Mechanisms for reducing photorespiration in submersed aquatic angiosperms

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Title:
Mechanisms for reducing photorespiration in submersed aquatic angiosperms
Uncontrolled:
Aquatic angiosperms
Physical Description:
xii, 117 leaves : ill. ; 28 cm.
Language:
English
Creator:
Salvucci, Michael Edward, 1956-
Publication Date:

Subjects

Subjects / Keywords:
Aquatic plants -- Physiology   ( lcsh )
Plants -- Photorespiration   ( lcsh )
Hydrilla   ( lcsh )
Myriophyllum   ( lcsh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1983.
Bibliography:
Includes bibliographical references (leaves 107-116).
Statement of Responsibility:
by Michael Edward Salvucci.
General Note:
Typescript.
General Note:
Vita.

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University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
oclc - 10039221
ocm10039221
System ID:
AA00011117:00001

Full Text












MECHANISMS FOR REDUCING PHOTORESPIRATION
IN SUBMERSED AQUATIC ANGIOSPERMS




BY


MICHAEL EDWARD SALVUCCI


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1983






























To my wife (spouse),

Cindy













ACKNOWLEDGEMENTS


I wish to acknowledge the members of my supervisory committee, Drs.

George Bowes, Richard Smith, Joseph Davis, Thomas Humphreys and Thomas

O'Brien,for contributing their time and effort in providing constructive

criticism, advice, and suggestions throughout my degree program. I sin-

cerely appreciate the patient guidance and support given to me by Dr.

George Bowes over the last four years. The time and experience that he

supplied so selflessly in supervising my research exemplified profession-

alism and I hope to emulate his example.

I wish to thank Dr. William Haller and Margaret Glenn for their

indispensable assistance in obtaining much of the plant material used in

the study. I am indebted to Dr. J.T. Mullins and Charlie Cottingham for

providing the equipment and expert advice required for the lyophilization

and electrophoresis procedures. I would like to express my gratitude to

Drs. Thomas Humphreys, Robert Ferl, and Jesse Gregory for furnishing some

of the chemicals and equipment used in the experiments, and to Dr. Jack

Ewel for supplying the expert services of Jake Fuller for the graphic work.

I would especially like to thank my parents for their constant sup-

port and encouragement. I appreciate the interest and enthusiasm expressed

by Richard and Marjorie Spaulding and the services of Richard Spaulding in

locating and collecting plant material. The loving support of my wife,

Cindy, provided much of the motivation for the expeditious completion of

this phase of my scientific career.













TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS. . .* .i

LIST OF TABLES. .. . ........... vii

LIST OF FIGURES . .. . viii

KEY TO ABBREVIATIONS .. .. .. .. ..... x

ABSTRACT. . ... . ....... xi

CHAPTER I: PHOTOSYNTHETIC CARBON ASSIMILATION AND
PHOTORESPIRATION IN SUBMERSED AQUATIC MACROPHYTES:
A REVIEW . . . ... 1

General Features of Submersed Aquatic Macrophytes. 2

Physiological Aspects. ... . .. 4
Photosynthesis and the Apparent Km(CO2) . 4
Bicarbonate Usage .. . ..... 7
Photorespiration . 9
CO? evolution into C02-free air (water)
in the light . . 10
Oxygen inhibition of photosynthesis ... 11
C02 compensation points . ... 12
True versus apparent photosynthesis . 13
Light/dark (L/D) ratios of CO2 evolution. 13
The Concept of Variable Photorespiration. .. 14

Biochemical Aspects. .. . 16

Photosynthetic and Photorespiratory Enzymes .. 17
RuBP carboxylase-oxygenase . .. 17
PEP carboxylase .. ........ .. 18
Other enzymes of C4 acid metabolism. .. .... 19
Carbonic anhydrase . 19
Photorespiratory enzymes . 20
Pathways of Photosynthetic Carbon Assimilation
and Metabolism . . 21
613C Values in Submersed Macrophytes. . 26








Page


The Role of C4 Acid Metabolism and Its Relation-
ship to the Photorespiration State .
Submersed Macrophytes with Low Potential for


a-Carboxylation .


Concluding Statement. . .

CHAPTER II: ETHOXYZOLAMIDE REPRESSION OF THE LOW
PHOTORESPIRATION STATE IN SUBMERSED ANGIOSPERMS

Introduction . .

Materials and Methods . .
Plant Species ........
Infrared Gas Analysis . .
Ethoxyzolamide Treatments .
Carbonic Anhydrase . .
PEP Carboxylase . .


Results. . ... .............

Discussion . .

CHAPTER III: TWO PHOTOSYNTHETIC MECHANISMS MEDIATING
THE LOW PHOTORESPIRATION STATE IN SUBMERSED AQUATIC
ANGIOSPERMS .. . .... ......


Introduction .... ..

Materials and Methods .
Plant Material .
Infrared Gas Analysis .
Initial Product and Pulse-Chase
Glycine Accumulation .
Extraction of Labeled Compounds
Separation of Labeled Compounds
Thin-Layer Chromatography .


Experiments
. .


Results . . .

Discussion . . . 80





* .


: : : : :


* *









Page


APPENDIX A: CHARACTERISTICS OF RIBULOSE-1,5-BISPHOSPHATE
CARBOXYLASE-OXYGENASE FROM MYRIOPHYLLUM AND HYDRILLA. .

Introduction . . .. .

Materials and Methods. ... ..
Preparation of 3H-RuBP and P-Glycolate Phosphatase.
Specificity Factor Determinations . .


Km Determinations .
Rocket Immunoelectrophoresis, .

Results and Discussion .

APPENDIX B: THE EFFECT OF IRRADIANCE ON THE
TION POINTS OF MYRIOPHYLLUM AND HYDRILLA.

Introduction . .

Materials and Methods .

Results and Discussion .

APPENDIX C: DIURNAL CHANGES IN THE SENSITIVE
PEP CARBOXYLASE TO MALATE INHIBITION. .

Introduction . .

Materials and Methods .

Results and Discussion .

BIBLIOGRAPHY . .

BIOGRAPHICAL SKETCH . .


CO2 COMPENSA-








ITY OF HYDRILLA
. .

.

. .

. .

. .

. .














LIST OF TABLES


Page


Table


Features associated with the occurrence of
B-carboxylation in submersed aquatic macrophytes .

Carbonic anhydrase activity in Myriophyllum and
Hydrilla in the low and high photorespiration (PR)
states, and in Nicotiana . ..


Effect of ethoxyzolamide on the CO2 compensation
points of Myriophyllum, Hydrilla, and Proserpinaca
in different photorespiration (PR) states, and of
Sorghum, Moricandia, and Nicotiana . .

Effect of ethoxyzolamide on net photosynthesis at 21
and 1% 02 and 330 pl C02/1, and on dark CO2 evolution
for Myriophyllum and Hydrilla in the low and high
photorespiration (PR) states . .

Effect of ethoxyzolamide (100 yM) on the distribution
of 1C among the photosynthetic intermediates of
Myriophyllum and Hydrilla in the low photorespiration
state . . .. ..

The effect of isonicotinic acid hydrazide (5 mM INH)
on the distribution of 1C among the photosynthetic
intermediates of Myriophyllum and Hydrilla in the
low and high photorespiration (PR) states .

Kinetic parameters of RuBP carboxylase-oxygenase from
Myriophyllum and Hydrilla in the low and high photo-
respiration (PR) states, and from Nicotiana .

Chlorophyll, soluble protein and RuBP carboxylase-
oxygenase (RuBPCO) protein content in Myriophyllum
and Hydrilla in the low and high photorespiration
(PR) states . . .

Effect of malate (2 and 10 mM) on PEP carboxylase
activity rapidly extracted from low photorespiration
state Hydrilla plants at three times of the day .


