PHQYORESPSRAYEGN AND RELEASE
OF GRQJXMC CARBGN IN SUBMERSED'
AQUARC VASCULAR PMNTS

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_ WCHMN SEMI-Z WWERSITY.
RICHARD AHTGR HWGH
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University

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This is to certify that the

thesis entitled

PHOTORESPIRATION AND RELEASE OF ORGANIC CARBON
IN SUEMERSED AQUATIC VASCULAR PLANTS

presented by

Richard Anton Hough

has been accepted towards fulfillment
of the requirements for

__Ph..D_._degree in BOtany and Plant
Pathology

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Major p fessor

Date June 22, 1973

 

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ABSTRACT

PHOTORESPIRATION AND RELEASE OF ORGANIC CARBON
IN SUBMERSED AQUATIC VASCULAR PLANTS

By

Richard Anton Hough

A llJ'C-assay for photorespiration (the light induced
uptake of oxygen and release of €02 resulting from glycolate
metabolism) was developed for use in submersed aquatic
plants both in the laboratory and lfl.§l§2- Laboratory

studies with axenic cultures of NaJas flexilis (Willd.)

 

Rostk. and Schmidt indicated that respired carbon dioxide
is refixed extensively in the light, similar to the activity
of plants with the Cu photosynthetic pathway, although
analyses of leaf cross sections and of first 140 fixation
products showed that N. flexilis is not a Cu plant. Hence,
to the extent that C02 is refixed in the light, the 140
photorespiration assay is a measure of net, rather than
gross, photorespiration. Respiration in the light in

.E- flexilis increased with increasing dissolved oxygen con-
centration, indicating presence and enhancement of photo-
respiration, and indicating that net photosynthesis would
decrease with increasing oxygen concentration consistent

with the Warburg effect.

Richard Anton Hough

_ln'§i£u experiments indicated that photorespiration
varies within a day's photosynthetic period in Naias
flexilis, in which afternoon decrease in net photosynthesis
was correlated with afternoon increase in photorespiration,
and a causal relationship was suggested. Photorespiration
and dark respiration were 10-fold higher in fall than in
summer in N. flexilis, which reflects senescence character-
istic of annual plants. Net photorespiration in the

perennial Scirpus subterminalis Torr. in the fall was similar

 

to that in N. flexilis in the summer, but was increased
somewhat under the ice in late winter.

The luC-assay evaluates total release of dissolved
organic carbon from submersed plants simultaneously with
respiration. Kinetics of release of organic carbon with
respect to light vs. dark and to oxygen concentration suggest
that glycolate was not excreted extensively by Naias
flexilis, consistent with the evidence for oxidation of
glycolate in photorespiration. Release of organic carbon
‘in ElEE was relatively low in N. flexilis in summer, but
increased 10-fold in the fall during senescence, which
suggests an increased source of substrate for microbial
metabolism and subsequent enhancement of fall phytOplankton
bloom phenomena. Release of organic carbon was relatively

low in Scirpus subterminalis in the fall but increased

 

somewhat under ice in the winter.

PHOTORESPIRATION AND RELEASE OF ORGANIC CARBON

IN SUBMERSED AQUATIC VASCULAR PLANTS
By

Richard Anton Hough

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1973

This work is dedicated to my father, Jack L. Hough,

and to the memory of my mother, Alice C. Hough.

ii

ACKNOWLEDGMENTS

Initially I must acknowledge the profound influence of
four individuals, without which this work would never have
been undertaken. Foremost is that of my father,

Dr. Jack L. Hough, who engendered my aptitude for natural
science and provided the opportunity for early shipboard
experience in aquatic science. My grandfather, the late
Dr. Anton J. Carlson, kindled my early interest in biology
and physiology. Dr. Lester Ingle provided outstanding
instruction and undergraduate laboratory experience in
biology. Dr. John C. Ayers joined my father in exposing me
to an intangible philosophy of aquatic science and to
numerous practical field techniques.

Early graduate work under Dr. George W. Saunders
resulted in my strong interest in the role of carbon
metabolism in the aquatic ecosystem.

For the direction of the present work I extend most
sincere thanks to Dr. Robert G. Wetzel, who constantly gave
unlimited time and effort in assistance with the work and
provided crucial encouragement of my ideas and approaches
to the problem. I thank the other members of my guidance
committee, Drs. Brian Moss, Clifford J. Pollard, and

N. Edward Tolbert, for helpful advice during the study. In

iii

particular, I gratefully acknowledge the help of Dr. Tolbert,
from whose work and ideas my interest in photorespiration
originally developed.

I thank Madelaine J. Hewitt, Karen E. Hogg, Jane Holt,
Evelyn M. Hough, and Jayashree Sonnad for able technical
assistance, and Mary Hughes and Evelyn Hough for help in
preparation of the manuscript. Numerous discussions with
Dr. Michael J. Klug, Dr. Bruce A. Manny, Kelton R. McKinley,
and W. Sedgefield White were most helpful to me. Assistance
of Dr. Kenneth W. Cummins and Robert C. Petersen in
scintillation counting techniques is gratefully acknowledged.
Dr. Steven Stephenson generously provided advice in
preparation of leaf sections with the Hooker microtome and
in interpretation of material.

The research was supported financially by subventions
from AEC Grant AT-(ll-l)-1599, COO-1599-71, and NSF Grants
GB-15665 and GB—31018X. I wish to thank Drs. George H.
Lauff and R. G. Wetzel for ensuring that generous support
was consistently available.

I have warmest appreciation for my wife, Lynn, who
shared and endured with me the ordeal, and for my sons

Jack Brian and Robert Anton, who flourished in spite of it.

iv

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . .
LIST OF FIGURES.
INTRODUCTION . . .

Productivity of Aquatic Macrophytes
Photorespiration . . . . . .
Background of Methods . . . .
Scope of the Study . . . . . .

MATERIALS AND METHODS , . . . . . .

14c-Assay for Photorespiration .

Variation of Dissolved Oxygen Concentration .
Variation of Total Carbon Dioxide Concentration
Comparison of Presence and Absence of Epiphytic

Microflora . . .

Characterization of Photosynthetic Type
First 140 Fixation Products . .
Analysis of Leaf Cross Sections .

In situ Field Analyses of Photorespiration and.

Organic Carbon Release . . . .
General Procedures . .
Najas flexilis . . .
Scirpus subterminalis .

 

 

RESULTS . . . . . . . . . . .

Light and Dark Respiration and Organic Carbon

Release in Axenic NaJas flexilis
Influence of Dissolved Oxygen .
Influence of Carbon Dioxide . .
Epiphytic Microflora. .

Characterizafiion of Photosynthetic Type
First C Fixation Products . .
Analysis of Leaf Cross Sections .

In situ Studies . . . . . . .
Naias flexilis . . , . . ,
Seirpus subterminalis , , , ,

 

 

 

Page
vii

ix

H

Page

 

 

 

DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 1
Net Photorespiration and Dissolved Oxygen . . . . 81
Carbon Dioxide and pH . . . . . . . . . . . . . . 86
Epiphytic Microflora . . . . . . . . . . . . . . 91
In situ Photorespiration. . . . . . . . . . . . . 92

Najas flexilis . . . . . . . . . . . . . . 92

Scirpus subterminalis . . . . . . . . . . . 95

Release of Organic Carbon . . . . . . . . . . . . 97
SUMMARY AND CONCLUSIONS. . . . . . . . . . . . . . . . 101

BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . 103

vi

LIST OF TABLES
Table Page

1. Relationship of net photorespiration, dark
respiration, and organic carbon release
to dissolved oxygen concentration in
axenic Naias flexilis. Determined as %
initial internal 14C evolved per hour from
relabeled plants 2150 lux, 22C, pH 8.1 -
.2, 0.7 mg C02 1‘ , flow rate 20 ml min-1). . 37

 

2. Relationship of net photorespiration, dark
respiration, and organic carbon release to
total C02 concentration in axenic NaJas
Ekexilis. Determined as % initial internal
C evolved per hour from prelabeled plants,
with mean * SD of duplicate data (2150 lux*,
22 01 pH 8.1 — 8.2, flow rate 14-16 ml
min‘ ) . . . . . . . . . . 51

3. Net photorespiration, dark respiration, and
organic carbon release in NaJas flexilis
in presence and absence of epiphytic
microflora. Carbon dioxide and organic
carbon release in light and dark expressed
as % initial internal l14C evolved per hour
from prelabeled plants, with mean * SD of
duplicates (4842 lux, 23 C, pH 8.1 - 8.2,
0.7 ms 002 1‘1, 9.5 mg 02 1'1, flow rate
13-15 ml min'l) . . . . . . . . 61

 

A. First luC fixation products of photosynthesis
in Najas flexilis and Scirpus subterminalis.
Radioactivity is expressed as a percentage
of the total in all products . . . . . 62

 

 

5. In situ net photorespiration, dark respiration
and organic carbon release in natural NaJas
flexilis (0.5 m depth) and Scirpus
subterminalis (l m depth) in Lawrence Lake,
Michigan. Carbon dioxide and organic carbon
release in light Ifid dark expressed as %
initial internal C evolved per hour from
prelabeled plants, with mean * SD of
replicate data. Flow rate 15-16 ml min"1 . . 72

 

vii

Table
6.

Characterization of encrusting material
removed from 1y0phylized lac-labeled
natural Najas flexilis following in
situ experiments .

 

viii

Page

75

Figure

1.

LIST OF FIGURES

The paths of oxygen and carbon in plant
photorespiration .

Apparatus for obtaining carbon diox de and
organic carbon released from 1 C labeled
submersed aquatic plants. Flow-through
chamber: length 15 cm, diameter 3 cm,
volume 110 m1 . . . . .

Cumulative carbon dioxide release from axenic
Najas flexilis in light and dark in
reIation to dissolved oxygen concentration.
Separate light and dark analyses performed
simultaneously at each oxygen concentration

 

Cumulative carbon dioxide release from axenic
Najas flexilis in the light at medium and
low dissolved oxygen concentrations. Lines
represent duplicate analyses performed
simultaneously at each oxygen concentration

Cumulative carbon dioxide release from axenic
Najas flexilis in light and dark at high
and low dissolved oxygen concentrations.
Lines represent duplicate analyses
performed simultaneously at each oxygen
concentration; all plants in light and then
in dark . . . . . . . . .

 

Cumulative carbon dioxide release from axenic
Najas flexilis in light and dark at high and
low dissolved oxygen concentrations.
Separate light and dark analyses performed
simultaneously, each at low and then high
oxygen concentration . . . . . .

 

Relationship of rate of respiration in axenic
Najas flexilis in light and dark to dissolved
oxygen concentration (O:= rates in light;

A: rates in dark; r = correlation co-
efficient) . . . . . . .

 

ix

Page

17

33

34

35

38

Figure

8. Relationship of lightzdark respiration
ratio in axenic Najas flexilis to
dissolved oxygen concentration
(r = correlation coefficient)

 

9. Cumulative organic carbon release from axenic
Najas flexilis in light and dark in
relation to dissolved oxygen concentration.
Separate light and dark analyses performed
simultaneously at each oxygen concentration

 

10. Cumulative organic carbon release from axenic
Najas flexilis in the light at low
dissolved oxygen concentration. Duplicate
analyses performed simultaneously . .

 

ll. Cumulative organic carbon release from axenic
Najas flexilis in the light at medium
dissolved oxygen concentration. Duplicate
analyses performed simultaneously . .

 

l2. Cumulative organic carbon release from axenic
Najas flexilis in light and dark at high and
low dissolved oxygen concentrations
Duplicate analyses performed simultaneously
at each oxygen concentration; all plants
subjected to both light and dark . .

 

13. Cumulative organic carbon release from axenic
Najas flexilis in light and dark at high
and low dissolved oxygen concentrations.
Separate light and dark analyses performed
simultaneously, each at low and then high
oxygen concentration . . . . .

 

l4. Cumulative carbon dioxide release from axenic
Najas flexilis in the light in relation to
total carbon dioxide concentration (high
C02 = 9.5 mg 1' ; low C02 = 0.7 mg 1' )

 

15. Cumulative carbon dioxide release from axenic
Naias flexilis in the light in relation to
total carbon dioxi e concentration (high
002 = 185.15 mg 1‘ ; low 002 = <o.5 mg 1-1)

 

Page

39

41

42

43

44

45

47

48

Figure

16.

17.

18.

19.

20.

21.

Cumulative carbon dioxide release from axenic
Naias flexilis in light and dark in
relation to total carbon dioxide con-
centration. Light and dark analyses
performed separately, each at low and
then high carbon dioxide concentration

(highm co 186. 74 mg 1-1; low C02 =

<0 gig-)C....

Cumulative carbon dioxide release from axenic
Najas flexilis in light and dark in

relation to carbon dioxide concentration.
Duplicate analyses performed simultaneously

at high and low carbon dioxide concen-
trations; all plants in light and then
dark (high 002 = 150.5 mg 1-1; low 002 =
<0. 5 mg 1-1)

Cumulative organic carbon release from axenic

NaJas flexilis in the light in relation to

t0tal carbon dioxide concentration (high
CO2 = 9.5 mg 1-1; low 002 = 0.7 mg 1-1)

Cumulative organic carbon release from axenic

Nagas flexilis in the light in relation to

o a carBon dioxide concentration (high

002 = 185.15 mg 1'1; low 002 = <0. 5 mg 1- 1) .

Cumulative organic carbon release from axenic

NaJas flexilis in light and dark in relation

to total carbon dioxide concentration.

Light and dark analyses performed separately,

each at low and then high carbon dioxide
concentration (high 002: 186.74 mg l“
lOW C02 = <0. 5 mg 1-1) .

Cumulative organic carbon release from axenic

Najas flexilis in light and dark in relation

to total carbon dioxide concentration.

Duplicate analyses performed simultaneously

Page

49

50

52

53

54

at high and low carbon dioxide concentrations;

all plants in light and then dark (high 002

150.5 mg 1'1; low 002 = <0.5 mg 1' ).

xi

Figure Page

22. Cumulative carbon dioxide release from
Najas flexilis in the light and dark
in presence and absence of epiphytic
diatom populations (bacteria-free
Gomphonema parvulum) . . . . . . 57

 

 

23. Cumulative carbon dioxide release from
Najas flexilis in the light and dark
in presence and absence of mixed
epiphytic microflora (bacteria, fungi,
algae) . . . . . . . . 58

 

24. Cumulative organic carbon release from
Najas flexilis in the light and dark in
presence and absence of epiphytic
diatom populations (bacteria-free
Gomphonema parvulum) . . . . . . 59

 

 

25. Cumulative organic carbon release from
Najas flexilis in the light and dark
in presence and absence of mixed
epiphytic microflora (bacteria, fungi,
algae) . . . . . . . . 6O

 

26. Cross sections of leaves of natural Najas
flexilis. a: Central portion 0 leaf
section with single vascular bundle
(v) and adjacent gas lacunae (l).
b: Leaf section with iodine stained
starch appearing as black bodies (left
lateral portion of leaf folded under
central portion as a result of sectioning
process) . . . . . . . . . 64

27. Cross sections of leaves of Scirpus subterminalis.
a: Section from mid-length of 163?.
b. Section from near distal end of leaf.
Both sections: gas lacunae (l) partitioned
by septa containing vascular bundles (v).
Iodine stained starch appears as black
bodies . . . . . . . . . 66

 

28. Cumulative carbon dioxide release from.natural
Najas flexilis in light and dark in situ
in littoral zone of Lawrence Lake, _Michigan,
at 1230 hrs on 28 July 72, 0.5 m depth
(simultaneous quadruplicate analyses). . 69

xii

Figure

29. Cumulative organic carbon release from
natural Najas flexilis in light and
dark in situ in littoral zone of
Lawrence Lake, Michigan, at 1230 hrs
on 28 July 72, 0.5 m depth (simultaneous
quadruplicate analyses) . . . .

