CH, from the ﬂowers only during the day
would support the hypothesis of mass ﬂow through these plants.

The effect of infestation by chironomid larvae on 0, transport in submersed plants
has not, to my knowledge, been examined. The results presented here suggest that stems
infected with chironomid larvae may not be capable of transporting 0, to roots and
rhizomes. These insects may therefore have a signiﬁcant impact of the physiology and

ecology of the plants they infect.