. 46



. 47



. 73


. 98


. .106













LIST OF FIGURES


Figure Page

1 The effect of CO2 concentration on the percent
inhibition of net photosynthesis by 21% 02 for
Myriophyllum and Hydrilla in the low and high
photorespiration states . . 37

2 The effect of C02 concentration on net photosyn-
thesis and percent inhibition of net photosynthesis
by ethoxyzolamide for Myriophyllum in the low and
high photorespiration state. .... . ... 39

3 The effect of CO2 concentration on net photo-
synthesis and percent inhibition of net photo-
synthesis by ethoxyzolamide for Hydrilla in the
low photorespiration state . .. 41

4 Incorporation of 14C into malate and aspartate for
Hydrilla in the low and high photorespiration states
during a pulse-chase experiment. . ... 64

5 Distribution of 14C among photosynthetic inter-
mediates of Hydrilla in the low photorespiration
state during the initial period of the pulse-
chase experiment . . 66

6 Distribution of 14C among the photosynthetic inter-
mediates (excluding C4 acids) of Hydrilla in the
low and high photorespiration states during an
extended pulse-chase experiment. . .. 69

7 Distribution of 14C among the photosynthetic inter-
mediates of Myriophyllum in the low photorespiration
state during an extended pulse-chase experiment. .... 72

8 The effect of isonicy9inic acid hydrazide (INH) con-
cen~ration on total C fixation and the incorporation
of 'C into glycine and serine for Myriophyllum in the
low photorespiration state at 20 yM H14C03- and 50% 02
(gas phase) . . 76


viii










Figure Page

9 The effect of 02 concentration on the incorporation
of 1'C into glycine for Myriophyllum in the low
and high photorespiration states during 15 min
photosynthesis at 20 pM H14C03- and 5 mM INH .. 79

10 The effect of the external 02/C02 concentration
ratio on the percent inhibition of net photosynthe-
sis by 02 for Myriophyllum plants in the low and
high photorespiration states . .. 82

11 Double reciprocal plots of RuBP carboxylase activity
versus NaHC03 concentration for high photorespira-
tion (PR) state Myriophyllum and Nicotiana (tobacco) 96

12 The effect of irradiance on the C02 compensation
points of Myriophyllum and Hydrilla plants in the
low photorespiration (PR) states. . 103













KEY TO ABBREVIATIONS


CAM Crassulacean Acid Metabolism
DNPH 2,4-dinitrophenylhydrazine
DTT dithiothreitol
EDTA ethylenediaminetetraacetate
EU Enzyme Units
Hepes [N-2-hydroxyethylpiperazine-N'-2-
ethanesulfonate]
IgG gamma immunoglobulin
INH isonicotinic acid hydrazide
IU International Unit
L/D Light/Dark
Mes 2-[N-morpholino]ethanesulfonic acid
PCO Photorespiratory Carbon Oxidation
PCR Photosynthetic Carbon Reduction
PEP phosphoenolpyruvate
3-PGA 3-phosphoglycerate
PR photorespiration
PVP polyvinyl pyrrolidone
RuBP ribulose-1,5-bisphosphate
RuBPCO ribulose-1,5-bisphosphate-carboxylase-
oxygenase
SP sugar monophosphate
SP2 sugar bisphosphate
TPP+ tetraphenyl phosphonium
Tris tris(hydroxymethyl) aminomethane
r CO2 compensation point













Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

MECHANISMS FOR REDUCING PHOTORESPIRATION
IN SUBMERSED AQUATIC ANGIOSPERMS

By

Michael Edward Salvucci

April 1983

Chairman: George Bowes
Major Department: Botany

The biochemical mechanisms regulating photorespiration in submersed

angiosperms were examined in Myriophyllum spicatum L. and Hydrilla verti-

cillata (L.F.) Royal. Oxygen inhibition of net photosynthesis at 330 pl

CO2/1 was 23 and 24%, respectively, and responded competitively to CO2 in

the high photorespiration state, but was 15 and 5%, respectively, and

independent of the CO2 concentration in the low photorespiration state.

Carbonic anhydrase activity increased by three-fold with the induction

of the low photorespiration state, but no change in the kinetic character-

istics of ribulose-1,5-bisphosphate carboxylase-oxygenase occurred.

In pulse-chase experiments, C4 acids accounted for greater than 50%

of the initial products in Hydrilla, but label in malate decreased during

the chase only in the low photorespiration state. Carbon assimilation was

almost exclusively via the Calvin cycle in Myriophyllum.

Ethoxyzolamide repressed the low photorespiration state in Myriophyl-

lum and Hydrilla as it reduced photosynthesis by 40%, and increased 02








inhibition, the apparent Km(CO2) of photosynthesis, the CO2 compensation

point and, for Myriophyllum, the fraction of label fixed into photores-

piratory intermediates. Net photosynthesis in high photorespiration
Myriophyllum and Hydrilla plants was inhibited only slightly by ethoxyzol-

amide, and their CO2 compensation points, and those of the terrestrial

plants,Sorghum, Moricandia and Nicotiana, were unaffected.

Treatment with isonicotinic acid hydrazide caused label to accumulate

in glycine, which then represented 25 and 47% of the labeled products in

low and high photorespiration Myriophyllum, respectively, and 9 and 14%

in Hydrilla, respectively. Glycine labeling and 02 inhibition of photo-

synthesis appeared to be uncoupled from the external [02]/[CO2] ratio in

low photorespiration Myriophyllum as both were affected by changes in the

02 but not in the CO2 concentration. Oxygen inhibition in the high photo-
respiration state responded to changes in both CO2 and 02.

It is suggested that Myriophyllum and Hydrilla exemplify two differ-

ent photosynthetic mechanisms but both represent strategies, though dif-

ferent, for concentrating inorganic carbon. Common to both is a require-

ment for carbonic anhydrase, possibly to enhance the usage of accumulated

CO2 for reducing photorespiration. These data suggest that submersed

angiosperms cannot be categorized in the presently recognized photo-

synthetic groupings.













CHAPTER I
PHOTOSYNTHETIC CARBON ASSIMILATION AND
PHOTORESPIRATION IN SUBMERSED
AQUATIC MACROPHYTES: A REVIEW


The pathway for carbon assimilation elucidated by Calvin, Benson,

Bassham and their co-workers is central to the incorporation of carbon

into the biosphere by photoautotrophic organisms (cf. Bassham and Calvin,

1957; Hatch and Boardman, 1981). Among photosynthetic organisms, atmos-

pheric or dissolved CO2 may be assimilated directly into the Photosynthet-

ic Carbon Reduction cycle (PCR or Calvin cycle) by the initial carboxyla-

tion reaction or indirectly, following the release of CO2 by an internal

decarboxylation reaction. In the former case, the initial event in the

fixation of CO2 is the carboxylation of CO2 with ribulose-l,5-bisphosphate

(RuBP), catalyzed by RuBP carboxylase, while in the latter case, this

carboxylation step is proceeded by an initial B-carboxylation reaction

catalyzed by phosphoenolpyruvate (PEP) carboxylase. In terrestrial

plants, the route of CO2 assimilation can ultimately determine what per-

cent of the carbon fixed flows through the Photorespiratory Carbon Oxida-

tion cycle (PCO or photorespiratory cycle). The PCO cycle is important

energetically for a plant, and it has major implications in regard to

growth, productivity, and even survival in a particular habitat (Bjbrkman,

1976; Osmond, 1981).