 

30. Cumulative carbon dioxide release from natural
Najas flexilis in light and dark in situ
in littoral zone of Lawrence Lake,
Michigan, 0.5 m depth. Duplicate
analyses in mid-morning, mid-afternoon,
and late afternoon on 15 September 72

 

31. In situ net photosynthesis and net photo-
respiration in natural Najas flexilis,
light intensity, and dissolved oxygen
concentration in littoral zone of
Lawrence Lake, Michigan, 0.5 m depth, on
15 September 72 (data points: mean t SD)

 

 

32. Cumulative organic carbon release from natural
Najas flexilis in light and dark in situ
in littoral zone of Lawrence Lake,
Michigan, 0.5 m depth. Duplicate analyses
in mid-morning, mid-afternoon, and late
afternoon on 15 September 72,

 

33. Cumulative carbon dioxide release from natural
Scirpus subterminalis in light and dark
in situ in littoral zone of Lawrence Lake,
'MIchigan, at 1440 hrs on 14 October 72,

l m depth. Duplicate analyses with
epiphytic microflora removed from plants
(dashed lines); duplicate analyses with
untreated plants (solid lines) ,

 

34. Cumulative organic carbon release from natural
Scirpus subterminalis in light and dark
in situ in littOral zone of Lawrence Lake,
'MIchigan, at 1440 hrs on 14 October 72,

l m depth. Duplicate analyses with
epiphytic microflora removed from plants
(dashed lines); duplicate analyses with
untreated plants (solid lines) , ,

xiii

Figure

35. Cumulative carbon dioxide release from
natural Scirpus subterminalis in
light and dark in situ under ice in
littoral zone of Lawrence Lake,
Michigan, at 1140 hrs on 28 February 73,
l m depth (simultaneous quadruplicate
analyses)

 

 

36. Cumulative organic carbon release from
natural Scirpus subterminalis in light
and dark in situ under ice in littoral
zone of LEWrence Lake, Michigan, at
1140 hrs on 28 February 72, l m depth
(simultaneous quadruplicate analyses)

 

37. Commonly observed variations in light intensity,
dissolved oxygen concentration, net
photosynthesis of natural submersed
macrophyte and phytoplankton populations,
and proposed concurrent variation in
photorespiration

xiv

INTRODUCTION

Productivity of Aquatic Macrophytes

 

 

In the long history of investigations of the ecology
and productivity of aquatic macrophytes (macroalgae and
aquatic vascular plants), the majority of the work has
involved descriptions of plant associations and their
distribution, with estimates of primary productivity based
on measurements of standing crop biomass, as reviewed
by Penfound (1956), Westlake (1963, 1965), Wetzel (1964,
1965) and Sculthorpe (1967). Early extensive work in
physiological aspects of macrophyte growth was performed and
reviewed by Arens (1934), Blackman (1895), Blackman and
Smith (1911a, 1911b), Gessner (1937, 1955, 1959), Gorski
(1929, 1935), Ruttner (1926) and others. Ecological studies
of these plants generally have not included sophisticated
physiological techniques, especially in coupled laboratory
and field studies with manipulative experimental approaches,
and controlling mechanisms in macrophyte productivity remain
poorly known (Wetzel and Hough, 1973). In particular, very
little is known of photosynthesis, respiration, and environ-

mental factors affecting this metabolism in these plants.

DO

The present work was directed toward aspects of these
questions in the light of increasing developments in the
area of plant photorespiration and its relationship to
photosynthetic efficiency and plant productivity.

Net photosynthetic carbon fixation in aquatic
macrophytes is reduced to varying decrees by losses of
respiratory 002 and secretion of soluble organic compounds,
and photosynthetic efficiency is influenced directly by the
rates of these processes. In calculations of primary
productivity, the respiration rate is usually assumed to
be the same in light as in the dark. Physiological
evidence increasingly suggests that such an assumption is
erroneous. The normal process of mitochondrial or dark
respiration may be inhibited in the light in some plants,
perhaps by suppression of glycolysis (Jackson and Volk, 1970).
Furthermore, refixation of respired C02 in the light may be
extensive in submersed aquatic plants (Wetzel and Hough,
1973). These processes would restrict loss of respiratory

C02.

Photorespiration

 

Alternately, the phenomenon of photorespiration, well
known in terrestrial plants (reviewed in Fock and Egle, 1966;

Gibbs, 1970; Goldsworthy, 1970; Hatch, gt a1., 1971; Jackson

and Volk, 1970; Tolbert, 1963; Zelitch, 1964, 1971), enhances
loss of C02 in the light and may be a significant factor in
reduction of photosynthetic efficiency of aquatic macro-
phytes. In this process 02 is consumed and 002 is generated
in the light as a result of synthesis and oxidation of
glycolic acid, a process directly associated with the C3
Calvin cycle of photosynthesis.

The precise pathways of oxygen and carbon dioxide in
photorespiration have been investigated intensively, but
remain in some dispute. Evidence from the laboratories of
Tolbert (Andrews, gt 31., 1971, 1973; Lorimer, gt 31., 1973;
Tolbert, 1971b) and Ogren (Bowes, gt 91., 1971; Bowes and
Ogren, 1972; Chollet and Ogren, 1972a, 1972b) strongly
suggests that glycolate is synthesized in chloroplasts from
phosphoglycolate resulting from oxygenation of ribulose
diphosphate (RuDP; Figure 1). In this reaction oxygen
competes directly with 002 for reaction with RuDP.
Alternative hypotheses for glycolate synthesis were
proposed by Gibbs (1970), involving oxidation of a
transketolase addition complex arising from a hexose
phosphate in (or produced by) the Calvin cycle, and by
Zelitch (1971), involving reductive condensation of C02. The
pathway depicted in Figure 1 appears to be consistent with
the widest range of physiological characteristics of
photorespiration (Andrews, gt a1., 1973).

Glycolate leaves the chloroplasts and, in higher plants,

enters the glycolate—glyoxylate shuttle in peroxisomes

4

02 C02

\/

RuDP <

Calvin
/\\ cycle

P-Glycolate PGA Sugars

 

Glycolate

te Excretion

Glyoxyla
(Algae)

Glycine

C02 J

Serine

Figure 1. The paths of oxygen and carbon in plant
photorespiration.

(Tolbert, 1971a), in which glycolate is oxidized by
glycolate oxidase, with uptake of 0?, to glyoxylate and
hydrogen peroxide. Tolbert (1971b) proposes that

glyoxylate is transaminated to glycine, which leaves the
peroxisomes and subsequently is condensed (perhaps in
mitochondria) to serine with loss of 002, presumably the C02
of photorespiration; Zelitch (1972) proposes direct
decarboxylation of glyoxylate. Serine then may enter a
series of interconversions leading to, among several
possibilities, phosphoglyceric acid (PGA).

The rate of glycolate metabolism (i.e. photorespiration)
is highly influenced by, and is proportional to, oxygen
concentration, light intensity, and temperature. Glycolate
metabolism is also enhanced when low 002 limits photo-
synthesis. The rate at which photorespired C02 is actually
lost from the plant depends on the efficiency of C02
refixation. Regardless of whether the C02 is lost from the
plant or recycled, considerable photosynthetic assimilatory
reducing power is consumed in photorespiration.

Terrestrial C3 plants, in which all green cells
photosynthesize by the C3 Calvin cycle, can lose up to 50%
of fixed carbon immediately through the glycolate pathway
(Tolbert, 1963). In C4 plants, C02 is fixed by the C4
carboxylation pathway (Hatch and Slack, 1970; Kortschak,
.gt.§1., 1965) in the meSOphyll cells and transported via
malic and/or aspartic acids to highly developed bundle sheath

cells, where the C02 is transferred by decarboxylation to the

C3 Calvin cycle (Johnson, et al., 1971). The mesophyll cells
lack photorespiration; they also efficiently refix photo—
respiratory C02 lost from the bundle sheath cells, and

little or no C02 is lost from these plants in the light.

C3 and Cu plants can be distinguished by the following
combination of characteristics,any one of which is reasonable
evidence for the distinction (adapted from Black, 1971, and
from review articles cited above): C4 plants have highly
developed bundle sheath cells, best observed in leaf cross
sections, with unusually high concentrations of chlorOplasts,
other organelles, and starch (elucidated by use of iodine-
potassium iodide stain); C3 leaves lack this "Krantz type"
anatomy. The first 002 fixation products are primarily malic
and/or aspartic acids in C4 plants, and PGA and sugar
phosphates in C3 plants. Photosynthesis is difficult to
light-saturate in C4 plants, while in 03 plants saturation
illuminance is in the range of 10,000 to 45,000 lux.
Photosynthetic temperature optima are 30° - 40° C in Cu
plants, 10° - 250 C in C3 plants. Photosynthetic C02

compensation points are low for 04 plants (0-10 ppm C02),
and high for C3 plants (30-70 ppm C02). Response of net

photosynthesis to 02 concentration is not detectable in
Cu plants, whereas C3 plants exhibit the Warburg effect
(Warburg, 1920), the progressive inhibition of net photo-
synthesis with increasing 02. Photorespiration is not
detectable in C4 plants at any 02 concentration, but is

detectable and is enhanced with increasing 02 in C3 plants

(the probable cause of the Warburg effect). Glycolate
synthesis and glycolate oxidase activity are low in Cu
plants and high in C3 plants; glycolate metabolism can be
inhibited by hydroxymethanesulfonates, with corresponding
reduction in photorespiration in C3 plants. An extensive
and growing list of Cu species exists (Black, gt al., 1969;
Downton, 1971), with obvious implications as to presence or
absence of photorespiration. However, the list consists
entirely of terrestrial plants,and little is known of the
role of photorespiration in higher aquatic plants.

Emergent hydrophytes are exposed to terrestrial
environmental conditions, and photorespiration undoubtedly
can be extensive. C3 metabolism and photorespiration have

been demonstrated in the cattails Typha anqustifolia and

 

T. latifolia (Forrester, gt g1., 1966b; McNaughton, 1966,

 

1969; McNaughton and Fullem, 1969). The C4 photosynthetic
system thus likely would be of adaptive value in many
emergent hydrophytes, particularly in regions of high
temperature and high light intensity, and also in situations
where salt content of the environment adversely affects
internal C02 and water balance (Bjorkman, 1971; Slatyer,
1971). In the latter context, S artina, the dominant plant
of the salt marshes of the east coast of the United States,
appears to be a Cu plant (Krenzer and Moss, 1969). Also
evidently of C4 type is a highly productive floating species

of Alternanthera (C. C. Black, personal commun.) of the

 

family Amaranthaceae, which includes numerous C4 plants.

H

Submersed hydrophytes in a given locality are exposed
to lower maximum 02, light, and temperature than are
terrestrial plants, and photorespiration correspondingly may
be generally of somewhat lesser magnitude in the submersed
plants. Also, the greater resistance of water to diffusion
of C02 relative to air (Raven, 1970) and presence of massive
internal gas lucanae may retard loss of 002 from submersed
hydrophytes and facilitate refixation of C02 regardless of

presence or absence of the C4 photosynthetic system. For

these reasons the Cu system may not be of major adaptive
value, and although few studies have been made, there are no
known C4 submersed hydrophyte species. 0n the other hand,
the glycolate pathway operates in all C3 plants, and it is
probable that photorespiration affects photosynthetic
efficiency in submersed plants in some circumstances,
especially in view of the complexity and variability of
environmental conditions in the littoral zones of lakes. If
photorespiration does exist significantly in some aquatic
plants, its magnitude may vary in different species in
relation to the C3 and C4 photosynthetic pathways and to
environmental conditions, with evolutionary and ecological
implications particularly in terms of competitive
capabilities under conditions of C02-, 02-, and temperature
stress, both in natural and culturally altered water bodies
(Hough and Wetzel, 1972). Such stress often reaches greatest

extremes in the latter situation.

Background 9f Methods

 

At the time that oxygen inhibition of net photosynthesis
was first observed by Warburg (1920), an associated
acceleration of respiration in light was suspected by
several workers (Meyer and Deleano, 1913; Spoer and McGee,
1923; reviewed in James, 1928). Strong evidence that 002
evolution in the light can exceed that in the dark in green
plants was reported initially by Decker (1955, 1957, 1959a,
1959b), who observed a "post-illumination burst" of C02 in
suddenly darkened plants, followed by greatly reduced C02
evolution in continued darkness. Similar effects were
reported by Krotkov's group (Krotkov, gt al., 1958;
Forrester, et al., 1966a, 1966b; Tregunna, gt 21., 1966),
along with other evidence that light and high 02
concentrations stimulate C02 production. Holmgren and Jarvis
(1967) and Moss (1966) devised measurements of steady state
light and dark C02 release into C02-free air using a gas
flow-through system and infrared 002 analysis. This system
was modified for use with l[TC-labeled plants by Goldsworthy
(1966), and further so by Zelitch (1968), in which ll‘LCOQ
evolving into COg-free air flowing past prelabeled leaf discs
is followed in the light and dark; this method is based on
the assumption that photorespiratory C02 is derived from the
most recently synthesized carbohydrates.

The use of 114C in measurements of photosynthesis has

been particularly valuable in studies of aquatic plants, as

first developed for phytoplankton by Steeman Nielsen (1952a).

10

The 140 technique in aquatic macrophytes was treated in
detail by Wetzel (1964), and was viewed as a superior
approach for studies of carbon metabolism in these plants.
Accordingly, the ll‘LC-assay of Goldsworthy and Zelitch
appeared to be well suited for investigations of photo-
respiration in submersed plants, with appropriate
modification to an aqueous flow-through system (Hough and
Wetzel, 1972). Flow-through systems have been used
previously for analyses of photosynthesis and respiration
in submersed macrophytes, initially by Blackman and Smith
(1910), and later by Meyer (1939), Westlake (1967), and
McDonnell (1971; McDonnell and Weeter, 1971). These studies
involved measuring changes in 02 or C02 content of water
flowing past plants; flow-through techniques do not appear
to have been used in conjunction with 114C in aquatic
macrophytes prior to the preliminary study of Hough and
Wetzel (1972). Investigations of 140 loss from prelabeled
macrophytes or algae by Carr (1969), Steeman Nielsen (1955),
and Ryther (1956)(cf. Discussion) were not performed with
flow-through systems. Recycling of respired 14002 in the
light, both internally and from the medium, is a serious
problem in the method (cf. Discussion) and a flow-through
system at least minimizes recycling of ll‘LCOQ leaving the
plants. Other problems associated with closed-bottle
techniques (Sculthorpe, 1967), especially the rapid changes
in 02 and C02 concentrations, also are avoided with a flow-

through system.

ll

Scope of the Study

 

In the initial stages of the present study, techniques

of the 14C photorespiration assay were developed as an

aqueous system. Najas flexilis (Willd.) Rostk. and Schmidt

 

was selected for the major portion of the work because
reliable techniques were available for obtaining axenic
(algal-and bacteria-free) cultures, to ensure that all data
would apply to the plants alone.