Higher terrestrial plant species can be divided into 4 major groups

(C3, C4, C3-C4 intermediate, and Crassulacean Acid Metabolism (CAM)) de-
pending upon their mode of photosynthetic carbon assimilation (Chollet and









Ogren, 1975; Rathnam and Chollet, 1980; Osmond and Holtum, 1981). The

biochemical, physiological and ecological aspects of photosynthetic car-

bon metabolism, in each of these groups, have been described in detail,

and several excellent review articles are available (Chollet and Ogren,

1975; Ehleringer, 1979; Rathnam and Chollet, 1980; Hatch and Boardman,

1981; Lorimer, 1981; O'Leary, 1981; Osmond, 1981; Tolbert, 1981; Somer-

ville and Ogren, 1982). In contrast to terrestrial plants, a relatively

limited amount of attention has been directed toward the study of photo-

synthetic carbon metabolism in submersed aquatic macrophytes and no reviews

on the subject have been conducted to date. A casual perusal of the liter-

ature, however, will reveal that, while there is no consensus view on the

subject, there is unanimity among investigators that submersed aquatic

macrophytes exhibit some physiological and/or biochemical features which

are not typical of any one of the recognized photosynthetic groups. It

is the purpose of this paper to review the literature on photosynthetic

and photorespiratory carbon metabolism in submersed aquatic macrophytes

from a mechanistic/biochemical perspective,underlining the fact that the

biochemical reactions of photosynthesis serve as a common basis from which

to compare the physiological responses of photosynthesis and photorespira-

tion in these organisms, with the same physiological responses in terres-

trial higher plants.


General Features of Submersed Aquatic Macrophytes

Submersed aquatic macrophytes are plants which are adapted physio-

logically and morphologically to grow and photosynthesize under water.

Included for discussion in this review will be species from charophytic









algae through submersed angiosperms, both freshwater and marine; but with

more emphasis on the freshwater species for which there is more informa-

tion available. Emergent plants such as Typha (cattail) and Spartina

(cordgrass), which photosynthesize primarily in the aerial environment,

will not be considered. Also excluded from discussion will be the aerial

or floating-leaf forms of heterophyllic aquatic macrophyte species such

as Myriophyllum brasiliense (parrot's feather) and Potamogeton amplifolius

pondweedd). These species are similar to emergent species in that they

exhibit few of the more unusual features characteristic of photosynthetic

carbon metabolism in submersed aquatic macrophytes (Hutchinson, 1975;

Lloyd et al., 1977; Salvucci and Bowes, 1982).

In general, submersed macrophytes are among the least productive of

plants and exhibit correspondingly low photosynthetic capacities (Cooper,

1975). Some seagrass species,however, are regarded as highly productive

(Williams and McRoy, 1982), although this is inconsistent with their rel-

atively low photosynthetic capacity (Beer et al., 1977). In spite of

their low productivity, freshwater macrophytes can rapidly form heavy

stands of vegetation in an invaded area and some species have become major

weed problems (cf. Van et al., 1978). The apparent prolific growth is due,

in part, to a lower requirement for structural biomass components in the

aquatic environment which, as evidenced by the low dry weight/fresh weight

ratio, provides for volume increases without substantial dry matter gain

(Westlake, 1975; Bowes et al., 1979). Low chlorophyll and soluble pro-

tein content, expressed on an area or fresh weight basis, are also char-

acteristics of freshwater submersed macrophytes due to their high (up to

95%) water content (De Groote and Kennedy, 1977; Holaday et al., 1983).

This consideration should be realized when comparing photosynthetic and









photorespiratory activity in submersed macrophytes to that in terrestrial

plant species.


Physiological Aspects


Photosynthesis and the Apparent Km(C02)

In spite of the use of a variety of methods for measurement, pub-

lished rates of net photosynthesis for a large range of freshwater macro-

phytes are remarkably consistent. At air-levels of CO2 (330 pl C02/1,

gas phase or 10 pM free CO2, aqueous), the light saturated rate of net

photosynthesis under ambient 02 concentrations is typically 1 to 20 pmol/

mg Chl-h whether measured by 02 evolution (Van et al., 1976; Beer and

Wetzel, 1981; Raven et al., 1982), 14C incorporation (Brown et al., 1974;

McCracken et al., 1975; Best and Meulemans, 1979; Holaday and Bowes, 1980)

or infrared gas analysis (Van et al., 1976; Lloyd et al., 1977; Browse et

al., 1979b;Keeley and Bowes, 1982; Raven et al., 1982; Salvucci and Bowes,

1982). Under saturating inorganic carbon concentrations, net photosynthet-

ic rates of approximately 50-60 pmol/mg Chl-h are typically measured in

02 electrode systems (Van et al., 1976; Beer and Wetzel, 1981); however,
somewhat higher rates of approximately 150 pmol/mg Chl-h have been meas-

ured with infrared gas analysis systems (Browse et al., 1979b;Salvucci

and Bowes, 1982).

Under saturating concentrations of inorganic carbon, rates of net

photosynthesis reported for marine angiosperms (seagrasses) are, in general,

very similar to those for freshwater macrophytes (Black et al., 1976;

Downtown et al., 1976; Beer et al., 1977). In a few species of seagrasses

from tropical waters, carbon uptake has been reported to be substantially

higher than in freshwater macrophytes (Williams and McRoy, 1982); however,









these rates are still considerably lower than the rates of most terres-

trial C3 plants (Cooper, 1975).

The concentration of CO2 required to saturate photosynthesis in sub-

mersed aquatic macrophytes is of the order of 350-600 vM or approximately

10-20 times greater than required for terrestrial C3 plants (Steeman Neil-

son, 1947; Raven, 1970; Hutchinson, 1975; Van et al., 1976; Beer et al.,
1977; Browse et al., 1979b). This high requirement for CO2 has long been

recognized as a feature characteristic of photosynthesis in submersed

macrophytes (Steeman Neilson, 1947; Raven, 1970). The apparent Km(CO2)

values for RuBP carboxylase-oxygenase from several submersed macrophyte

species are somewhat higher than in terrestrial C3 plants (Yeoh et al.,

1981), although similar to the values of the enzymes from C4 plants and

green algae (Jordan and Ogren, 1981b; Yeoh et al., 1981). Although higher,

the difference in the Km(C02) values between aquatic plants and terres-

trial C3 plants is less than two-fold, and thus insufficient to account

for the very high apparent Km(C02) values for photosynthesis typically
reported in submersed macrophytes (Van et al., 1976; Browse et al., 1979b;

Black et al., 1981).

The high apparent Km(C02) for photosynthesis in submersed macrophytes

has been evaluated in terms of the component resistances to CO2 transfer

and is attributed most frequently to a slow rate of CO2 diffusion through
the aqueous environment (Steeman Neilson, 1947; Raven, 1970). It is now

well-established that, under unstirred conditions, the major resistance

to CO2 assimilation is diffusion through the boundary or unstirredd" layer,

which can be 100-500 um in distance (Steeman Neilson, 1947; Raven, 1970;

Browse et al., 1979b; Smith and Walker, 1980; Black et al., 1981). Under
well-stirred conditions, both the apparent Km(C02) for photosynthesis and








the boundary layer decrease (Browse et al., 1979b); however, the residual

resistance is still considerable as compared with photosynthesis in ter-

restrial plants (Van et al., 1976; Ascencio, 1979; Browse et al., 1979b;

Smith and Walker, 1980; Black et al., 1981).

The bulk of the remaining resistance under nonphotorespiratory con-

ditions has been attributed to diffusion within the boundary layer which

is thought to persist as the major limitation to CO2 assimilation even

under turbulent or well-stirred conditions (Browse et al., 1979h;Black

et al., 1981). Evidence for this is available in a study of photosynthe-

sis in the amphibious moss Polytrichum formosum (Gessner, 1955), for

which the apparent Km(CO2) for photosynthesis was reduced by measurement

in air. However, in a more recent study, the high CO2 requirement for

net photosynthesis in four submersed angiosperm species could not be over-

come by measurement in a water-saturated aerial environment (Lloyd et al.,

1977). These results are confirmed by similar, but independent studies

(Ascencio, 1979; Salvucci and Bowes, 1982) which report high apparent

Km(C02) values (122 and 70 vM) for net photosynthesis measured in air

for Myriophyllum (at 21% 02) and Hydrilla (at 1% 02), respectively.