Major emphasis was placed on the effects of variation
of dissolved oxygen concentration on rates of respiration in
the light and dark. Influence of carbon dioxide concen-

tration was also tested, partly as a prelude to in situ

 

studies in the field. Effects of presence of epiphytic
algae and other microorganisms also were examined, inasmuch
as these organisms are always present on naturally growing
macrophytes and are highly active metabolically.

To aid the assessment of photorespiration, the status

of the plants used in the study as C3 or C4 was examined,

both in terms of first 14C fixation products and leaf cross
section anatomy.

.£§.§itg lL‘LC photorespiration assays were initiated on
natural plants in Lawrence Lake, southwestern Michigan.
Lawrence Lake is a small (4.9 ha) marl lake in the outwash
apron of a glacial moraine system and receives water of very
high calcium carbonate content. As in most hard-water marl
lakes, rates of phytoplanktonic production are moderate to

low, largely as a result of several nutrient interactions

12

associated with calcium carbonate precipitation (Wetzel, 1965,
1966a, 1966b, 1968, 1972, 1973). The littoral zone is well
developed, and while diversity of submersed macrophytes is
low, the species present dominate the total primary

production of the lake (Rich, gt al., 1971). Najas flexilis,

 

a common annual rooted angiosperm in marl lakes, is fairly
widely distributed in the littoral zone of this lake,
largely at depths less than 1 meter. The perennial rooted

angiosperm Scirpus subterminalis Torr. completely dominates

 

the macrophytic growth between 1 and 7 meters depth, below
which no macrophytes are present. .In situ studies were
initiated with natural N. flexilis as an extension of the

work in axenic cultures, and with S. subterminalis because

 

of its major role in the productivity of the lake. Photo-
respiration assays of two marine angiosperms performed at
this writing are summarized in the Discussion.

Of particular interest in the in gitu work was the
initial testing of a hypothesis (Hough and Wetzel, 1972)
concerning possible variations in photorespiration during
a day's photosynthetic period as an explanation for the
"afternoon depressions" in net photosynthesis in freshwater
macrophytes and phytoplankton, a phenomenon observed by
several workers (cf. Discussion). Inasmuch as net
productivity often is maximal in the mornings, and decreases
thereafter regardless of constant or increasing light

intensity, photorespiration may increase through the day

13

with increasing light intensity, oxygen tension, and
temperature, and possible decreasing CO2 availability.
Conditions are known to develop in this manner particularly
in littoral zones with dense macrophytic growth (Sculthorpe,
1967), especially in calm weather with little turbulence,
and these are precisely the conditions conducive to
accelerated photorespiration. Accordingly, multiple lfl;§l£2.
photorespiration assays were performed in a single day with
concurrent measurements of net photosynthesis and environ-
mental conditions.

The 12.§lEE studies were performed in summer, fall and
late winter, which allowed consideration of seasonal aspects
of respiration.

The 1”C photorespiration assay in an aqueous system
automatically provides data on total released dissolved
organic carbon as well as 002. The release of organic carbon
may be significant in terms of glycolate metabolism. A large
body of literature exists concerning glycolate metabolism
in algae (e.g. Hess and Tolbert, 1967; Bruin, gt gl., 1970;
and many others) which is useful in planning and interpreting
experiments on photorespiration in higher aquatic plants.
However, important differences between glycolate metabolism
in algae and that in vascular plants have been demonstrated.
In particular, some algae oxidize glycolate via a dehydro-
genase enzyme in a reaction which does not use 02 as the
terminal electron acceptor, as opposed to the oxidase

universally present in terrestrial vascular plants (Bruin,

l4

.EE.§l-: 1970; Nelson and Tolbert, 1969). Activity of
glycolate dehydrogenase in these algae is low in relation
to the oxidase in higher plants, and much of the glycolate
is excreted rather than oxidized. Glycolate excretion by
algae, first reported by Tolbert and Zill (1956), has
received much attention by workers in phytoplankton ecology
(reviewed in Fogg, 1971). While this aspect of the glycolate
pathway has not been documented in higher aquatic plants,
the loss of various amounts of recently synthesized
unidentified organic carbon as secreted dissolved organic
compounds has been demonstrated for a few submersed and
floating angiosperms (Khailov, 1970, 1971; Khailov and
Burlakova, 1969; Wetzel, 1969; Wetzel and Manny, 1972;
Hough and Wetzel, 1972). This loss of organic carbon
represents a significant reduction in net photosynthetic
efficiency similar to the effects of photorespiration, and
may involve glycolate excretion to varying degrees,
depending on plant biochemistry and environmental conditions.
Tolbert (personal commun.) recently has found glycolate
dehydrogenase in several marine macrophytes.

The release of dissolved organic matter by submersed
hydrophytes enhances the development of a productive
epiphytic microflora (Allen, 1971; Wetzel and Allen, 1972),
with resulting symbiotic nutrient interactions of much
greater magnitude than exist in the more diluted planktonic
regime. Physiologically the release of organic matter may

represent an incomplete adaptation to the aquatic environ-

lb

ment, but the resulting community interaction may be viewed
in an evolutionary context as a community adaptation to
environmental conditions imposing physiological stress on
the macrophytes (Wetzel and Hough, 1973). Hence, attention
was given in this study to release of organic matter,
including its relation to respiration and photorespiration,
environmental variables, and epiphytic microbial

interactions.

MATERIALS AND METHODS

l4C Assay for Photorespiration

 

Photorespiration was evaluated in terms of C02 loss in
the light in a lLAC-assay adapted from the Zelitch (1968)
technique of following 14C02 evolution from prelabeled leaf
discs. The assay was modified from the flowing gas system
employed by Zelitch to a flowing aqueous system (Hough and
Wetzel, 1972). In each case plant material was incubated
in the light in aqueous medium containing NaHluC03 to develop
a ll‘LC--1abeled cellular organic carbon pool. Plants then
were rinsed thoroughly and placed in specially constructed
tubular glass flow—through chambers (Figure 2) situated
between fluorescent light tubes. The chambers were equipped
with removable opaque jackets and with Tygon tubing at the
intake and outlet ports. Aqueous media were pumped
unidirectionally through the chambers at constant rates,
during which the effluent medium was collected in five-minute
fractions for 30, 50, or 55 minutes and radioassayed for
lACOQ and acid stable organic compounds released by the
plants. Carbon dioxide was separated from organic carbon by
acidification (pH 2.5) and N2 purging.

Laboratory experiments were performed with axenic

cultures of Najas flexilis (Willd.) Rostk. and Schmidt, a

 

submersed, rooted, freshwater angiosperm of the family

Najadaceae. Methods of axenic culture were as described in

16

17

gas manipulation

 

 

 

 

mewum
reservou'

l.___=fl

 

 

 

 

 

sglass
fin

 

 

 

fmsk

Figure 2. Apparatus for obtainiflg carbon dioxide and

organic carbon released from

C labeled submersed

aquatic plants. Flow-through chamber: length 15 cm,
diameter 3 cm, volume 110 ml.

18

Wetzel and McGregor (1968), in which seeds are collected
from natural populations, surface-sterilized, and germinated
(after cold treatment) in sterile growth medium. Cultures
of ca. 60 to 100 seedlings were maintained in erlenmeyer
flasks containing 200 m1 defined synthetic lake water growth
medium at 250C and 5500 lux (16 hr day) in controlled
environmental chambers. Experience enabled the visual
recognition of contaminated cultures, because the relatively
large amount of organic buffer in the medium provided
sufficient substrate for rapid bacterial growth, resulting
in cloudiness of the medium. Sterility tests with Difco
nutrient broth, as well as microscopic examinations, were
performed periodically to support the visual examinations.
Prelabeling for the 140 photorespiration assays was
accomplished by injecting an appropriate volume (usually
1 ml) of known high specific activity (88.6 or 97.2 pCl mi'l)
aqueous carrier-free NaHluCO3 into the culture flasks and
incubating for 30, 60, or 190 minutes at 25°C and 5500 lux.
A final isotope concentration of about 0.5 to 1 p01 ml'1
and an incubation time of 60 minutes resulted in optimum
plant radioactivity.

A COQ-free environment is recommended in the lug

photorespiration assay to avoid dilution of the labeled
cellular organic carbon pool by 120 fixed in the light
during the analysis (Zelitch, 1968). Experimental media

were prepared by omitting additions of carbonate, autoclaving

19

to remove dissolved gases, and then allowing contact only
with COQ—free N2 or 02. Infrared analyses (Beckman 215A)
of 002 purged from acidified medium samples collected from
the flow-through system under N2 showed presence of

0.19 t 0.02 mg C (inorganic) 1‘1, or ca. 0.7 mg 002 1'1.
This value was higher than expected, and exceeded the

theoretical concentration of C02 in water at equilibrium

with air (ca. 0.5 mg 1‘1 at 22c; Raven, 1970); the system
of C02 analysis was operating near its lower limit of
sensitivity, and the figure may be somewhat high. Because
of the high solubility of C02 in water, it is difficult to
render and maintain water absolutely free of C02. Media
treated for C02-removal thus are designated "low-C02" in
this study.

In initial experiments the medium consisted of phosphate
buffer (0.025 M monobasic and dibasic potassium phosphate);
in later laboratory experiments a modified synthetic lake
water growth medium was used containing (in mg 1’1):

CaClg (54), NH4C1 (3.82), MgSOQo7H20 (100), KCl (30),

Kgnpoh (0.56), NTAl (20), and tris buffer? (500). All media
were prepared with deionized, glass distilled water. Adjust-
ments of pH (8.1 - 8.2) were made with 1N NaOH or HCl by
syringe through the stopper in the medium reservoir, or by

pipet, with N2 in the space over the medium. Dissolved

 

l nitriolotriacetic acid
2 tris (hydroxymethyl) aminomethane

20

oxygen concentrations in the media were determined by the
unmodified Winkler titrimetric method (Welch, 1948).

At the beginning of an experiment, the chambers and
tubing were flushed with N2, and aqueous medium was allowed
to flow by gravity into the lower portion of the chamber
(Figure 2); the flow was then stopped and the prelabeled
plants placed in the chamber. The upper portion of the
chamber was seated on the lower portion (silicone-greased
ground-glass fitting), and the flow of medium was resumed.
Rates of flow were controlled at ca. 15 ml min"1 with a
multi-channel peristaltic pump. The rates were similar to
those found by James (1928) to be optimal for measurements
of photosynthesis and respiration.

In initial experiments, the five-minute effluent

fractions were acidified and purged with N2 in a closed
system in which the gas was passed through 10 ml hyamine-OH
to absorb evolved C02. After each fraction was purged,
a 1 ml aliquot of hyamine was collected and radioassayed in
14 ml PPOl/toluene (5 g/l) scintillation mixture with a
Beckman LS-l50 scintillation counter. Purged fractions were
radioassayed for non-volatile lb’C organic carbon by
evaporating 3 ml aliquots on planchets and counting with a
Nuclear-Chicago G-M counter (D-47) of known efficiency.

In later experiments, radioassay of the fractions was

accomplished in a simpler procedure, in which two-ml

 

l 2,5-Diphenyloxazole

21

aliquots of each fraction, and of each purged fraction,

were radioassayed directly by liquid scintillation in a
mixture of 7 ml Triton X-100 and 6 ml butyl PBDl/PBBOE/
toluene (8 g 1-1, 0.5 g 1’1). Counting efficiency was
calculated, using external standard ratios, with an
efficiency regression for chemical quenching obtained by
radioassay of aqueous 14C standards in the scintillation
mixture. Carbon dioxide radioactivity was calculated by
subtracting organic 1ac counts min‘l(cpm) in purged fractions
from total cpm in non—purged fractions.

To relate isotope released in each experiment to total
activity in the prelabeled plant material (allowing
comparison of performance of plants in separate assays),
radioactivity of the plants was determined at the end of
the experiment by combustion of lyophilized plants with a
Packard tritium-carbon oxidizer and radioassay of C02 evolved
(into ethanolamine) in 15 m1 PPO/bis-MSB3/toluene (15 g 1‘1,
1 g 1'1) scintillation mixture. Total cpm released in the
experiment were added to the final radioactivity of the
plants to give initial plant radioactivity, which also
allowed calculation of 14C fixation rates during prelabeling.
Carbon dioxide cpm present in each effluent fraction were
added consecutively, as were organic carbon cpm, to give

cumulative cpm released at each time interval. Cumulative

 

l 2- z4-'-t-Butylphenyl)-5-(4"-biphenyl)-l,3,4,-oxidazole
2? 2— Zl'-Biphenyl)-6-phenol-benzoxazole
3 P-bis- (o-Methylstyryl) -Benzene

cpm at each time interval were then divided by initial
plant radioactivity and multiplied by 100 to give %
initial internal lac released.

Experiments were usually performed both in the light
and in the dark, so that dark respiration could be

accounted for in observations of C02 release in the light.

Variation_g§ Dissolved Oxygen Concentration

 

 

The first series of experiments was performed using

axenic Najas flexilis cultures, with oxygen content of the

 

medium as the experimental variable. Oxygen concentration
was kept low in the medium after autoclaving by allowing
contact only with N2 or was raised to higher levels by
aerating the medium with 02.

In three 30-minute analyses, separate light and dark
chambers were attached in parallel to the same medium
reservoir, with 02 maintained at <5, 14.98, and 21.0 mg 1"1
Two duplicated analyses were performed entirely in the

1

light, one at 3.04 mg 02 l- , and the other at 10.85 mg 02

Further duplicated analyses were performed, one at

0.56 mg 02 1’1 and one at 25.5 mg 02 1'1, in which each
chamber was in the light for 30 minutes, followed by dark-
ness for another 20 or 25 minutes. Finally, an experiment
was performed in which separate light and dark chambers
received medium containing 0.98 mg 02 l"1 for 30 minutes

immediately followed by medium (from a second reservoir)

23

containing 23.1 mg 02 l"1 for another 25 minutes. Other
experimental conditions were constant and are given in

Table 1.

Variation 9: Total Carbon Dioxide Concentration

 

 

 

The effect of presence of relatively large amounts of
total 002 in the medium on the 1LAC photorespiration assay
was evaluated in several experiments using axenic cultures

of Najas flexilis.

 

Two chambers were operated in the light for 50 minutes
in the first analysis (C-l), one receiving the normal
low-C02 medium and the other receiving the same medium to
which had been added 20 mg Na2003 1"1 and which had been
aerated with 0.03% 002 giving 9.5 mg total 002 1’1.
Dissolved oxygen was adjusted to 8.66 mg 1'1 in both
reservoirs.

Two 50-minute replicated assays were performed in the
light (C-2) using HA Millipore filtered (pore size 0.45 um)
water from Lawrence Lake, Michigan, described above. CO2
and carbonates were removed from a portion of the filtered
lake water by acidification and N2 purging (90 minutes);
pH was restored with 1N NaOH, facilitated by addition of
150 mg tris buffer 1'1. Total 002,as determined by
alkalinity titrations and calculations as in Golterman
(1969), was 185.15 mg 1‘1 in the untreated filtered water.
1

Total 002 in the purged water was assumed to be <0.5 mg l-

(i.e. below atmospheric equilibrium), although alkalinity

24

titration indicated 23.28 mg l-l. Tris buffer interferes
with the titration, and its presence in the treated water
likely is responsible fon the relatively high value
obtained. Oxygen was adjusted to 15.9 mg 1-1 in both media.
Other experiments (C-3) were performed using filtered
lake water, in which separate light and dark chambers
received cog-purged water for 30 minutes, immediately
followed by non-purged water (CO2 and carbonates present)
from a second reservoir for another 25 minutes. Alkalinity
titrations indicated total C02 concentrations similar to
those in the previous experiments. Oxygen was adjusted to

l in the

12.0 mg 1"1 in the purged water and to 10.75 mg 1'
non-purged water.

Synthetic lake water medium was used in further
experiments (C-4) in which 002 was removed by acidifying
and N2-purging the distilled water prior to adding the salts,
chelator, and buffer; final pH was 8.1 with no adjustment
necessary. Duplicated assays were performed simultaneously,
one with the C02-free medium (<0.5 mg C02 1'1), and one
with the same medium containing 150.5 mg C02 1"1 added as
NaHCO3 and as atmospheric 002, Equivalent NaCl was added
to the CO2-free medium to balance Na content. Oxygen was
adjusted to 8.14 in both media. All chambers were indubated
in the light for 30 minutes, followed by darkness for 25

minutes. Other experimental conditions in the experiments

were constant and are given in Table 2.

Comparison 2: Effects_g£ Presence and Absence 9f Epiphytic
Microflora

 

 

 

 

Axenic cultures of Najas flexilis were inoculated with

 

axenic Gomphonema parvulum Kutz., an alga which is a common

 

epiphytic diatom in Lawrence Lake, obtained as an axenic
culture from the Indiana University algal culture collection.
Epiphytic growth of the diatoms was allowed to develop in
the N. flexilis cultures in the controlled environmental
chamber for about 4 weeks, at which time cultures still
bacteria-free (tested by nutrient broth inoculation and
microscopic examination) were assayed for photorespiration
and organic carbon release, along with axenic N. flexilis
control cultures of the same age. Experimental and control
chambers, all receiving low C02 synthetic medium from a
single reservoir, were operated in the light for 30 minutes
followed by darkness for 25 minutes.

Axenic N. flexilis cultures also were inoculated with
material scraped from the surface of natural plants in
Lawrence Lake to establish a mixed microbial epiphytic
growth. After about 3 weeks, microscopic examination
revealed dense epiphytic growth of bacteria and fungi of
many kinds, including motile and non-motile unicells, short
chains, and long filaments. No identifications were
attempted. Very few diatoms and other algae were present.
Experimental cultures and axenic control cultures of the

same age were assayed for respiration and organic carbon

26

release simultaneously in the light for 30 minutes, followed
by darkness for 25 minutes. Environmental conditions in

both experiments are given in Table 3.

Characterization of Photosynthetic Type

 

 

First l“C Fixation Products

Procedures for analysis of the 140 fixation products
were essentially those developed by Benson, et a1. (1950),
with the modification of Laing and Forde (1971) in which
chromatographic separation is limited to one solvent system
(butan-l-ol-propionic acid-water), which effectively
separates the initial C3 and C4 14C products when not
confounded by products of subsequent metabolic transforma-
tions.

In each analysis, several axenic Najas flexilis cultures

 

were combined, with a total dry weight of about 200 mg, and
incubated for 10-15 seconds in 300 ml synthetic growth

medium containing 1.5 uCi ml"1

at 4850 lux (22 c). The
plant material was then quickly rinsed in distilled water
and placed in 150 ml boiling 80% ethanol for 15 minutes to
halt photosynthesis and to extract organic products. The
extract was reduced in volume to 2 ml by flash evaporation
at 38 C, and spotted in 2, 5, or 10 ul quantities on
Whatman No. l chromatographic paper adjacent to, and in

h, 14

combination wit C marker standards of glucose-6-

phosphate, representing the C3 phosphate esters, and malic

27

and aspartic acids, representing the first C4 products.
Duplicate chromatograms were prepared for each extract
obtained. The paper was subjected to one-way descending
chromatography, using a butan—l-ol-propionic acid-water
(10:5:7, v/v/v) solvent system. Positions of radioactive
materials were identified by autoradiography (Kodak RPR
medical X-ray film; two-week exposure).

Several analyses were carried out with N. flexilis

cultures. Subsequently, samples of Scirpus subterminalis

 

were collected from Lawrence Lake and analysed similarly,
using filtered lake water as the labeling medium. Epiphytic
material was stripped from the samples as thoroughly as

possible with cheesecloth and cold running tap water.

Analysis 2: Leaf Cross Sections

 

Fresh thin cross sections of leaves of axenic and

natural N. flexilis and of natural S. subterminalis were

 

obtained with a Hooker plant microtome (Lab-Line/Hooker;
prototype unit used). Using starch accumulation, identified
by iodine-potassium iodide staining, as an index of C3
metabolism, the fresh sections were examined in wet mounts
for presence or absence of Cu anatomical system under a
Zeiss phase contrast microscope. Photomicrographs were

taken concurrently with visual observations.

_LQ situ Field Analyses g: Photorespiration and Organic Carbon
Release

 

 

General Procedures

 

For in gltu analyses of respiration and organic carbon
release, four flow-through chambers were clamped vertically
in parallel on a tubular aluminum frame. Lake water was
used directly as the medium; each chamber intake port was
equipped with a filter unit containing a disc of Nitex
plankton netting (diam. 20 mm; mesh size 75 um) to exclude
potentially clogging plankton and debris. Sufficient
lengths of tubing were attached to the outflow ports to reach
the pump and collection bottles at the surface of the lake
from the experimental depth.

All field experiments were performed in the littoral
zone of Lawrence Lake, described above. In summer and fall,
a small boat was used; the bow was moored against the shore,
and the stern, from which the respiration chambers were
lowered, was anchored over 1 meter of water. In winter
equipment was operated through the ice.

Plants were carefully removed from the bottom sediment
with roots intact, and prelabeled in gitu in stoppered flasks
containing lake water and NaHluC03. Plants were then
thoroughly rinsed in fresh lake water and placed in the
flow-through chambers. In all manipulations, exposure of
the plants to direct sunlight was avoided. The chambers were

lowered to the site of natural growth, and the peristaltic

pump was activated. Electricity (110V AC) was provided

by a portable gasoline—powered generator. Five-minute
effluent fractions were collected as usual; after 30
minutes the rack was raised briefly to pull the dark jackets
into place and returned to depth for the remaining 25
minutes. Plants and fractions then were returned to the
laboratory for radioassay.

After the plants were lyophilized, prior to combustion
and radioassay, encrusting material composed of CaCO3 and
ephiphytic microflora was removed from the plant surfaces.
On three occasions this material was weighed and radioassayed
in the same manner as were the plants. A portion of the
material was exposed to concentrated HCl fumes to remove
carbonates, allowing radioassay of residual organic carbon.

During each experiment, environmental conditions were
determined as follows: Temperature was measured with a YSI
tele-thermometer with underwater probe. Light intensity at
depth was measured as per cent intensity at the surface
with an underwater photometer designed by Rich and Wetzel
(1969); absolute incident light intensity at the surface was
measured with a Weston illumination meter (Model 756). Water
samples were collected in a Van Dorn sampler for determi-
nation of pH, total inorganic carbon, and oxygen (techniques

as described above for laboratory experiments).

30

Najas flexilis

 

The initial lfl.§l§E analyses of photorespiration and
organic carbon release in N, flexilis were performed in
quadruplicate on 28 July 1973 at noon. Plants of similar
size and appearance were selected from a healthy bed of
N. flexilis at 0.5 m in depth. The weather was warm and
clear, with 0 wind and 0% cloui cover.

On 15 September 1972 three additional in gitu assays
were performed in duplicate; one in mid-morning, one in early
afternoon and one in late afternoon. Technical difficulties
prevented inclusion of a dark period in the morning assay.
Plants were in early stages of senescence, consistent with
annuals at this time of year, so that careful handling was
necessary to avoid fragmentation. The weather was cool
and clear, with a light breeze and intermittent (25%) cloud
cover.

Environmental conditions during all analyses are

presented in Table 3.

Scirpus subterminalis

 

Photorespiration and organic carbon release in situ in

S. subterminalis were assayed initially in quadruplicate on

 

14 October 1972 in mid-afternoon. Four plants were selected
from a healthy bed at 1 m depth. Epiphytic growth was
removed as much as possible from two of them by gently
stripping them with wet cheese cloth until the brown

diatomaceous material no longer appeared on the cloth; the

31

remaining two plants were left with epiphytic microflora
intact. The weather was cool and clear, with 0 wind and
10% cloud cover.

On 28 February 1973 assays were performed with

.S. subterminalis in quadruplicate at 1 m depth under 21 cm

 

ice cover. Loosely attached epiphytic material was removed
from the plants by hand-stripping. Weather was cloudy-
bright with 10% cover.

PhotoreSpiration assays of S. subterminalis were

 

performed with flow—through chambers 30 cm in length and

220 ml in volume.

RESULTS

Light and Dark Respiration and Organic Carbon Release

in Axenic Najas flexilis

 

Influence of Dissolved Oxygen

 

 

Cumulative C02 release from axenic Najas flexilis

 

generally was enhanced by increased dissolved oxygen
concentration in the light. In Figure 3, the lowest rate of
C02 loss in the light is at.<5 mg 02 1-1- C02 1033 in the

’

dark was relatively independent of 02 concentration, except

at 21 m8 02 1-1, where it appeared somewhat depressed.

Figure 4 indicates a similar effect of oxygen on CO2 loss in
the light. Experiments in which each plant was assayed both
in the light and dark (Figure 5) also resulted in Og-enhance-
ment of C02 loss in the light but not in the dark. The
response of reSpiration in the light to sudden increase from
very low [03] to very high [03] was inducible within a few

minutes (Figure 6).

Cumulative C02 release vs. time in each experiment
(Figures 3-6) was subjected to linear regression and slopes
used to express rate of CO2 release in terms of % initial

internal 1”

C released per hour (Table 1). Correlation of
rates in light and dark with dissolved 02 concentration

(Figure 7; includes data from the 02 series and from control

32

33

 

 

 

l l l l l l
DARK
3 ,_ <5 mg 02 Id —
2 _ —<
LIGHT
]_ —l
0 ’//(’—*—’d
V
2,0 ‘ i l l l l
< DARK
(312
g E 3 __ 14.98 mg 02 I" _.
a l—
(I) Z LIGHT
32' .1
u S2 b 5
O l:
0 .2:
o\° ‘ ” ‘
O i i i l i t
2 __ 21 mg 02 l'| LIGHT a

 

 

 

l l l l l

 

20
MINUTES

Figure 3. Cumulative carbon dioxide release from axenic
Najas flexilis in light and dark in relation to dissolved
oxygen concentration. Separate light and dark analyses
performed simultaneously at each oxygen concentration.

 

34

 

    

 

 

 

A15 I I I I I
1;) 10.85 mg 02 I"
_J
‘z‘
D
LLJ CI 1.0 - r
cr UJ
o: 5
(L3 _
cr 2‘
N :05 I— 3.04 mg 02 I-I —
O E .. .......
O - :::::
0 I l l 1 l
10 2O 30 40 50
MINUTES

Figure 4. Cumulative carbon dioxide release from axenic
Najas flexilis in the light at medium and low dissolved
oxygen concentrations. Lines represent duplicate analyses
performed simultaneously at each oxygen concentration.

35

 

 
    

 

 

 

 

2.5 I I I I
‘4»-
20 P _25'5 mg 02 I'l _.
8 ....... 0.56 mg 02 H
3
2’
E§E§15__ ! A
E '_ 2'.
33 :2.
C: _l
5 E 1.0_ fl
0 2
c,\°
05 _ d
if"... ....... .
IO 20 30 40 50
MINUTES

Figure 5. Cumulative carbon dioxide release from axenic
Najas flexilis in light and dark at high and low dissolved
oxygen concentrations. Lines represent duplicate analyses
performed simultaneously at each oxygen concentration;
all plants in light and then in dark.

 

 

 

 

 

 

 

‘0 T I I I
0.98 mg I'| 23.1 mg I"
02 02
I— <— —-> _.
DARK

10- —
8
I

._J r— .4
g E
at. £5
a P

(I) 22.0- -
l“ _
(I _J
,.S
p.

8 2 — ~
39

1.0? "

lJGHT
1 1 I 1 1
° 20 40 60
MINUTES

Figure 6. Cumulative carbon dioxide release from axenic
Najas flexilis in light and dark at high and low dissolved
oxygen concentrations. Separate light and dark analyses
performed simultaneously, each at low and then high oxygen
concentration.

 

37

«Saga 3 ”:32 s.

 

 

 

 

*wm.H mm.o om.o HN.~ wH.N om.mm hfl.om HmH.H Ho.o~
«co.H ¢N.o om.o ms.~ oo.~ om.m~ om.oa HwH.H mo.mo
oH.o -- . hm.H -- NH.MN mo.mHH HwH.H 0H.wm
-- so.o so A -- sa.~ NH.m~ mH.Ha fimH.H om.mm
NH.m -- no.H 0H.m -- NH.HN om.m~ Han.o mH.om
-- om.H -- Hm.m NH.HN mm.o¢ Hmm.o qw.mo
«m.~ -- o.o ¢~.m -- om.¢H mm.ae Hmn.o Hm.HN
-- mm.m H -- mm.m oa.¢H mo.n~ Hmm.o om.oo
-- NH.o -- -- H¢.H mm.oH mN.NHH HmH.H mq.wo
-- mo.o -- -- N~.H mw.oH so.mHH HmH.H Nn.Nm
-- mm.H .. -- Hm.o «o.m sq.wm amm.o om.mm
-- o~.H -- -- wo.o so.m N¢.mm Hmm.o wm.oq
mH.N -- mm.o Ho.m -- mv mm.a¢ «no.o oq.Hm
-- mo.H -- NH.H mv ~¢.ma mmo.o N¢.Ho
mm.H -- . mm.m -- mm.o mo.wHH HmH.H ofl.wm
-- ¢H.o ma 0 -- no.0 no.0 mH.H¢ HwH.H om.mm
«oh.o oo.o aN.o oa.~ ow.o om.o um.mm HwH.H H~.o~
*N~.H oq.o qH.o om.m w¢.o om.o Hm.on HwH.H wa.oo
an“: N HIHSIMI owumu an»: N an“: N aIH we any: firm alas H0 1 we
xumn paw“; a n a xymm unwfig cocoa Ho 1 cocoa o. .u: sun
nonumo ownmmuo acaumuwmmom mo .me o: Hmnmamym unmam

 

.Afl.cfia.Ha.o~ mum“ aon .fl.H moo we 5.0 .~.m - H.m mm .o NN .x5H omHNV muamfia eufimpmfioua
Scum ~50: you wo>~o>o o: Hangman“ Hmwuaaw N no co:w8houmo .maawxoam mmfiwz oaflwxw aw cowuwuuawuaou cowmxo
vo>~ommav ou ommoamu conumu uwcmwuo paw .aowumuwnmmu Juan .coHuwuHmmououoam you no magmaoaumamm .H oaama

 

38

 

-I
d

DARK

      
  

(fl -O.46)

8
I

RATE OF RESPIRATION
N
o

(%IN|TIAL INTERNAL “c EVOLVED AS 002 PER HR)

   

LIGHT
(r: 0.75)

 

 

l l l
10 20 30
MG DISSOLVED 02 PER LITER

 

0

Figure 7. Relationship of rate of respiration in axenic
Najas flexilis in light and dark to dissolved oxygen
concentration (C = rates in light; A: rates in dark;
r = correlation coefficient).

 

39

 

1.2 I I T I I

,O
O

LIGHT:DARK (RATIO)
9

 

 

 

I I l

 

l
10 20 30
MG DISSOLVED 02 PER LITER

Figure 8. Relationship of lightzdark respiration ratio in
axenic Nalas flexilis to dissolved oxygen concentration
(r = correlation coefficient).