These high values indicate that, under conditions which minimize the

boundary layer resistance, the internal resistance to CO2 assimilation

is still considerably greater in submersed macrophytes than in terrestrial

or amphibious species (Lloyd et al., 1977; Salvucci and Bowes, 1982).

Components of this high resistance include carboxylation resistance,

photorespiration, and dark respiration (Ascencio, 1979),indicating that

these physiological processes offer substantially more resistance to CO2
assimilation in submersed macrophytes than in terrestrial plants (Jarvis,

1971). Under so-called natural conditions,however, boundary layer









resistance can be extremely high and is probably the most important fac-

tor limiting CO2 assimilation (Raven, 1970; Hutchinson, 1975; Browse et

al., 1979b;Smith and Walker, 1980).

Bicarbonate Usage

The ability of submersed macrophytes to utilize HCO3 as a source

of inorganic carbon for photosynthesis is widely recognized and well-

documented (cf. Steeman Neilson, 1947; Raven, 1970). It should be empha-

sized,however, that the species of carbon that is directly incorporated

during photosynthesis is determined by the carboxylase enzymes, free CO2

for RuBP carboxylase (Cooper et al., 1969) and HCO3- for PEP carboxylase

(Cooper and Wood, 1971), regardless of the species of carbon that is

taken up by the plant. Furthermore, it is the pH at the active site of

carboxylation that dictates the relative abundance of free CO2 and HCO3

for use in photosynthesis, and equilibrium concentrations of these two

species may be maintained, in accord with the pH, by the enzyme carbonic

anhydrase (Maren, 1967). Carbonic anhydrase activity has been reported

in virtually all submersed macrophytes that have been examined (Graham

and Smillie, 1976; Van et al., 1976; Weaver and Wetzel, 1980).

The utilization of HCO3- as an exogenous source of inorganic carbon

has been discussed extensively (cf. Raven, 1970; Smith and Walker, 1980)

and it is generally accepted that most submersed macrophyte species

utilize HCO3 for photosynthesis (Steeman Neilson, 1947; Van et al.,

1976; Beer et al., 1977; Browse et al., 1979b;Bain and Proctor, 1980;

Lucas, 1980; Prins et al., 1982a). Distinctions in the past of plants

as "users" and "nonusers" of HC3- may in reality be one of degree rather

than an absolute (Allen and Spence, 1981). It has been suggested that









the ability to utilize HCO3 may be a determining factor in the distribu-

tion of freshwater macrophytes in waters of different hardness and pH

(Hutchinson, 1975; Bain and Proctor, 1980), though the evidence is not

conclusive.

Under natural conditions, HCO3- may be the predominant form of carbon

available in the environment (for example in seawater) and likely supplies

the bulk of carbon assimilated in photosynthesis (Raven, 1970; Van et al.,

1976; Beer et al., 1977). However, when HCO3 and free CO2 are present

in equal molar concentrations, free CO2 appears to be the preferred spe-

cies of carbon utilized (Steeman Neilson, 1947; Raven, 1970; Van et al.,

1976; Browse et al., 1979b;A1len and Spence, 1981) even for marine macro-

phytes (Beer et al., 1977; Benedict et al., 1980). Thus, the relative

importance of HCO3 as a source of carbon for photosynthesis in submersed

macrophytes is perhaps more dependent upon the environmental factors which

determine the availability of CO2 versus HCO3" in the aquatic environment,

than upon the efficiency of the plant to take up HC03 .

Bicarbonate utilization by submersed macrophytes is generally con-

sidered to be an active process (Steeman Neilson, 1947; Raven, 1970; Prins

et al., 1982b), possibly electrogenic (Lucas et al., 1977), and exhibits

polarity in some species, in that OH" ions are extruded from the adaxial

side of the leaf and the inorganic carbon species enters via the abaxial

surface, while in others it is nonpolar (Steeman Neilson, 1947; Lucas,

1980; Prins et al., 1982a, 1982b). Possible mechanisms for HCO3- uptake

or utilization have been discussed in detail (Lucas, 1980; Walker, 1980;

Prins et al., 1982b) based primarily on studies with a few selected

species (i.e., Chara and Potamogeton). A fundamental difference between

the proposed mechanisms concerns the question of which species of inorganic









carbon actually crosses the plasmalemma. One hypothesis is that HCO3

can be taken up directly in a uniport or synport (H /HC03 ) mechanism

(Lucas, 1980; Lucas et al., 1982). There is evidence,however, based on

pH changes at the leaf surface, to support an alternative hypothesis;

that is that HCO3- utilization results from the conversion of HC03- to

CO2 near or in the cell wall due to active H+ extrusion. This results in

CO2 uptake by passive diffusion into the cell (Ferrier, 1980; Walker et

al., 1980; Prins et al., 1982b). As is discussed below, in some macro-

phyte species, the utilization of HCO3 may require certain biochemical

modifications in photosynthetic carbon metabolism and further study of

this relationship may help resolve some of the mechanistic details of

HCO3- uptake/utilization.

Photorespiration

While the absolute rate of photorespiration is difficult to quantify,

several methods have been used with terrestrial plants to provide esti-

mates of the extent of photorespiratory activity (Chollet and Ogren, 1975).

As most of these methods involve infrared gas analysis measurements, some

additional limitations are imposed when applying these methods to plants

in an aqueous environment (cf. Salvucci and Bowes, 1982; Holaday et al., in

press). Estimates of photorespiration in submersed macrophytes are also

complicated by the relatively high rate of dark respiration (as compared

with net photosynthesis) in these plants (Lloyd et al., 1977; Bowes et

al., 1978; S0ndergaard and Wetzel, 1981; Salvucci and Bowes, 1982). As

in terrestrial plants, dark respiration in submersed macrophytes is sat-

urated by 1-2% 02, gas phase (Lloyd et al., 1977; Salvucci and Bowes,

1982), and the contribution of this respiratory process can be accounted








for in any measure of photorespiration by determining whether the meas-

ured response is 02 sensitive (Chollet and Ogren, 1975).

CO2 evolution into C02-free air (water) in the light. The rate of

CO2 evolution into C02-free air is a useful estimate of photorespiratory

activity in terrestrial plants when the rate is evaluated with respect

to the net photosynthetic rate (Chollet and Ogren, 1975; Lloyd and Can-

vin, 1977; Holaday et al., 1982). Unlike for most terrestrial species,

the rate of CO2 evolution in the light in submersed macrophytes includes

a relatively large 1% 02 component, which should be accounted for when

making comparative estimates of photorespiratory activity based on this

method (Lloyd et al., 1977; Bowes et al., 1978; Salvucci and Bowes, 1982).

In the few studies in which the rate of CO2 evolution has been measured,

CO2 evolution in the light, corrected for the rate at 1% 02, was 5 and 17%

of the net photosynthetic rate at 350 pl C02/l for Myriophyllum and

Potamogeton,respectively, in one study (Lloyd et al., 1977) and was less

than 2% and greater than 15% for Myriophyllum in another (Salvucci and

Bowes, 1982). The high rate of CO2 evolution measured for Myriophyllum

and Potamogeton in comparison to net photosynthesis conflict with the

widely published notion that lacunal air spaces in submersed macrophytes

contribute to an enhanced internal refixation (Hough and Wetzel, 1978;

Sondergaard, 1979; Spndergaard and Wetzel, 1981). Instead, these rates

were comparable to those obtained for terrestrial C3 plants (cf. Lloyd
and Canvin, 1977; Holaday et al., 1982), indicating that photorespiratory

activity in submersed macrophytes can be similar in magnitude to that in

C3 plants. However, for some plants, the low rates that can be obtained
approximate the level measured in C3-C4 intermediate or even C4 plants

(Morgan and Brown, 1979; Holaday et al., 1982), species with reduced








photorespiration. It has been postulated that refixation of photorespired

CO2, by accumulating in the lacunae, would account for these low rates

(S~ndergaard and Wetzel, 1981). However, not all submersed plants with

low rates of photorespiratory CO2 evolution have extensive lacunae and

even those with a well developed lacunal system do not always show any

differences in the rate when the lacunal effect is removed by splitting

the leaves (S0ndergaard and Wetzel, 1981; Keeley and Bowes, 1982).