 

40

portions of subsequent experiments in which experimental
conditions were identical to those in the 02 series)

indicated that respiration in the light was positively

related to 02 (r significant at P<0.0l), with approximately
2-fold increase in respiration rate with lO-fold increase
in 02 concentration, whereas respiration in the dark was
relatively unaffected by 02; the appearance of a slight
inverse correlation with 02 was not significant,

Except at very high 02 concentrations, rates of 002
release in the light were lower than in the dark, as
reflected in the light to dark (LzD) ratios (Table 1).
Light to dark ratios correlated positively with 02
concentration (Figure 8; r significant at P<0.0l), with
maxima of about unity at highest 02 concentrations.

Rates of release of organic carbon were calculated
from regression slopes in the same manner as those of CO2
release. Rates of release of organic carbon in each
experiment generally were lower than rates of 002 release
but varied widely from Just slightly lower to over lO-fold
lower than corresponding CO2 release rates (Figures 9-13;
Table 1). Organic carbon release in the dark tended to be
more rapid than in the light. In particular, when plants
were subjected to sudden darkness after an initial light
period (Figure 12), rates of organic release increased
immediately by more than 2—fold followed by a reduction to

rates slightly greater than those in the light.

41

 

 

 

 

   

 

 

 

L2 I I I I T I
- c g. DARK
< 5 mg 02 I"
08? ‘
LIGHT
0.4L- / _
O l L I I l 4
' ' ' ' ' 'UGHT
A ‘1” DARK-
52-:
(n er2~ —
(I m
4 If
0 g 0.8 s —
‘2) _.
< S
CDEZOA~ i
c: 2
O 2
(E 0 I l I I I l
I
DARK
‘2 ,_ 21.1 mg 02 I.I —I
LIGHT
Q8- “
04- _
I I l I I I
0 IO 20 30

MINUTES

Figure 9. Cumulative organic carbon release from axenic
Najas flexilis in light and dark in relation to dissolved
oxygen concentration. Separate light and dark analyses
performed simultaneously at each oxygen concentration.

LI2

.Namdoocmpaseflm possompom mommawcw opmoflagsm
.CoapmspCoocoo Cmmkxo po>aommflp 30H pm pgmfla osp CH mflafixoam

wmwmz oHCoxm Eogm ommoaop Cooamo oflcmwso o>HpmHSESQ .OH mgsmflm

 

 

 

 

 

meDZ__2
om ON 0. o
a m _ I4 d a
W.
IInu
I NH
4 mm
Wm
Dado
Huwv
NnHu
Hqu
I nu
.I .I
mnvmcchvm “WM”
7_ fly
a _ r _ _ _ .WO

43

 

 

 

 

020 I I I I I
K185rng<D§P'
0.16— _
G
.3.
(Z) 3‘ 0.12 —- —+
m z
“:a:
< LU
LJF—
9 Z 0.08 ”‘ ‘
Z ._I
5 S
+—
m —
0 Z 0.04~ ..
c,\<>
I I I I I
0 10 20 30 40 50
MINUTES

Figure 11. Cumulative organic carbon release from axenic
Najas flexilis in the light at medium dissolved oxygen
concentration. Duplicate analyses performed simultaneously.

 

LILI

 

   
 

 

 

 

 

 

03 ' ' IDARK ‘ '
G 06- _. ’ -
(Z) 3’ 0.4~ -
80: z
< E 0.2— /—.a d
O E 0’? c f c T 4 I
g 3 0 ‘ T [DARK T l
2: --€P'
<1 , - -
5 ,: 06 25.5 mg 02 I-l
85 Z 0.4~ a
O
i. 0.2~ -
l L l l l
0 10 20 30 40 50
MINUTES

Figure 12. Cumulative organic carbon release from axenic
Najas flexilis in light and dark at high and low dissolved
oxygen concentrations. Duplicate analyses performed
simultaneously at each oxygen concentration; all plants
subjected to both light and dark.

 

LIB

 

£3
00

I T I
0.98 mg 02 I" 23.1 mg 02 I"

 

 

 

 

 

r~ -<h-——» -———q>-
9
2"_l I— ._
8<
(:22
<:&:
LU
OI-
Oz04— g
-_J
is
o:
g; _ _
o\°
0 60

 

MINUTES

Figure 13. Cumulative organic carbon release from axenic
Najas flexilis in light and dark at high and low dissolved
oxygen concentrations. Separate light and dark analyses
performed simultaneously, each at low and then high oxygen
concentration.

 

#6

Neither rate of release in light nor that in dark
corresponded consistently to dissolved oxygen concentration
in the experiments (Table 1), although the transient rapid

increase in sudden darkness was somewhat greater at

25 mg 02 1‘1 than at 0.56 mg 02 1'1 (Figure 12, Table 1).

Influence gf Carbon Dioxide

 

 

In the first analysis (C-l) using synthetic media,
respiration in the light was similar at low and high C02
concentrations (Figure 14; Table 2).

Respiration in the light was 30-40% lower at high [002]

than at low IEOéI in experiments using filtered lake water
(C—2 and C-3, Table 2); the slower rates appeared to develop
lO-15 minutes after exposure to high [COé] (Figures 15, 16).
Dark respiration was not affected by change in [COé].

In the final analyses (C-u), using synthetic medium,
mean light respiration rates at high [COé] were slightly
lower than at low [Coé] , but the same occurred in the dark,
and L:D ratios were similar at both levels (Figure 17;

Table 2).

Rates of release of organic carbon (Table 2) did not
vary consistently with CO2 concentration, showing
contradictory responses including enhancement in.high CO2
(C—2 and C-4; Figures 19 and 21), decrease in high [COé]
(C-3; Figure 20), and little or no effect (C-l; Figure 18).

47

.AHIH we 5.0 u moo 30H mHIH we m.m u moo nwflsv CompwhpCoocoo
ooHROfio coohgo Hoooo 0o coeogaog 2H pcmfla gee :H mHHonHm

mammz oflCon sogm ommmaog ooHXOHo Congmo o>HpmHSSdo

.JH thMHm

 

mmHDZE/i
on ov Om ON 0—
N00 :9:
- Noo 264
r _ _ — _

 

I I
V. “I
C) C)

I
0.
C)

 

oaaldsaa Zoo

°‘?
0

 

(OM TVNHSINI TVII INI 0/6)

48

.AH-H ms m.ov u moo 20H m IH me mH.mmH u Noo emflev coaeosocoozoo
goHROHo cooggo Hop o oo cofiogaoh CH ocmfla one cH mflfiflxoao
mmwmz oHCoxm Eosm ommoaog momXONU consmo o>HpmHSEdo .ma ossmflm

 

 

 

 

352:2 m
on ow on on o_ o 0|
u u u — + NO
no
- N _ Imo va
munv Timu_ri 1.
- Ixo mm
a ammo
I 00 >>o._ :3 mm
vm
_ _ _ p _ ”.0 .I
w.
0

49

 

2!) I I I I
[JDVV FHCBH

14C»

G
I

 

002 RESPIRED
(% INITIAL INTERNAL

 

 

 

 

60

MINUTES

Figure 16. Cumulative carbon dioxide release from axenic
Najas flexilis in light and dark in relation to total
carbon dioxide concentration. Light and dark analyses
performed separately, each at low and then high carbon
dioxide concentration (high C02 = 186,74 mg 1'1; low 002:
<0.5 mg 1'1).

SO

 

  

 

 

 

 

22 I I

5:3 — HIGH C02

" ------ LOW C02

2; 1.6— a
C32
“1m
E LIJ
a.#
«)2 »— a
“J-
O: .1
«*3
Qt:
L)gos— T

o\°

0

60
MINUTES

Figure 1?. Cumulative carbon dioxide release from axenic
Najas flexilis in light and dark in relation to carbon
dioxide concentration. Duplicate analyses performed
simultaneously at high and low carbon dioxide
concentrations; all plants in light and then dark

(high co2 = 150.5 mg 1-1; low 002 = <0.5 mg 1‘1).

 

.e-o .uoxm ca HLdoHoa omn.o ”m- .N- .HIo .muaxm a“ Ante Ho: Nwm.o sauce 0: Honofioum m

51

.q-o .uaxm ca on News I

 

 

 

 

 

 

 

 

mo.mmm.o No.+om.o oowgom.o mm.Meo.N mo.uNA.A
oN.o om.o oo.o Na.m mo.H m.omH so.oH om.om mo.om
mm.o Ho.o mm.o no.m No.o m.omH eo.oH He.om No.o¢
Snood 83.3.; Sui-.26 oN.IHNm.m oomemq
mm.o oe.o om.o oH.m oN.H m.ov oo.oH eN.HN oo.em
oN.o om.o hm.o no.m o~.A m.ov oo.oH o~.o~ o~.om o-o
oo.o - hm.o oo.H - en.ooa eR.oH mm.mo oo.oo

- oH.o - mm.o eh.ooa mR.oH No.5o om.oo
mm.o - oe.o Ho.A - m.ov mo.AA mm.mo oo.oo

- oN.o - oa.o m.ov oo.HA No.5e om.oo m-o
- NH.o - - me.o mH.moH oo.mH mo.ee om.mm

- mo.o - - og.o m.ov oo.mH om.~o on.mm N-o
- oH.o - - mm.o m.o oo.o ou.mm Ho.H~

- oH.o - - hm.o n.o oo.o oo.oe oN.oo H-o
HIN: N any: N owumu NIH; N flIuL N NIH we HIH we aqu HIw ma uaoE
xugo “swag o u a xtgo uswgq cocoo socoo go a .03 man Iaumaxm

copumo omcmwuo coflumuwamom moo No 0.xwm: ucmam

 

 

.I .AA-afla Ha oH-oH
mama 30am .N.w I a.w ma .0 mm .sxSH omHmV moon oumoflaasn mo om HFsmoE LuHB .mucmHa owaonmamua
scum “so: you po>Ho>o u Hmcuoucw Hmfluwcw N mm pmcfieumuoo .mflaflonm mmflmz owcoxm cw cofiumuucmocou moo

kuou ou ommoaou conumm ogcmwuo paw .cofiumuwamou xuwc .coHumufiamououosa uoa mo QHSmcoHumHom .N oHan

 

52

.AHIH we N.o n mom 30H m Ia we m.m n moo zmficv COHQMngoocoo
moflxoeo oopggo ngoe my coapoaog CH pcmfia who CH mHHHNon
mmwmm vasoxm Eogm ommoama copamo oflcmmso m>mpmHSESo .ma ohzmmm

mmt-DZ:>_

ow ON on om op
_ _ A _ i

 

O

I
I
8
(o l‘IVNtIEIlNI 'lVlllNl 96)

I N00 :9: I

N00 39 I

 

 

'Noeavo OINvoao

 

I—
p—
I—
“I
O

53

.AH-H ms m.ov n N8 33 ”TH me 3.9% u Noo :35 8320888
moHNOHo coogmo Hdep 0p coapmamg CH pnwfia 0:9 CH maafixoam
mmwwz oaCoxm Eosm mmmmams Conamo omcwwso m>HpmHSESo .mH ohsmwm

mmE-DZ__)_
on 0v On ON 0—

, ’ ‘

IIIIIQIIIICIIIII I
I II - N00 30-. I

C)

 

w N00 10.: I

h — p p _

NOBHVO OINVDHO

 

 

 

T
I

g- a

(1),,l "IVNHEIINI TVIIINI %)

54

.AHIH ma m.ov.n moo 30H MHIH we 35.0wa u moo cmfinv
CowpmhpCoocoo ooflxomo Copamo swag Cocp ocm 30H pm comm
«zaopmhmmom coehomgom mmmhamcm xsmu 6cm pswflq .COdeHPCmocoo
momKOHo Conhmo HMPOp op compmaog cm Mgmo oflm pnwma CH mmHHXon
wwwwz omfioxm Sogm ommoaoh Coohwo oflcmwso o>mpmaseso .om ohsmmh

mmHDZE;
00 CV 0N
_ _ _ _

O

 

FIG:

I
I
c!
O

I vE<O I I

NOEIHVO OINVDEIO

 

 

‘V
C5
(OI-("IVNHELLNI 'IVIIINI 96)

 

55

.AHIH m8 m.OV

n moo 30H m IH we m.oma u Noo nmflcv gage gasp one pnmfia

CH mpcmam Ham mmMOprhpCmocoo oanomo Conpmo 30H cam smms pm

NHmSOocmpHseHm cospomsog mommamcm mpmomamdm .COHpmngoocoo

ooflROHo congoo Hgooo Op cogogaoh cg ggoo one pzmfia ca meaexoam
mmmmz oflCoxm Sosm mmmoamg Conamo oflcmwso o>flpmassdo .Hm mazmflb

 

 

 

 

mmHDZEz
oo ow on o
_ _ _ _ A
I Iuo %
N I v.0 WW
I IAIII nXU >>AV4 HmHv
_ _ ¥m<0 L _ W W
a _ H _ o IIAJ
N”
flu
I I «d M v
m m
I I V. O
. o w N
nXu Imv2i . N
I onvmw
xm<o
— p P _ ”IO

 

 

 

56

Epiphytic Microflora

 

Mean rates of light respiration in plants with
epiphytic diatoms (bacteria-free) were slightly higher
than those in axenic controls; dark rates were slightly
lower in presence of the diatoms than in controls.
Consequently, mean L:D ratio in presence of diatoms was
2-fold greater than in axenic controls (Figure 22; Table 3).

Mean rates of light respiration in plants with a mixed
epiphytic microflora (fungi, bacteria) were slightly higher
than in axenic controls. Dark rates were also higher in
presence of the microflora than in controls, with resulting
identical L:D ratios (Figure 23; Table 3).

Mean rates of organic carbon release were about 2-fold
higher in presence of epiphytic diatoms than in axenic
controls both in light and in dark (Figure 24: Table 3).

Mean rates of organic carbon release in plants with a
mixed epiphytic microflora were similar to those in axenic
controls both in light and in dark, except for the first
ten minutes of darkness, during which release rate was
slightly lower in presence of microorganisms than in controls

(Figure 25; Table 3).

Characterization g: Photosynthetic Type

 

 

First lLIC Fixation Products

 

In radiochromatograms of lO-15 second l)‘IC fixation

products of Najas flexilis, no detectable labeling appeared

 

57

 

 

.A83H5>pmg
mEoCOSQEOU mogm-dmpopomnv mCOHpmHSQog 80pmmo oflpzsmflgo mo

 

mommmnm ocm moCmmoaQ CH xsmo ocm pzmfia 029 CH mflafixmam

 

 

mmmwz 80am ommoaoh momxomo Coogmo m>wpmHSESO .mm oaswmm
mwhDZE‘
o: on on o. o co on on o. o
a m 4 a q d

W
1T 3 IMIO
lnv
WI.
18
Ir Maw
mum
8%
NO

I I. w

w<04< N

U_wa< U:>IQ.QN 3

4T
III III
¥m<O xme
p h F P F — NN

 

 

 

 

 

58

 

 

 

 

 

T
' T, DARKl
-—->
0".
3° "6” ........ EPIPHYTIC MICROFLORA 0.33:?"
.3.
2’ — AXENIC "
8 Z _
E E “
0.. I—
(022
u, _
0: .1
NS 0.8 '_ —
C)F—
0 Z
o\° _ _.
0 60

 

MINUTES

Figure 23. Cumulative carbon dioxide release from NaJas
flexilis in the light and dark in presence and a sence
of mixed epiphytic microflora (bacteria, fungi, algae).