Oxygen inhibition of photosynthesis. Photorespiration, unlike dark

respiration,is stimulated by increasing 02 concentrations through 100%,

and this stimulation, along with a direct competitive effect of 02 on CO2

fixation (Laing et al., 1974), results in an inhibition of net photosyn-

thesis by 02 (Chollet and Ogren, 1975). The ability of 02 to effect a

change in net photosynthesis has been used to indicate the presence and

estimate the extent of photorespiration in terrestrial plants (Forrester

et al., 1966a; Chollet and Ogren, 1975).

Oxygen inhibition of net photosynthesis is the most frequently cited

evidence for the occurrence of photorespiration in submersed macrophytes

(Brown et al., 1974; Hough, 1974; Black et al., 1976; Downton et al., 1976;

Van et al., 1976; Lloyd et al., 1977; Bowes et al., 1978; Hough and Wetzel,

1978; Sondergaard, 1979). However, inhibition of photosynthesis under high

02 concentrations (i.e., above ambient) may be caused by effects on proc-

esses unrelated to photorespiration (cf. Chollet and Ogren, 1975; Shelp

and Canvin, 1980) and so the degree of inhibition caused by ambient (versus
low) 02 is potentially the most reliable way to estimate photorespiration

when measuring 02 inhibition.
For terrestrial plants, the degree of inhibition is relatively con-

stant within the various photosynthetic groups; approximately 30% in C3








plants (Forrester et al., 1966a; Chollet and Ogren, 1975; Holaday et al.,

1982), 21-25% in C3-C4 intermediates (Morgan and Brown, 1979; Rathnam and

Chollet, 1980; Holaday et al., 1982), and little or no inhibition by 21%

02 is measured in C4 plants (Forrester et al., 1966b; Chollet and Ogren,

1975). In contrast, although some inhibition of net photosynthesis by 21%

02 can be measured in virtually all submersed macrophytes, the degree

varies considerably from 4 to 30% (Black et al,, 1976; Downton et al.,

1976; Van et al., 1976; Lloyd et al., 1977; Ascencio, 1979; Salvucci and

Bowes, 1981; Sondergaard and Wetzel, 1981), even for the same species.

CO? compensation points. The CO2 compensation point can be used re-

liably as a single determinant in categorizing terrestrial plants as C3,

C4, or C3-C4 intermediate species (Chollet and Ogren, 1975; Rathnam and

Chollet, 1980). With the appropriate precautions (cf. Holaday et al., in

press), the CO2 compensation point of submersed macrophytes can be used

similarly as a valid measure of the photosynthesis/photorespiration ratio;

evidenced by the correlation of CO2 compensation point values with photo-

synthetic and photorespiratory estimates determined by other methods

(Lloyd et al., 1977; Salvucci and Bowes, 1982). The CO2 compensation

point values reported for submersed aquatic macrophytes range from high,

>150 to 60 pl C02/1 (Lloyd et al., 1977; Ascencio, 1979; Spndergaard,

1979; Black et al., 1981; S~ndergaard and Wetzel, 1981; Holaday et al.,

1983),through intermediate, 50 to 30 (Van et al., 1976; Lloyd et al.,

1977; Browse et al., 1979b; Salvucci and Bowes, 1981),to very low, 30-0

(Stanley and Naylor, 1972; Hough, 1974; Van et al., 1976; Bowes et al.,

1978; Browse et al., 1980; Salvucci and Bowes, 1981; Raven et al., 1982),

sometimes for the same species (Bowes et al., 1978; Ascencio, 1979; Sal-

vucci and Bowes, 1982; Holaday et al., 1983). The continuous range of









possible CO2 concentration points in submersed macrophytes span the range

of values in terrestrial plants: C4, 0-5; C3-C4 intermediate, 12-25; and

C3, 40-60 (Chollet and Ogren, 1975; Morgan and Brown, 1979; Rathnam and

Chollet, 1980).

True versus apparent photosynthesis. A modification of the elegant

system developed by Ludwig and Canvin (1971) for determining the extent

of photorespiration in terrestrial plants, as the difference between true

and apparent photosynthesis, has been used with the submersed angiosperm,

Scirpus subterminalis (Sondergaard and Wetzel, 1981). Photorespiration,

estimated by this technique, occurred at rates of 8% of the net photosyn-

thetic rate at ambient 02 (8 mg/l), 0-4% at 1 mg 02/1, and 15-30% at 30

mg 02/1 (S6ndergaard and Wetzel, 1981). Unfortunately, a total inorganic

carbon concentration of 500 vM, pH 6.7 was used, and so a direct comparison

with measurements of terrestrial plants at air-levels (10 uM) are not

possible. It is uncertain whether this technique can be readily adapted

for use with submersed macrophytes at low inorganic carbon concentrations,

because the generally low rates of photosynthesis in these aquatic plants

limit the attainment of high specific activities in the plant (cf. Lloyd

et al., 1977). Furthermore, because this technique relies on rapid esti-

mations of 14C and 12C uptake and evolution, boundary layer effects on

CO2 diffusion will be a major source of interference.

Light/dark (L/D) ratios of C02 evolution. In several studies with

submersed macrophytes, the ratio of 12C0 or 14C02 evolved in the light

versus the dark has been used to estimate the extent of photorespiratory

activity (Hough, 1974, 1976; Hough and Wetzel, 1972, 1978; S0ndergaard,

1979; S0ndergaard and Wetzel, 1981). The original method was developed

as a way to estimate photorespiration in terrestrial plants (Zelitch, 1968);









however, its theoretical basis has been criticized (Chollet and Ogren,

1975; Chollet, 1978) and these criticisms are valid when the method is

used with submersed macrophytes (cf. Salvucci and Bowes, 1982). In addi-

tion, direct comparisons of L/D ratios between submersed macrophytes and

terrestrial plants, as is often done, can be misleading even for indicat-

ing the simple presence or absence of photorespiration, since the rela-

tively higher rate of dark respiration in submersed macrophytes is not

usually considered (Salvucci and Bowes, 1982; Holaday et al., in press).

The Concept of Variable Photorespiration

It is obvious from the discussion above that, while there appears to

be a consensus view that submersed macrophytes exhibit some photorespiratory

activity, reported levels vary considerably even within a particular species.

Thus, the classification of submersed aquatic macrophytes as a group, as

either C3, C4, or even C3-C4 intermediates based upon the level of photo-

respiratory activity, is not possible.

In order to explain changes in the CO2 compensation point that occur

naturally or as a result of specific laboratory growth conditions, reports

from this laboratory have suggested that variable photorespiration may be

a characteristic feature of submersed macrophytes (Bowes et al., 1978;

Holaday and Bowes, 1980). In recent investigations, the concept of vari-

able photorespiration (in effect a continuum of possible photorespiratory

states) has been documented for all 11 freshwater macrophytes examined,

including a charophytic alga and an aquatic moss (Salvucci and Bowes,

1981; Holaday et al., in press).

Two freshwater angiosperms, Myriophyllum and Hydrilla, have been

examined in detail with respect to the gas exchange features of their low









and high photorespiration states (Bowes et al., 1978; Salvucci and Bowes,

1981, 1982). In the low photorespiration state, the CO2 compensation

points of the plants can be as low as 10 ul C02/l, rates of CO2 evolution

in the light are usually less than 2% of net photosynthesis, and the de-

gree of inhibition of net photosynthesis by 21% 02 is low (5-15%) and in-

sensitive to changes in the CO2 concentration. Plants in the high photo-

respiration state exhibit a lower rate of net photosynthesis and a two- to five-

fold higher apparent Km(C02) for net photosynthesis than plants in the

low photorespiration state. Photorespiration in the high state is measur-

able and comparable to that in terrestrial C3 plants; as indicated by CO2

compensation points greater than 50 l1 C02/1, rates of 02-sensitive CO2

evolution of 15% of the net photosynthetic rate, and a 25-30% inhibition

of net photosynthesis by 21% 02 at air-levels of CO2.
The contrasting gas exchange characteristics of the high and the low

photorespiration states in submersed angiosperms resemble differences in

photorespiratory activity observed for unicellular green algae grown at

high, as opposed to low, concentrations of CO2 (cf, Berry et al., 1976;

Hogetsu and Miyachi, 1979; Badger et al., 1980; Beardall and Raven, 1981).