59

oo

 

.ASst>hdm
mEoCOsmaoc mongmHNopompv mCOHpmHSQom Bowman oflpznmflmo mo
monomom com monomopm CH mamo pom pswma 05p CH mmaaxoam

 

 

 

 

 

 

 

mmmwz Sosm ommmaos Coogmo owcmwso m>mpwHSSSo .zm madmam
mmeazzz
0V ON 0 I,
a . q A _ %
I L mm
mv
I . I to I W
M O
I. \ O_zmx< I L l 3
I ....... 0.. asuv
.......... ...-... e m<o-_< w %
I o ........... o. O_ >In:am ....... I
. ....... o AI wd .N m
_ xm<o _ _ n

6O

00

.Aodmam ammCdm .wfisopompv whoamONOHE oflpmnmfimo ooxfie mo
ooCmmpm Ugo monomosm CH tho Ugo psmfia 039 CH mHHmeHm

mmwmz Eogm mmmoaop COQNMo oflsmmgo o>mpmassdo

meDZ__2

 

9.0

end

 

 

    

IAIII
vE<O

P

<m04mOmOzz QF>IQEm

_

CD

9.0

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(On—IVNHBINI TVIIINI 96)
NOSE-IVS OINVOHO

6].

.NIHS Nu: mqq.o cocoa o: Honmfioum .mwme paw .chsm .mfiumuomm

 

 

 

 

 

 

 

 

 

 

 

 

 

w
.HIHE H01 Nwm.o cocoa oi Hmnmaoum .Aoouw mfluwuomnv 85H3>uma mamconaaow abumwu oauhsmumm m
. .xump aw mmuaaflfi OH umufim umuu< m
.xumv cw mousafia 0H umuwm H
mo. I36 2. M35 oo. Mono E Ho. M35 Ho. ...-olfio
mH.o om.o oA.o oN.o oo.m oo.o oo.o~ mo.om .mguNsa
oo.o Nm.o oA.o H~.o oo.m AN.o mo.o~ oH.Ho Iaag\s
oo..IeA.o no. wan.o oo. moA.o No. Hom.o No. moo.m No.anm.o
HH.o mm.o os.o Nm.o Am.m No.o oN.m~ om.eN
NH.o Nm.o oH.o oH.o oN.N mm.o om.o~ om.om uaagxa
oo. wood S. we: oo. wood 2. Memo 2. Moog om. Hoe;
eo.o «H.A oo.o No.o om.~ No.A mA.oH oA.oo .Mgusna
oo.o em.H eo.o oo.o mo.m Ho.A mo.mA mo.mo Iaag\s
oo. Memo om. H25 3. Memo oo. IRZo 24%;... No. ...-”S; .
o~.o mo.o ow.o mo.o om.m NH.A NA.oA mo om
Ho.o oo.H oe.o Hm.o oN.m oo.H He.oA oo.Hm aflagx<
A-u: N HIM: N AI»; N oaumu A-N: N AI“: N AI»: HIw we coauao
mxumn Axumo ucmga a u S xugo unwaa No a .uz mun -aoo
conumo owsmmuo cowumuwmmmm Nam 03 ucmam uamam

 

 

.AHICNE a8 mHImH mum» 30Hw .aIH moo we N.o .m.@ I H.w ma .0 mm .NSH quqv moumowflasp mo om Mucous

nuNB .mucwam coamnwamum souw use: Mom po>Ho>m u: gospoucw wauwcfl N mm oommoumxo xuwp can usmfla
cw ommoaou conumo owcmwuo paw opwxoflv conpmo .mpoHMONUNE owumnafiao mo mucomnm new monomoua CH

mwawxoam mmfimz aw mmmmHmu conumo oflcmwuo cam .cowumuwamou xumo .aoHumuwamououosa uoz .m manna

 

62

in the positions of malate and aspartate; all activity

appeared as single or closely connected double spots of

10W Rf corresponding to positions of the C3 phOSphate-
esters (Table 4). Proper separation of products was checked
by co-chromatography with marker standards and a marker dye
with samples.

Table 4. First 14C fixation products of photosynthesis

in Najas flexilis and Scirpus subterminalis. Radioactivity
is expressed as a percentage of the total in all products.

 

 

 

 

 

 

'fi, flexilis S, subterminalis
C3 P—esters > 99% > 90%
Ch Acids - - O-lO% ?
Other - - 0-10% ?

 

Scirpus subterminalis also initially fixed lI‘LC

 

predominantly in compounds corresponding in Rf to C3
phosphate-esters. A small amount of activity moved further
to one or two positions of higher Rf; the identity of this
material was not evident in the one-way system, but further
characterization was considered unnecessary inasmuch as it

was clearly not among the major first fixation products.

Analysis 9: Leaf Cross Sections

 

Cross sections of Najas flexilis (Figure 26) revealed

 

a single, centrally located vascular bundle, surrounded by

waTcH Tor LHJP
(Tm

 

63

.Ammmoomm maficoapomm mo padmm»

m mm coapgom Hmppcoo Novas oooaom mama mo soaphog Hmampma
pmmav mmwoop xoman mm mcfipmoaam nogmpm omcfimpm oCHUOH spa:
cowpomm mood no .AHV mmCSomH mom pdoommow ocm A>V oaocsn

pmHSomm> mamcfim Spa: Coapomm mama mo soappom ngpcoo "w
.mHHmeam mmwmz HmHstc mo mm>mma mo mCOHpomm mmopo .mm ohzmflm

 

 

.mm ohdwam

NW .mmfioop xomap mm mammmmm :ohwpm oochpm
mcHoOH .A>V mmHUCSQ amazomm> wchNMNcoo mpaom an cocoap
Iapgmm AHV omCSomH mow "chHpoom npom .mmoa mo cam Hmpmfio
ammo Eopm coapoom ”n .mmoa mo numCoHIofie Sony soapoom "m
.mHHmCHshmpnzm msmwaom mo mo>mma mo mcofipoom mmoao .Nm oasmfim

66

 

.Nm ogsmfim

67

a single layer of large cells. Two moderately large gas
lacunae are present adjacent to the vascular bundle, each
also surrounded by a single layer of large cells. The
remainder of the leaf consists of a double layer of small
cells extending laterally from each gas lacuna. The cells
surrounding the vascular bundle are not surrounded by
extensive mesophyll tissue, nor do they resemble the highly
developed bundle sheath cells of C4 plants. Furthermore,
starch production appears to occur substantially in all

cells and is not largely restricted to bundle sheath cells
as in Cu plants.

Scirpus subterminalis has 3 to 5 vascular bundles

 

Figure 27), also surrounded by a single layer of cells, and
separated by large gas lacunae. The remainder of tissue
consists of a single layer of small epidermal cells with an

inner layer of large cells. As in Najas flexilis, the cells

 

surrounding the vascular bundles are not highly different-
iated, nor are they surrounded by mesophyll tissue. Also,
starch production is almost entirely restricted to the

epidermal cells.

'In situ Studies

 

Najas flexilis

 

In situ rates of respiration in Najas flexilis at mid-

 

day in July (Figure 28) were lower in the light than in dark,

with rates and L:D ratios (Table 5) similar to those found

68

in axenic laboratory cultures at medium oxygen
concentrations. Rates of organic carbon release

(Figure 29: Table 5) were 1/3 to 1/6 respiration rates,
generally similar to rates of organic release in axenic
cultures, but were somewhat greater in light than in dark,
contrary to results in axenic cultures.

In September, respiration rates in the light were
greater than in the dark (Figure 30), with corresponding
L:D ratios greatly exceeding unity (Table 5). Both light
and dark rates exceeded those in July about lO-fold. The
mean rate in the light in September was lowest in the
morning and highest in the late afternoon (Figure 31).

Mean rate of net carbon fixation was highest in the morning
and lowest in the late afternoon. Dissolved oxygen
concentration was lowest in the morning and rose slightly
through the day, as did temperature. Light intensity was
highest in early afternoon.

Rates of release of organic carbon in September
(Figure 32) were about 3 to 6-fold those in July (Table 5).
Rates were highest in early afternoon.

Epiphytic encrustation on Najas flexilis increased in

 

dry weight by 2-fold from July to September (Table 6).
Microscopic examination revealed diatoms and other algae and
microorganisms in an amorphous matrix. Radioactivity in

the material in September was also nearly 2—fold that in

July. The relative distribution of 140 in organic matter

59

 

2.4 I I I T

      

Najas flexIlIs
Inmm

 

0.8 —

C02 RESPIRED
(O/o INITIAL INTERNAL 14C)

 

 

O l l 1 1
20 4O 60

MINUTES

 

Figure 28. Cumulative carbon dioxide release from natural
Najas flexilis in light and dark_in situ in littoral zone
of Lawrence Lake, Michigan, at 1230 hrs on 28 July 72,
0.5 m depth (simultaneous quadruplicate analyses).

 

70

.Amomzadcm opmomagdhomsw mSomCmpHSEHmv Cpmou 8 m.o

«mp Nash mm Co wCC bmma pm «meHCOHE nomad ooCoCBmQ mo

oCoN ACCOPPHH CH Spam Ca Mame qu psmma CH mHHonHC mmflmz
Hmhdpmc EOCC ommoama Conhmo omCmmCo w>fipmasaSo .mm oasmfim

 

wm-FDZ__2
00 0». ON

 

   

 

 

 

%

_ _ _ _ 0 WM

_ m

I I Hv
T

I IvtomM
stw c. Ii

3

I 9:on 8.62 I H

N”

_ _ xm<o- _ _ de

@-

NOBHVD OINVOHO

71

 

     

 

 

 

I I l I
20_ DARK _
Najas erXIIIs
in mtu
16— -
A //I
.0 — / —
:-
8:
EMIZ— -
1F
(02
LL]—
0:_, '//o
65 7 / ‘
I—
”Z
o\° 8_ _
'— / / , 7
I," /
I," n
"'4 // I..-
4._ c / _-
/ ,c
,/ -------- I015-ms hrs
——— 1345-1445hrs
” — I630-I730hrs ‘
- - I I .
° 20 40 60

MINUTES

Figure 30. Cumulative carbon dioxide release from natural
Najas flexilis in light and dark i_n situ in littoral zone
of Lawrence Lake, Michigan, 0.5 m depth. Duplicate
analyses in mid-morning, mid—afternoon, and late afternoon
on 15 September 72.

72

 

 

 

 

 

 

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.an«a as cauma mum» 30A»
Hmwuacg N am commuuaxo xunv vac uzwfia CH wmwodm» conuwo oacuxuo was «cwxoflv conuqu

.ms now mN co al~a «01 omn.o uawuxu .mucoaauoaxm Haw :« alga “on coo.o cu:00 u: donwfiuums
o~.QHno.o m~.QflOo.o m~.Qflmo.~ os.uflom.o hprMwe.n~ Ammwmwho.u
mm.o ¢~.o -.H mo.- qo.o~ oo.m ~o.~h~
on.o mo.o mc.~ am.o w~.o mm.~ no.0ml
on.o mo.~ oo.~ m¢.~ Mm.¢~ -.~ om.wml
oo.H o~.~ cm.H Ho.- om.a~ No.“ mfi.oc~ mo.o mq.ho~ memo mm.» m.m osfifi ms sou mm mmmwfiww
co.qu~.o ea.qwmm.o NH.Qqu.~ mo.QHNn.H HH.~Iom.~ hm.qwsn.o
“H.o o~.o so.H do.o 50.0 qum wo.hm~
o~.o mq.o fiH.~ oo.m c¢.m sq.» ~o.qo~
¢~.o om.o o~.~ ~m.~ m«.~ om.a H~.¢-
o~.o e~.o ow.o om.~ qm.~ an.“ cm.moH oo.» mn.¢~l meson so.» a.m~ ocsfi «a “00 ca mmummmw
mo.gflmm.o ¢~.QHQN.~ ml.QHsm.~ o~.mm@m.a mo.mmmm.m~ mm.lmmo.~
Nm.o “a.” o~.~ mo.oH qo.- ~H.o m~.qo~
mo.o wm.~ oc.N wo.o mm.m~ om.“ oh.mm~ mo.o mm.na~ ~q- an.» o.- one“
no.qwmm.H hfi.qwn~.~ oN. I ~.~ cm.fiweq.o cm.MMme.«H o~.qfl0m.o
nq.~ qum oe.~ «H.m no.- N .o q~.o-
m~.~ mH.~ ofi.~ es.“ n~.oH oo.o q~.~n~ mm.» qm.mmH ~o~mq on.“ m.H~ mqmd
oc.afiuo.~ mo.mMom.- mq.mflmm.-
-- 5.0 -- -- mo.w oo.¢~ qw.~n~
-- om.~ -- -- mo.¢~ “c.o~ 00.0mfi oo.w No.oq~ mm~m~ oo.“ o.o~ mood N“ mom ma .mmqmm
ooo.gfl¢~.o ooo.qfieq.o 0H.QHoo.o Ho.qwo~.~ m¢.Qfl~c.l a~.¢uo<.o~
m~.o Nm.o mm.o ow.~ o.~ H~.o~ ca.o~l
o~.o mm.o o«.o no.“ Hw.o om.o¢ No.-
¢~.o q¢.o nw.o so.~ om.“ ~o.c~ nn.ow
m~.o om.o m~.o om.~ om.~ mm.o~ oo.nm mo.w sm.wo~ comes oo.“ m.m~ OMNH Na Han ”N madam
”In; N "nu: N cauw» anus N Hun: N an»; Hum we Aug we ~a~ we xau u up:
sumo unwfiq a H A sumo ucwwg Mu: .uz Aha cucou cucou .ucoucH xn name has an «ace usuam
conuwu oacmmuo noduwuamwom «xwm u ucmdm we now uzwfiq wads

.wuav ouauwdamu mo cm H cmwe so“: .mucmda vmdwnmfioua Beau uno; uoa va>~o>o m chuwucfi
.cwwfizufiz .oxmq occupana ca Azuawc 5 Av manCaeuwunam

 

.m canoe

73

 

 

 

 

 

 

I I 1 ‘
m
H' .
1:9 4a.. ------- .u d
x .........
9 ....
.1320? d
_J x.
l J J l
' ‘ I I
U); I ...........
°'\‘O+- ........... In“ ‘
D—{D ........
- ""1
UJO 5P ‘
21
J l l l
l I 1 t
CK ".‘
Q520_ ..... .I
.c """
F" I ................ I.
LLJO '- ...... d
I I l 1
I I t '
Z
Lu— 9” "O ----------------- . ..
C)\ . ...................
>05
XE 8 ‘
C) ‘g

 

l 1 l 1
1000 I200 1400 1600
TIME OF DAY

Figure 31. ‘ln situ net photosynthesis and net photo-
respiration in natural Najas flexilis, light intensity,
and dissolved oxygen concentration in littoral zone
of Lawrence Lake, Michigan, 0.5 m depth, on 15 September 72
(data points: mean * SD).