The level of CO2 used during growth can also influence the photorespira-

tion state in submersed macrophytes (Holaday et al., 1982). Other factors

which influence the induction of the low photorespiration state are sub-

mergence (Salvucci and Bowes, 1981, 1982), temperature and photoperiod
(Bowes et al., 1978; Holaday and Bowes, 1980; Holaday et al,, in press).
These latter two parameters vary seasonally and are the likely cause of

seasonal changes in the photosynthetic/photorespiratory activities that
have been documented for some freshwater macrophytes (Bowes et al., 1978;
S0ndergaard, 1979; Holaday and Bowes, 1980). It is unknown whether a









similar seasonality in the extent of photorespiratory activity exists for

marine macrophytes such as seagrasses or, for that matter, whether marine

macrophytes exhibit variable photorespiratory activity. It is generally

recognized that certain freshwater habitats may subject submersed plants

to conditions of low CO2 availability and high 02, light, temperature and

pH; a situation in which a mechanism to minimize photorespiratory CO2 loss

would be most advantageous (Brown et al., 1974; Van et al., 1976; Hough

and Wetzel, 1978; Bowes et al., 1979; Browse et al., 1980). Similar con-

ditions can occur in some marine habitats, for example in coastal estuaries

or salt marsh pools, and a mechanism adapted to reduce photorespiration

would be advantageous for marine macrophytes photosynthesizing under these

environmental conditions.


Biochemical Aspects


Biochemical characteristics of photosynthetic carbon metabolism in

submersed macrophytes have been examined in only a comparatively small

number of studies and, consequently, detailed information is limited.

Many of the investigations that were conducted intended to determine

whether submersed macrophytes were C3 or C4 plants (cf. Stanley and Naylor,

1972; Beer et al., 1980; Beer and Wetzel, 1982b). In retrospect, it should

be realized that the concept of C4 photosynthesis requires the integration

of specific biochemical reactions with a specialized anatomical arrange-

ment (Bjdrkman, 1976; Hattersley et al., 1977; Perrot-Rechenmann et al.,

1982), the latter of which is lacking in the two- to four-cell layered

leaves of most submersed macrophytes (Sculthorpe, 1971; Doohan and Newcomb,

1976; Hough and Wetzel, 1977). From recent evidence it appears that the









physiological characteristics of photosynthesis in freshwater and marine

submersed macrophytes are representative of neither the C3 nor C4 group,

although certain biochemical features in some species may be quite analo-

gous to the pathway of C4 acid metabolism in C4 plants.

Photosynthetic and Photorespiratory Enzymes

RuBP carboxylase-oxygenase. Ribulose-1,5-bisphosphate (RuBP)

carboxylase-oxygenase activity has been measured in several submersed

freshwater angiosperms (Van et al., 1976; De Groote and Kennedy, 1977;

Salvucci and Bowes, 1981; Beer and Wetzel, 1982b; Valanne et al., 1982)

and in some seagrasses (Beer et al., 1980). Enzyme activity in submersed

macrophytes is low in comparison with most terrestrial plants (cf. Ku et

al., 1979), although it is generally comparable to the maximum photosyn-

thetic rates (50-150 umol/mg Chl-h) reported for these plants (Van et al.,

1976; Beer et al., 1980; Salvucci and Bowes, 1982). Van et al. (1976)

have attributed the low photosynthetic rates of submersed macrophytes,

in part, to these low RuBP carboxylase activities; however, the opposite

interpretation, that low enzymatic capacity is the result rather than the

cause of the high resistance to photosynthesis in submersed macrophytes,

has also been presented (Black et al., 1981).

In the 12 species of freshwater macrophytes in which it has been

determined, the Km(C02) of RuBP carboxylase-oxygenase is higher than for

the enzyme from terrestrial C3 species, but similar to the Km(C02) of the

enzyme from C4 plants and green algae (Yeoh et al., 1981). The substrate

specificity factor and the Km(C02) for RuBP carboxylase-oxygenase from

Myriophyllum or Hydrilla were virtually identical in the low and high

photorespiration state (APPENDIX A). The specificity factor, V K /V K

(Jordan and Ogren, 1981a, 1981b), for these species is in the lower range









of the values reported for terrestrial plants (Jordan and Ogren, 1981b;

APPENDIX A), indicating that the relative potential of RuBP carboxylase-

oxygenase for carboxylation and oxygenation in these submersed angiosperms

species is similar to that for the enzyme from terrestrial plants. The

amount of RuBP carboxylase-oxygenase protein in the leaves of Hydrilla

and Myriophyllum, estimated by rocket immunoelectrophoresis, coincides with

the low specific activity (per mg Chl) in these plants (Salvucci and Bowes,

1981). On a chlorophyll or fresh weight basis, RuBP carboxylase-oxygenase

protein in Hydrilla and Myriophyllum is two- to four-fold less abundant

than in terrestrial C3 plants (Ku et al., 1979; Rejda et al., 1981); the

difference is mainly a function of a lower level of total soluble protein

in submersed macrophytes (De Groote and Kennedy, 1977).

PEP carboxylase. The activity of PEP carboxylase in some submersed

macrophyte species may be relatively high when evaluated with respect to

the total carboxylase activity, RuBP + PEP carboxylase. This situation

is clearly the case for some freshwater macrophytes and marine seagrasses

(De Groote and Kennedy, 1977; Bowes et al., 1978; Ascencio, 1979; Beer et

al., 1980; Salvucci and Bowes, 1981; Beer and Wetzel, 1982b) although in

other species, PEP carboxylase activity is low when considered in relation

to RuBP carboxylase activity (Van et al., 1976; Salvucci and Bowes, 1981;

Valanne et al., 1982). Seasonal variations in the ratio of RuBP/PEP

carboxylase activity have been reported (Bowes et al., 1978; Beer and

Wetzel, 1982b; Holaday et al., 1982) and, for some species, a decrease

in the RuBP/PEP carboxylase ratio can be correlated with an induction of

the low photorespiration state (Bowes et al., 1978; Ascencio, 1979; Sal-

vucci and Bowes, 1981). Thus, there are at least two subgroups in regard

to the major carboxylase enzyme in submersed macrophytes. In one of these









groups, PEP carboxylase activity may be quite high; however, plants in

this group are not necessarily C4 plants just because they exhibit high

PEP carboxylase activity.

Other enzymes of C4 acid metabolism. Several enzymes associated with

C4 acid metabolism also increase in activity upon induction of the low

photorespiration state in Hydrilla, the only freshwater macrophyte for

which information is available (Salvucci and Bowes, 1981). A similar

increase in C4 enzyme activities occurs in facultative CAM plants, upon

induction of the CAM-mode (Kluge and Ting, 1978; Holtum and Winter, 1982).

When considered in relation to the maximum photosynthetic rate (Van et al.,

1976), the increased activities of C4 enzymes upon induction of the low

photorespiration state of Hydrilla are comparatively high. These enzymes

include NAD-malic enzyme, malate dehydrogenase, aspartate aminotransferase

and pyruvate Pi dikinase; the activities of which provide an increased

enzymatic potential for C4 acid decarboxylation, interconversion, and PEP

regeneration in the low photorespiration state (Salvucci and Bowes, 1981).