 

.mh pmpSmemm ma :0

Coocsmpmm mpma pom «Coonsmpmwuvfle «mcHCAoenofle CH mmmhamcw

mpdoflagzm .spmmc E m.o «Cmmflnoflz umqu mocmsqu mo

msoN HdhOppHH CH Spflm mm.xsm© nod pSMHH CH mHHmeHm mmflwz
ampspwc Scam mmdmams Coosmo oacwmso m>fipwazfiso .mm msswfim

 

 

 

 

 

kaDZE‘
00 0V on o 01 ON e 01 ON 3
T a . u J . . q 4 a J a . 1
I 11 1.! 13
1- 1w. 1' l
I 1. LT 1 3%
.4 mm
7 . : i- . u v
w w
.l 1.! 1.! l | O
N.— N
l. O
3 V
I 1T .I - a 8
Nos
Vnu
1 ...: 11 L0.— _] N
H
O
.l .41.. 11 l
35 c.
1 f .1 2:8: 862 lod
j ‘l LI ‘I it L
xmdd xm<o
» p _ h . p _ r L r ‘N

 

 

 

 

 

 

 

_ — -
mmI Gnu—lone. mmI nVV—Invm— mmI nz—lnpop

75

and carbonates in the material reversed from July to

September, with a relative increase in organic lac in

September.
Table 6. Characterization of encrusting material removed

from lyOPhylized luC-labeled natural Najas flexilis
following in situ experiments.

 

 

 

Dry Wt. Radioactivity 140 Distribution
% plant wt. % plant activ. %organic % inorganic

 

 

July 55 34 27 73
Sept. 113 50 67 33

 

Scirpus subterminalis

 

In situ rates of respiration in Scirpus subterminalis

 

in early afternoon in October (Figure 33; Table 5) were

similar to those in Najas flexilis in July except that rates

 

were the same in light as in dark in Scirpus subterminalis,

 

with corresponding L:D ratios of about unity. Variance in
rates was least in plants from which epiphytic material had
been removed (Figure 33). Rates of organic carbon release

(Figure 3h; Table 5) were similar to those in Najas flexilis

 

in July, and similarly were 1/3 to 1/6 respiration rates.
Rates were slightly greater in light than dark.
Dry weight of epiphytic encrustation on Scirpus

subterminalis in October was equivalent to 58% of plant dry

 

weight. Radioactivity of the material (“Ci g'l) was

76

equivalent to 34% of plant radioactivity. Forty % of the
activity in the material was in organic carbon, and 60%
in carbonates.

In February, respiration rates were greater in light
than in the dark (Figure 35). Variance in rates between
plants again was high, but L:D ratios were relatively
uniform (Table 5). Rates in the light in February were
nearly lO-fold those in October; rates in the dark in
February were about 5-fold those in October. Rates of
organic carbon release in February were 3-fold those in
October, and were somewhat greater in light than in

dark (Figure 36).

77

 

     

 

32 r I l I
DARK
-——§-
Scirpus subtermmalus
2.4_ m Sltu _,
O
2‘.
:‘z *‘ ‘
C32
“Jm
Eui
dr-
(021.6%— ._
UJ—
0: .1
«Vs
C):
02 - _
2§
08b _
O

 

 

 

60

MINUTES

Figure 33. Cumulative carbon dioxide release from natural
Scirpus subterminalis in light and dark in situ in
littoral zone of Lawrence Lake, Michigan:—at 1440 hrs
on 14 October 72, l m depth. Duplicate analyses with
epiphytic microflora removed from plants dashed lines);
duplicate analyses with untreated plants solid lines).

 

78

.Ammcfia UHHOmV mpcmam popdmspcs

Spas momhadcm mpdoaamdv mAmoCHH oocmmpv mpcmam Eosw co>oams
wsoamomofle Osznmfigo Spas mmmhawcm mprHHQSD .cpgoc S H

«mu homepoo :a Go wsmIO::H pm «Cmmfinoflz noxmg cosmhzmq wo mCON
HasOppHH CH Spam CH gasp and psmfia CH mHHmCHEAmeSm mfimnflom

 

 

Hmsdpmc 809m mmdoamh Consmo oacmwso o>HpmHSESO .zm mhsmflm

 

 

 

 

mmFDZE;
on 0 Wm
M
I I m
v
“I
I I do M
i
3
I. U
I N
35 c. . w
I 9698558 msotomn vo On
_ _ xm<o _ _ (

 

NOEHVOCNNVOHO

79

 

‘6 I I 1 I
DARK

F- ‘

Scirpus subterminalis
in situ

 

d

N
#
1

C02 RESPIRED
(% INITIAL INTERNAL "0)

 

 

 

 

8 1- a
/
‘ P- -I
I. -.
i 1 J 1
O 20 40 60
MINUTES

Figure 35. Cumulative carbon dioxide release from natural
Scirpus subterminalis in light and dark in situ under ice
in littoral zone of—Lawrence Lake, Michigan, at 1140 hrs
on 28 February 73, 1 m depth (simultaneous quadruplicate
analyses).

 

8O

 

 

 

 

 

 

I I I I
DARK
10" -—' .I

A SCIrpus subtermmalIs

L) In Sltu

1’- 0.8»- _
Z _J
C>‘<
CD:Z
air:
3 If O.6- -

Z
2 7 /
z —J
< 3 0.4— _
CJI—
m:‘—
c>§§

O /

§:()2~ ///’ .

l l I l
0 2o 40 60
MINUTES

Figure 36. Cumulative organic carbon release from natural
Scirpus subterminalis in light and dark in situ under ice
in littoral zone of Lawrence Lake, Michigan, at 1140 hrs

on 28 February 73, l m depth (simultaneous quadruplicate
analyses).

DISCUSSION

Net Photorespiration and Dissolved Oxygen

 

While the initial laboratory experiments indicated that
loss of C02 was measurable in the light using the 14C-assay,
it was striking that the rates in the light were never
appreciably greater than in the dark, contrary to results
of Goldsworthy (1966) and Zelitch (1968) with terrestrial
plants in which rates in the light were 2 to 5-fold those
in dark. Indeed, C02 loss in the light in axenic fialas
flexilis was less than in dark (except at very high 02

concentrations), which indicated extensive refixation of

respiratory 002 in the light. There is a possibility that
dark respiration was inhibited somewhat in the light,
although such an inhibition was not observed at high 02
concentrations. Some evidence exists for low-level light
inhibition of glycolysis in green plants (e.g. Kok, 1947;
Hoch, et al., 1963; Hirt, gt al., 1971), but this effect is
apparently saturated at very low light intensities, and is
discounted by other workers (e.g. Gessner, 1937; Warburg,
et al., 1949; Zelitch, 1971). Refixation of C02 in the
light is likely of greater significance here, especially
in view of the diffusive resistance of water to 002, which
is 105 times greater than that of air (Hutchinson, 1957;
Raven, 1970).

81

82

The extensive 002 refixation by Najas flexilis is

 

reminiscent of Cu plants. However,the analyses of leaf
cross section anatomy and first 140 fixation products
indicate that it is not a Cu plant; Donaldson and Tolbert
(unpublished) have more detailed data on fixation products
of E. flexilis, also indicating C3 metabolism. Furthermore,
the enhancement of 002 loss in the light by increased 02
concentration is characteristic only of C3 plants.

Evidence for refixation of respiratory 002 in the
light in aquatic plants also appeared in studies of 14002

loss in the submersed angiosperm Ceratophyllum.demersum

 

(Carr, 1969) and Myriophyllum amphibium (Steemann Nielsen,

 

1955), and in the marine flagellate Dunaliella euchlora

 

(Ryther, 1956). Refixation of 002 in vascular hydrophytes
is probably enhanced by internal gas lacunae, often of
massive size, present in nearly all species (Sculthorpe,
1967). Oxygen produced in photosynthesis builds up in
these spaces, and respiratory and photorespiratory C02
undoubtedly diffuses into them as well, perhaps more readily
than into the water. The evidence for 002 refixation casts
some doubt on the assumption (Steeman Nielsen,l955;

Wetzel, 1964) that, in short-term 140 primary productivity
measurements for macrophytes, incorporated 14C would not

be respired and recycled appreciably. The possibility of
recycling was subsequently reconsidered (Wetzel, 1965),

and it is now clear that the 140 technique for primary

83

productivity in aquatic plants underestimates photosynthesis
to some extent.

Refixation of C02 in the light also causes under—
estimation of true photorespiration as measured by C02
evolution, which is a major criticism of the 12+C photo-
respiration assay (Zelitch, 1971); the assay thus measures
apparent or net photorespiration. Zelitch (1971) estimates
that 50 to 67% of photorespired C02 must be recycled and
missed by the 11+C-assay. A similar underestimation of
photorespiration likely occurred in the present study.

Apparent photorespiration in axenic Najas flexilis increased

 

only 2-fold with lO-fold increase in 02 concentration.

True photorespiration may have increased by lO-fold
internally, inasmuch as glycolate metabolism is directly
Proportional t0 02 concentration, especially at relatively
low concentrations (Andrews, gt al., 1973). If so, true
photorespiration may have been underestimated by as much as
80%, and corrected rates (8 to 10% hr'1 at medium, i.e.
normal aquatic, 02 concentrations, rather than 2% hr‘l)

and resulting new L:D ratios (3 to 5, rather than 0.5),

are similar to those found by Zelitch (1968) and Laing

and Forde (1971) in terrestrial C3 plants. While accurate
estimates of true photorespiration cannot be made from these
data, the 02 enhancement of C02 evolution in the light, but
not in dark, constitutes evidence for presence of photo-

respiration in g. flexilis. A similar 02 enhancement of

84

C02 evolution in the light (but not in dark) was
demonstrated with the luC-assay in the marine angiosperms

Cymodocea sp. (angustata?) and Halophila sp. (ovalis?)

 

 

in the Barrier Reef region of Australia (Hough, 1973).
The 02 enhancement of photorespiration parallels and
is assumed to cause the Warburg effect, the 02 inhibition
of net photosynthesis. Oxygen inhibition of net photo—
synthesis has been demonstrated in several submersed aquatic

angiosperms, including Elodea sp. and Sagittaria sp.

 

(Donaldson and Tolbert, unpublished), Elodea canadensis

 

(Kutyurin, gt al., 1964), Sagittaria sp. (Bjorkman, 1966),

 

Ranunculus pseudofluitans (Westlake, 1967), and the marine

 

plant Cymodocea sp. (W. J. S. Downton, personal commun.).

 

Downton also found the Warburg effect in the marine macroalga

Halimeda cylindracea. Hough (1973) did not find a

 

corresponding 02 enhancement of CO2 evolution in the light

in_fi. cylindracea; glycolate may have been excreted rather

 

than oxidized (although this was not reflected in the
organic carbon data), or much of the excess C02 was bound
into the CaCO3 continuously formed internally in this plant.
Normal concentrations of dissolved 02 in freely mixing
natural waters are 10Ll times lower than in air (8-10 ppm
compared with 21%) and in themselves are unlikely to
support rates of photorespiration as high as commonly found
in terrestrial plants. However, buildup of 02 within the

submersed plants during active photosynthesis likely

85

forces considerable additional photorespiration
(cf. discussion below).
The lack of enhancement of dark respiration in

Najas flexilis, Cymodocea, and Halophila by increased 02

 

 

 

concentration is contrary to results of other workers, who
have demonstrated an apparent 02 enhancement of dark
respiration in a variety of other aquatic macrophytes
(Gessner and Pannier, 1958; Kutyurin, gt al., 1964;
McIntire, 1966; McDonnell, 1971; McDonnell and Weeter, 1971;
Owens and Maris, 1964; Pannier, 1957, 1958; Westlake, 1967).
However, none of the latter studies were done using axenic
plants, and all of them were done using measurements of

02 uptake. The 02 technique measures respiration of the
entire community of macrophyte and epiphytic algae, bacteria,
and fungi. The epiphytic community can cause greater 02
fluxes than the macrophytes themselves (Sculthorpe, 1967),
and it is possible that much of the increased 02 uptake
occurred in the epiphytic microflora. Gessner (1959) and
Gessner and Pannier (1958) found that dark 02 consumption
in several algae was influenced by 02 concentration;
McIntire (1966) demonstrated 02 enhancement of respiration
in benthic periphyton communities (microflora attached to
rocks, etc.). Dark respiration in higher terrestrial plants
generally is not affected by 02 concentrations above 2%
(Goldsworthy, 1966; Jackson and Volk, 1970; Martin, gt_al.,
1972).

257.

Carbon Dioxide and pH

 

As reviewed extensively in Hutchinson (1957), Raven
(1970), and Stumm and Morgan (1970), CO2 exists in water
in a complex, dynamic equilibrium system composed of free
dissolved 002, hydrated C02 (carbonic acid), bicarbonate,
and carbonate. Total amounts of inorganic carbon in a lake
depend on the magnitude of dissolution of atmospheric 002,
the amounts of bicarbonates and carbonates entering the
water from the terrestrial watershed, and biotic respiratory
C02 production. The relative concentrations of the
components of the equilibrium system depend mostly on pH,
as is well known, with free 002 favored by low pH. In
freely mixing surface waters, the partial pressure of total
free and hydrated 002 is in equilibrium with the atmosphere,
at a concentration of about 10 “M or 0.5 mg 171, and is
independent of pH. Lakes containing very high concentrations
of bicarbonate and carbonate (>150 mg 1'1) are often
supersaturated with free C02 (Hutchinson, 1957), as is true
of Lawrence Lake (Otsuki and Wetzel, 1973).

Several aquatic plants appear to require free
(dissolved) 002 for photosynthesis (James, 1928; Steemann
Nielsen, 1947, 1951, 1952b; Osterlind, 1950; Briggs and
Whittingham, 1952; reviewed in Raven, 1970), on the basis
of C02 fixation experiments in which pH and total 002 content
are varied, with low pH favoring photosynthesis. Using this

technique, Wetzel (1969) demonstrated such an apparent

87

affinity for free 002 in Najas flexilis at several total

 

Cog concentrations, including those comparable to levels
in hardwater lakes, both in cultures exposed to air and
in those sealed from atmospheric contact. Argument has

been made (Raven, 1970; Wetzel, 1972) that the dehydration

Of H2C03 within the C02-carbonate equilibrium complex may
be slow enough to limit the availability of C02 for
photosynthesis, but this should not be relevant in water
at equilibrium with the atmosphere or supersaturated with
free 002. Strictly from the standpoint that the partial
pressure of free dissolved 002 in equilibrium with air is
independent of pH, and is supersaturated in presence of
high concentrations of bicarbonates and carbonates,
photosynthesis should not be affected by varying pH; the
results of Wetzel and others suggest that not all of the
free C02 is instantaneously available at the plant surface,
perhaps as a result of the low diffusion rate of 002 in
water. Raven (1970) suggests that diffusion resistance

of water should be added to that of cell wall and cytoplasm
in estimates of diffusive resistance to 002 fixation in
aquatic plants. It is not clear how pH would affect
diffusion rate in the water. Alternatively, the pH effect
on photosynthesis can be explained in that the pH Optimum
for RuDP carboxylase activity (7.8) is lower than that for
RuDP oxygenase activity (9.3-9.5) (Andrews, et_al., 1973),

with lower pH favoring photosynthesis and higher pH favoring

88

photorespiration. Within a pH range of 7.3 to 8.8,net
photosynthesis in axenic N. flexilis is greatest at
pH 7.3-7.9 and lowest at 8.5-8.8 over a wide range of total
002 concentrations (Wetzel, 1969). A causal relationship
between the enzymatic pH Optima cited above and the
response of net photosynthesis in N. flexilis can be
assumed only if pH of the water significantly influences
pH in the chloroplasts of intact submersed plants, which
is unknown. In any case, it remains unresolved whether,
over the range of pH and carbonate alkalinity in fresh-
waters, photosynthesis in aquatic plants is COQ-limited to
a greater or lesser extent than it is in terrestrial plants.
In view of the above, it is difficult to predict the
influence of ambient C02 on photorespiration in aquatic
plants in terms of what is known of terrestrial plants.

Effects Of C02 on the lLPG-assay were tested in the

laboratory partly to facilitate interpretation of in situ
studies in Lawrence Lake. The assay normally is performed
under COg-free conditions, to avoid dilution of the 140
labeled organic carbon pool by fixation of 120 during the
experiment (Goldsworthy, 1966; Zelitch, 1968). The removal
of C02 (including bicarbonate and carbonate) from lake water
involves acidification, gas purging, and reinstatement of
original pH with the aid of a buffer, which drastically
alters water chemistry and is not desirable ecologically.

Influence of 002 was tested also because photorespiration

itself is highly influenced by 002, presumably as a result

89»

of the direct competition of CO? and 02 for reaction with
RUDP. Cog-free conditions thus would favor unusually high
rates of photorespiration by allowing unrestricted
oxygenation of RuDP and glycolate synthesis.

Presence of large amounts of 12002 in the lac—assay
would be expected to lower the apparent rate of photo-
respiration both by decreasing the rate of glycolate
synthesis and by diluting the 1“C organic substrate pool.

The relative magnitudes of these effects would be difficult
to determine, but they probably are similar since they are
part of the same process (002 fixation). Thus in performing
the assay in presence of 12C02, the 12C dilution effect
(causing some degree of underestimation of photorespiration)
replaces the unnatural effect of zero 002 (causing some
degree of overestimation of photorespiration), and the former
is probably no more serious than the latter.

The results with changing [COé] were variable,
depending on experimental conditions. Presence of C02 in its
natural forms in lake water did appear to cause lower net
photorespiration rates relative to 002 purged lake water,
although water chemistry other than 002 concentration was
unavoidably different in the two treatments. Also, the
axenic plants had not been grown in lake water prior to the
experiments. The apparent lag-time in the reduction of rates
after exposure to high C02 may reflect extensive recycling
of internal 002, which would reduce the rate of 120 dilution

of the 140 pool. In synthetic media, to which the axenic

90

plants were acclimated, additions of bicarbonate or
carbonate to levels similar to those in Lawrence Lake
water did not affect the results of the assay appreciably.
In general the results indicated that the assay could be
performed lfl.§l£2 without drastic complications resulting
from ambient C02.

Carbon dioxide concentration also is important with
respect to plant 002 compensation point, or the minimum
C02 concentration at which the plant can continue to remove
CO2 from the environment. Rapidly photorespiring plants
have high 002 compensation points. The compensation point

of Najas flexilis is rather low (Donaldson and Tolbert,

 

unpublished). The same is true of Myriophyllum spicatum L.

 

(Stanley and Naylor, 1972). Myriophyllum spicatum was

 

characterized in terms of fixation products as a C3 plant,

as iS.H- flexilis. Stanley and Naylor assumed that the low
compensation point was the result of extensive C02 refixation,
regardless of the lack of Cu carboxylation. This argument
may be inaccurate: if the plant were rapidly photorespiring,
the compensation point would be high regardless of extensive
refixation, since refixation of a given amount of photo-
respired 002 would preclude fixation of a similar amount of
external 002. Thus, while refixation is certainly extensive
in these plants, low 002 compensation point probably should
be regarded as evidence for relatively low rates of

photorespiration.

91

Epiphytic Microflora

 

The community of epiphytic microflora on naturally
growing aquatic macrophytes is a highly active, and often
major, component of the metabolism of the littoral and
overall lake systems (Allen, 1971; Wetzel, 1964; Wetzel
and Allen, 1972; Wetzel, et al., 1972). The intimate
association of macrophyte surface and the epiphytic biota
undoubtedly provides for extensive exchange of 002 and
organic carbon between the two. Any 002 photorespired by
the macrOphyte would be available for use by epiphytic
algae, and any Of this 002 fixed by the algae would not be
measured by the 14C photorespiration assay. On the other
hand, the epiphytic algae are prelabeled along with the
macrophyte, and of course are respiring and releasing organic
carbon themselves during the assay. Epiphytic bacteria and
fungi likely utilize some of the organic carbon released by
the macrOphyte, and respire some of it as well. All of
these processes potentially confound the results of the
14C-assay when applied to natural plants.

Effects of epiphytic diatoms and mixed epiphytic
microflora generally were minimal in the laboratory experi-
ments, which would suggest that in situ ll‘LC-assays would not
be confounded seriously by the epiphytic community.
However, the dense epiphytic growth common on naturally
growing plants was not obtainable on the axenic_Naia§

flexilis seedlings without deterioration of the plants and

92

resulting incomparability with controls, especially in the
case of the axenic diatoms, and experiments were performed
with less than maximal epiphytic growth.

Net photorespiration and organic carbon release were
enhanced somewhat in presence of the diatoms, which
apparently were releasing 14C in excess of any that they
were obtaining from the host plants.

The effect of the mixed epiphytic community was largely
an enhancement of dark respiration (Figure 23), which may
reflect mineralization of labeled organic substrate
released from the host, although total release of organic
carbon (Figure 25) was not reduced in presence of the
epiphytes except possibly during the first 10 minutes of

darkness.

In situ Photorespiration

 

Najas flexilis

 

The relatively low rates of net photorespiration in

natural Najas flexilis in July were unexpected, in view of

 

the high light intensity. In thiscontext, light intensities
in the laboratory epiphyte experiments were over 2—fold

those in the 02 and C02 experiments, but at similar 02
concentrations there were no appreciable differences in net
photorespiration. The light intensity in Lawrence Lake
during the July EE.§$EE assays was over lO-fold those in the

laboratory; if this intensity was actually causing high rates

93

of true photorespiration, then 009 refixation rates must
have been high as well.

In September, net photorespiration exceeded dark
respiration for the first time in the study, even though
light intensity was lower than in July. However, the
plants were beginning to undergo senescence, and were losing
lO-fold more carbon than in July, both in light and in dark.
Capability of refixing 002 likely was reduced, allowing
more photorespired carbon to be released. Carboxylation
may be diminished relative to oxygenation of RuDP as a result
of changes in chloroplast chemistry (including loss of
chlorophyll) during senescence, enhancing photorespiration.

The variation of net photorespiration during the day in
September was consistent with the hypothesis that afternoon
depression of net photosynthesis in aquatic plants is
associated with increase in photorespiration. The afternoon
depression in net photosynthesis, depicted schematically in
Figure 37, has been demonstrated consistently in several
submersed angiosperms and phytoplankton populations (Doty
and Oguri, 1957; Hartman and Brown, 1967; Hartman, gt al.,
1965; Meyer, 1939; Steeman Nielsen and Wium-Andersen, 1972,
and Ohle in discussion thereof; Verduin, 1957; K. F. Walker,
personal commun.; Wetzel, 1965). Various explanations for
the phenomenon have been offered, including narcosis or
"sugar glut", photooxidation by surplus light energy,

protection against photooxidation by inactivation of the

94

 

I r j

 

 

 

 

Photorespiration

    

RELATIVE MAGNITUDE

 

 

 
 

 

 

 

______~
Dissolved Oxygen
l l l
0000 1200 1000

TIME OF DAY

Figure 37. Commonly observed variations in light intensity,
dissolved oxygen concentration, net photosynthesis of
natural submersed macrOphyte and phytoplankton popu-
lations, and prOposed concurrent variations in photo-
respiration.

()5

photochemical reaction, or poisoning by Cu++ contamination
in 14C stocks (Strickland, 1960; Steemann Nielsen, 1962;
Steeman Nielsen and Wium—Andersen, 1972); most of these
propositions are somewhat vague, and no specific mechanisms
have been demonstrated in the natural situation. Photo-
respiration is an alternative explanation, inasmuch as
conditions conducive to it can develop during the day.

Indeed, net photorespiration in Najas flexilis in September

 

increased through the day (Figure 31), while net photo-
synthesis decreased through the day. These processes, as
well as light intensity and oxygen, generally followed the
patterns predicted in Figure 37. Dissolved oxygen did not
increase sufficiently to fully explain the increase in
photorespiration. However, the increase of dissolved 02
may reflect a much greater increase in 02 tension within
the plants. Hartman and Brown (1967) demonstrated that

dissolved 02 in the water surrounding Elodea canadensis

 

fluctuated with internal 02 tension, and in one case both
were greatest in late afternoon; the internal 02 concen-
tration was generally similar to that in air, i.e. much

higher than in water.

Scirpus subterminalis

 

The leaf cross sections and first 1“C fixation products

indicated that Scirpus subterminalis is a C3 plant, and

 

photorespiration was expected. However, the massive gas

lacunae were expected to engender extensive internal CO?

96

recycling, with resulting lower rates of CO2 evolution

in the light than in the dark. On the contrary, rates in
light were the same as in dark. The photosynthetically
active epidermal cells are one cell removed from the gas
lacunae, and thus 002 may diffuse into the water as readily
as through the inner cells into the lacunae, especially

if the partial pressure of 002 in the lacunae is already
high relative to the water. The rates of respiration were

generally similar to those in Najas flexilis in July,

 

showing no evidence of the senescence reflected in rates
in N. flexilis in September. Rates of carbon fixation
(Table 5) were lower than in N. flexilis in July, however,
similar to those in N. flexilis in September, perhaps
because temperature was substantially lower.

Rates of carbon fixation in S. subterminalis were

 

drastically lower under the ice in February than in October,
undoubtedly because of very low temperature (3 C) and low
light. Surprisingly, rates of respiration (in terms of %

of fixed carbon) were high, similar to the September rates
in N. flexilis. Extremely low temperature may reduce the
rate of carbon fixation more than it does the rate of
respiration, with corresponding apparent increase in
respiration relative to fixation. 002 refixation might then
be reduced as well, with resulting relative increase in
release of photorespired C02, reflected by the L:D ratios

of greater than unity.

97

Release of Organic Carbon

 

The lack of response of organic carbon release from

axenic Najas flexilis to increased 02 concentration in

 

the light suggests that glycolate is not a major component

of the material released, since excretion of glycolate

should parallel its synthesis, which was undoubtedly enhanced
by high 02, The apparent post—illumination surge of organic
carbon also does not suggest involvement of glycolate,
because oxidation vs. excretion of glycolate is not a
function of light. The burst is reminiscent of the sudden
rise of PGA in chloroplasts upon sudden darkness, as

demonstrated in Chlorella pyrenoidosa by Bassham (1971),

 

in which the light-dependent reduction of PGA ceases
immediately while PGA synthesis from RuDP carboxylation

(a dark reaction) continues briefly until the RuDP pool is
exhausted. The excess PGA is gradually metabolized to

amino acids and fatty acids in g. pyrenoidosa. Release of

 

PGA in large quantity has not been reported in algae or
higher aquatic plants, but this would be a highly transient
phenomenon. However, the apparent enhancement of the burst
by high oxygen (Figure 12) suggests that PGA is not involved,
inasmuch as oxygenation of RuDP produces half as much PGA

as does carboxylation, and the PGA buildup would be

inhibited by high 02 rather than enhanced. The data are

14

based on C tracing, of course, and it was assumed that the

actual amount of organic matter released in this short period

98

was too small to characterize. Special efforts to obtain
relatively large quantities for isolation may be desirable,
however.

The ecological significance of the post—illumination
excretion is questionable inasmuch as sudden darkness is
not likely to be common under natural conditions, although
rapidly intermittent cloud cover might have a similar
effect. In any case, the data provide additional evidence
that excretion of organic carbon is a metabolic alternative
in aquatic macrophytes under certain environmental
conditions. The post—illumination excretion burst was not
observed in any of the in_situ photorespiration assays;
immediate microbial utilization of the excreted carbon is
an obvious possibility. The reduction of the burst by
mixed epiphytic microbes in the laboratory (Figure 25) was
minimal, but these organisms may not have been prepared
metabolically for the compound(s) involved, particularly in
view of the high concentration of organic buffer in the
cultures, providing a plentiful organic substrate.

The rates of organic carbon release in natural
_1\1. flexilis in July (<l% initial internal 140 hr '1) do not
support the estimate of Wetzel, £3.2l' (1972) that 4% of
photosynthetically fixed carbon in macrOphytes is released
as dissolved organic carbon. However, rates of release in
September were greater C>2% hr'l), especially in early

afternoon, and allowing for differences in analytical

99

methods, the estimate of Wetzel, et a1. (as a mean rate of
release over a year) is not diSputed here. The increased
rate of release in September reflects, as does the increased
respiration, the senescence of the plants at this time. The
relative increase of 12+C labeling in the organic fraction of
the encrusting material in September (Table 6) may be a
result of the increased release of organic matter from the
plants, some of which likely was assimilated by micro-
organisms or adsorbed by calcium carbonate; the data are
confounded by fixation of 1”002 by algae within the material
during prelabeling, however. The data indicate that
unusually large quantities of soluble organic carbon become
directly available to aquatic microorganisms in the fall,
without the necessity of microbial decomposition of the
plants. Furthermore, since lake circulation reaches a max-
imum at this time of year, much of this dissolved organic
matter is likely to be dispersed in pelagic waters, and
subsequently enter the dynamic organic-inorganic-microbial
interactions which are among the fundamental controllers of
overall lake metabolism (Saunders, 1957; Wetzel, 1968, 1971;
Wetzel and Allen, 1971; Wetzel, gt al., 1972). In this
manner the enhanced release in the fall may contribute to
fall phytoplankton blooms. Rates of release of organic

carbon from Scirpus subterminalis remained low (<0.5 % hr7l)

 

in the fall, but increased 3-fold in winter. The carbon

released in winter may accumulate to some extent as a result

100

of low temperature (i.e. low microbial metabolism) and
then become dispersed during Spring lake circulation,
with chemical and trophic involvement similar to that in

the fall.

SUMMARY AND CONCLUSIONS
A 14C-assay for photorespiration has been developed for
use in submersed aquatic plants both in the laboratory and
_in sign. Advantages of the technique include avoidance of
problems associated with closed-bottle techniques, ease of
manipulation of experimental conditions, and simultaneous
evaluation of total released organic carbon. Problems
include imposing a current on plants normally growing in
relatively still water (less serious than imposing small-
volume stagnation), and internal recycling of 14002, which
appears to be more serious in aquatic plants than in
terrestrial plants. All methods of measuring photo-
respiration have inherent difficulties resulting in under-
estimation of true photorespiration (Zelitch, 1971), and
comparisons of methods in the same plant species result in
varying estimates (Martin, gt al., 1972). Within the
limitations of the 14C-assay, the following conclusions

emerged from this study:

1. Net photorespiration is low in C3 submersed aquatic
plants in relation to that in terrestrial 03 plants, but

is enhanced by high oxygen concentration, indicating net

101

102

photosynthesis can vary with changes in dissolved oxygen

in these plants.

2. True photorespiration may be somewhat lower in
submersed plants than in terrestrial plants because of lower

environmental oxygen concentrations.

3. Much of true photorespiration is not measured by

the 14

C-assay because of extensive internal recycling of
C02,partly or largely accounting for low rates of net

photorespiration.

4. Net photorespiration varies within a day and
seasonally in submersed plants, and may account for after-

noon depressions in net photosynthesis.

5. Kinetics of release of dissolved organic carbon do
not indicate that glycolate excretion is extensive in

Najas flexilis, which is consistent with the evidence for

 

glycolate oxidation, i.e., 02 enhancement of CO2 production

in the light.

6. Release of organic carbon is relatively low in

natural Najas flexilis in summer, but increases in the fall

 

during senescence. Release of organic carbon from Scirpus
subterminalis is low in fall and increases in winter. The
patterns of release may be associated with seasonal aspects

of planktonic productivity.

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