It is also possible that the activity of C4 enzymes may also increase in

other submersed macrophytes, specifically those in the Hydrocharitaceae

(cf. De Groote and Kennedy, 1977; Browse et al., 1980), upon induction of

the low photorespiration state (Browse et al., 1980; Salvucci and Bowes,

1981).

Carbonic anhydrase. Despite the preponderance of HCO3- in the aquat-

ic environment, in only a few studies has the carbonic anhydrase activity

of submersed macrophytes been measured. In freshwater species, carbonic

anhydrase activity is considerably lower than in either emergent or ter-

restrial species (Van et al., 1976; Weaver and Wetzel, 1980; CHAPTER II),

but this activity is probably in excess of that required for photosynthetic









HCO3- usage (Van et al., 1976). In Hydrilla and Myriophyllum, carbonic

anhydrase activity increases by two- to three-fold upon induction of the

low photorespiration state (CHAPTER II). An analogous increase in carbonic

anhydrase activity occurs in unicellular green algae when transferred from

high (5%) to low (0.03%) CO2, although the increase is of a much greater

magnitude in the algae (Graham et al., 1971; Hogetsu and Miyachi, 1979).

Relatively high levels of carbonic anhydrase activity have been re-

ported in marine seagrasses (Graham and Smillie, 1976), although other

investigators have found little or no activity in these plants (Beer et

al., 1980). The low levels,however, may be due to the use of Tris-SO4

in the extraction medium, as sulfonamides are potent inhibitors of car-

bonic anhydrase (Maren, 1967).

Photorespiratory enzymes. The activities of the enzymes of the photo-

respiratory carbon oxidation pathway in the few submersed macrophytes in

which they have been measured coincide with the generally low activity

of photosynthetic enzymes in these plants. Detailed information concern-

ing the enzymatic capacity of the glycolate pathway in submersed macro-

phytes is not available since the activity of only two major enzymes, P-

glycolate phosphatase and glycolate oxidase, has been measured and then

only in a few species (Frederick et al., 1973; Stanley and Naylor, 1973;

Van et al., 1976; Salvucci and Bowes, 1981). Glycolate dehydrogenase, an

alternate glycolate oxidation enzyme in some algae, has also been assayed

for a few freshwater macrophyte species, but activity has not been

detected (Frederick et al., 1973; Salvucci and Bowes, 1981). Activity

of this enzyme has been reported in extracts from two seagrass species,

Cymodocea rotundata and Thalassia hemprichii, but it is not ubiquitous

to seagrasses in that Halophila ovata possesses only glycolate oxidase








activity (Tolbert, 1976). The significance of glycolate dehydrogenase

activity in some but not other marine angiosperms is unknown. Similarly,

the significance of this enzyme in certain algae, where it replaces gly-

colate oxidase, is also an enigma. Glycolate dehydrogenase activity has

not been reported in higher plants, from terrestrial environments, but

there is insufficient data to speculate as to whether or not this enzyme

may provide some advantage in an aquatic habitat.

Pathways of Photosynthetic Carbon Assimilation and Metabolism

Although it is clear that submersed macrophytes are not C4 plants,

in some species the C4 acids, malate and aspartate can be major initial

products of photosynthesis. Several species of freshwater macrophytes

exhibit considerably greater C4 acid fixation (20-50% of the initial prod-

ucts) than C3 plants (cf. Rathnam and Chollet, 1979) and high levels of

PEP carboxylase activity have been reported in many of these same species.

Table 1 is a compendium of the results from a number of workers which lists

for purposes of comparison the taxonomic status, the extent of C4 acid

versus C3 product formation, the PEP carboxylase relative to RuBP carboxy-

lase activity, and the polar versus nonpolar HCO3 use of several submersed

aquatic macrophytes. Direct comparison of the extent of C4 acid labeling

reported by different investigators is not always possible since the condi-

tions used for equilibration and 14C fixation are usually not identical.

For example, a greater fraction of the total label incorporated enters C4

acids when the concentration of free CO2 is reduced either by decreasing

the total inorganic carbon concentration (Browse et al., 1979a; Holaday

and Bowes, 1980) or by increasing the pH of the solution (Browse et al.,

1979a). In addition, considerable variation in the percentage of C4 acids

formed may occur as a result of changes in the photorespiration state of

the plant (Browse et al., 1980).















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..0 "to/ "o C S- U
0 r-r- 4. r- E 0 W 0


o o tC r_ m u
r-- V B V J S- V 10 a
U)4 3C CM Il CM N I 1-




4) w w0 a w a. a) O a- 3r- (A





S0 U -


C r- C C EO4



0 0 *- = C



.a +j o Z
0 4 04- I C) mw








I n > ') 0 s .
O O c0 0 S.
C *





03 E a0.) 4 >W
X t,- t.,- C










'a o --t
4- U U U t* -
I 0 0 0V r>4 W 4
L3. V .3- u r(4





( 0 0 0 O




.34-
wo t o ) 0 -
0s 4-s 44- 0 fa3 0





S- 1 o- *i- O 4-
0 0 V
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Ljv 1-0) 0.1- '4- >0o
0.UC OC O > .
) 0 4.) r 4P s- E





L S 4-VU V3U 0-V c V- .C

S- o -5- > O- 0 C


u c r_ toto x O
4J m- a) E u -0




00 + I*(~ I* I I CU)
.00- V U 4 U 0 0 M -0o0
(Da L = = m rO (S O
)L O *0
Eo+ E E Ux
*3 *-.
1 m co to 40 1+--0


VL 0- VMUL
.3- <* 0 0 043

0> I- 4 3
S0 C 0 *Lr U)*- r-O *-
0CC E Cr M0 S
C 0 43L LOR
*.) 0 C EIE Co 43 E
C C I3-1i-J3 V 43 0.-0
C 4 *r -r- > *r- 0
0 0 0 4 *oo- 00 0-mE4
40 S. r- Ul U- 0'v-00

0) 0 0o0 4 0 3a3
S *- -- 5-5- L O t *r o
3 0 0 00 C zo 0 + c*-
.0 43 o C.-)jE 0 0 0 D.C 0 C
r U 1 U ) 10.









Some dicotyledonous species of submersed freshwater macrophytes

clearly lack the enzymatic potential for substantial B-carboxylation and

appear to fix CO2 almost exclusively via RuBP carboxylase-oxygenase (Table

1). One of these species, Myriophyllum spicatum, has been examined by

several investigators and has been shown to possess both low levels of

PEP carboxylase activity and pulse-chase labeling patterns that are simi-

lar to terrestrial C3 plants, even when the plant is in the low photores-

piration state (Salvucci and Bowes, 1981). It is interesting to note from

Table 1 that dicotyledonous macrophytes,such as Myriophyllum, are nonpolar

HCO3 users and non-C4 acid former while monocotyledonous, C4 acid form-

ing species that have been examined are recognized as polar HC03 users.

Polar or nonpolar HC03 usage is no doubt a function of the characteristic

differences in leaf anatomy between these submersed monocotyledonous and

dicotyledonous species (Sculthorpe, 1971; Prins et al., 1982a). For this

reason, there may also be a relationship among leaf anatomy, polar HCO3

usage and the occurrence of C4 acid metabolism in submersed macrophytes

(see discussion below).

The C4 acids have been reported as major products of photosynthesis

in some seagrass species, but not in others (Table 1). All seagrasses

are monocotyledonous, and therefore the apparent relationship between

monocots and C4 acid formation found in freshwater species does not per-

tain to these marine angiosperms. Conflicting reports occur for several

genera concerning the extent of C4 acid fixation (Benedict and Scott,

1976; Andrews and Abel, 1979; Beer and Wetzel, 1982a). For example,

values for C4 acid labeling as high as 50% and as low as 6% have been

reported by different workers for the seagrass Thalassia., It is unknown

whether these differences are due to different conditions used in the 14C









fixation experiments (cf. Andrews and Abel, 1979) or perhaps because of

natural variability such as found in freshwater species (Holaday and

Bowes, 1980).

6 13C Values in Submersed Macrophytes

The ratio of 13C/12C is used with terrestrial plants to determine

the principle pathway for carbon assimilation based on the fact that C3

and C4 plants exhibit differing capacities to discriminate between these

two isotopes, largely because they possess different initial carboxyla-

tion enzymes (for a discussion of carbon isotope fractionation see O'Leary,

1981). Thus, 613C values for C3 plants range from -23.2 to -34.3 o/oo,

while C4 plants fractionate to a lesser extent with values ranging from

-11.5 to -18.3. Although the a13C values of several submersed macrophytes,

especially seagrasses, have been determined, seldom do the values (-5.6

to -15.5 o/oo) correlate with data from labeling studies (Benedict and

Scott, 1976; Andrews and Abel, 1979; Beer et al., 1980; Benedict et al.,

1980; Smith and Walker, 1980). For example, C4-like 613C values of -14.5

and -6.8 have been reported for two seagrass species that exhibit C3-type

labeling patterns (Andrews and Abel, 1979). It is now recognized that
13C/12C ratios of submersed macrophytes are influenced by factors which

effect carbon isotope fraction that are peculiar to their environment
and physiology. These factors include HCO3 utilization, large unstirredd"

layers, variations in the 13C/12C ratio of the source water, and even the

turbidity of the water. As a result, 613C values of submersed macrophytes

are not strictly related to fractionation at the carboxylation reaction,

but instead can be manifestations of isotope discrimination at other

stages of carbon assimilation (Andrews and Abel, 1979; Smith and Walker,









1980; Osmond et al., 1981; Faquhar et al., 1982; Raven et al., 1982).

Consequently, for submersed aquatic macrophytes, 613C values are not

necessarily valid indicators of the carbon assimilation pathway. In

this respect, they are similar to aquatic algae.


The Role of C4 Acid Metabolism and Its Relationship to
the Photorespiration State

On the basis of evidence from pulse-chase experiments, many investi-

gators regard submersed aquatic macrophytes as C3 plants (Hough and Wetzel,

1977; Beer and Waisel, 1979; Beer and Wetzel, 1982b; Helder and van

Harmelen, 1982; Raven et al., 1982), including those which exhibit sub-

stantial B-carboxylation. This interpretation would appear to be errone-

ous because it ignores the possibility of dual carboxylation and also the

potentially significant, but not readily apparent, contributions of malate

and aspartate to photosynthetic carbon assimilation in several submersed

macrophyte species. The turnover of label in submersed macrophytes is

generally slower than in terrestrial plants (De Groote and Kennedy, 1977;

Browse et al., 1980; Holaday and Bowes, 1980), and in general, if chase

periods are of a relatively short duration (seconds), the fate of C4 acids

produced in photosynthesis cannot be determined (De Groote and Kennedy,

1977; Holaday and Bowes, 1980; Beer and Wetzel, 1982b).

From the few studies in which the chase period has been extended,

there appear to be at least two possible patterns for malate metabolism

in submersed species in the family Hydrocharitaceae, which exhibit sub-

stantial 8-carboxylation:

1. The fraction of the label fixed into malate may be quite high

during the initial pulse with 14C and actually increase during an extended

chase period of several hours with 12C (Browse et al., 1980; CHAPTER III).









2. The high percentage of label initially fixed into malate may be-

gin to turnover within the first minutes of an unlabeled chase period

until almost all of the label is chased out of this compound within 30-60

minutes (De Groote and Kennedy, 1977; Holaday and Bowes, 1980; CHAPTER III).

In the first situation, malate acts more like an end-product of photo-

synthesis than an intermediate in that the malate pool expands only slowly

in the light and is metabolized during the dark (Browse et al., 1980).

Browse et al. (1980) suggest that this pattern of metabolism may be an

indication that malate functions in these plants as an ion-balance to

mediate cytoplasmic pH regulation or electroneutrality. The accumulation

of malate during excess cation uptake is a widely-recognized phenomenon

in a number of plant tissues (cf. LUttge and Higinbotham, 1979). Recently

it has been described in the Hydrocharitacean, submersed species, Valisneria

spiralis, upon exposure to K2SO4 in the light (Helder and van Harmelen,

1982). Moreover, light-driven K uptake is a major mechanistic feature

of HCO3- utilization in those submersed macrophytes that have been ex-

amined (Lucas, 1980; Walker, 1980; Prins et al., 1982b), and it is thus

likely that malate formation in these plants may be associated with their

HCO3 utilization mechanism.

It is interesting that malate accumulates in the light for high

photorespiration state Egeria and Hydrilla (the photorespiration state

of Valisneria was not reported) but this C4 acid quickly turns over in

the light in Hydrilla plants in the low photorespiration state (Browse et

al., 1980, CHAPTER III). It thus appears that Hydrilla plants in the low

photorespiration state are representative of the second situation described

above. The conditions in freshwater under which the low photorespiration

state is induced are rather severe and include daytime pH values in excess








of 10 (Van et al., 1976). At this pH, free CO2 levels are negligible and

CO3-, an inhibitor of HCO3 usage in submersed macrophytes (Lucas, 1980;

Walker, 1980; Prins et al., 1982a), replaces HC03- as the predominant

species of inorganic carbon in the environment. In general, C03- ions

cannot be used as a direct inorganic carbon source for photosynthesis

(Raven, 1970). Thus, low photorespiration state plants, besides being

exposed to conditions of low inorganic carbon availability, are also

subjected to an environment in which HC03~ utilization is inhibited, and

probably operates only to a very limited extent.

One possible adaptation to these daytime conditions of severely re-

duced carbon availability is dark fixation. This process has been shown

to occur to a considerable degree in Hydrilla plants in the low but not

the high photorespiration state (Holaday and Bowes, 1980), and also has

been reported for the lower vascular plant Isoetes (Keeley, 1981) and

the submersed angiosperm, Scirpus subterminalis (Beer and Wetzel, 1980).

This situation is reminiscent of terrestrial CAM plants in which CO2 is

fixed in the dark into malate, and this C4 acid is stored in the vacuole

for decarboxylation during the day (Kluge and Ting, 1978). Several other

CAM-like features can occur or be induced in these submersed macrophytes,

including a diurnal rhythm in titratable acidity or malic acid levels

and net CO2 fixation in the dark (Beer and Wetzel, 1980; Holaday and Bowes,

1980; Keeley, 1981; Keeley and Bowes, 1982; Holaday et al.,in press). Al-

though speculative, it is quite possible that the induction of increased

activity of C4 acid enzymes that occurs in the low photorespiration state

(Salvucci and Bowes, 1981) is largely for the purpose of metabolizing

malate accumulated in the dark. The continued synthesis and turnover of

malate in the light that is observed in the low photorespiration state









(CHAPTER III) suggests that, unlike in high photorespiration plants,

malate formed during the day is rapidly metabolized by the high enzymatic

capacity induce for matabolizing malate formed in the dark. Since high

02 and temperature enhance photorespiratory CO2 loss during conditions of
low CO2 availability, decarboxylation of C4 acids formed in the light may

be especially advantageous if the CO2 derived from the C4 acids can be

accumulated internally to provide an endogenous source of inorganic car-

bon and reduce photorespiration (Salvucci and Bowes, 1981). This accumu-

lation could conceivably be accomplished if outward diffusion of CO2 is

restricted by the resistance imposed by the large boundary layer due to

the surrounding water. The method for formation and utilization of C4

acids in the light is analogous to C4 acid metabolism in C4 plants and

may explain the C4-like gas exchange characteristics of the low photo-

respiration state in some macrophyte species (Bowes et al., 1978; Hola-

day and Bowes, 1980; Salvucci and Bowes, 1981).

Submersed Macrophytes with Low Potential for B-Carboxylation

Submersed freshwater macrophytes such as Myriophyllum are reminiscent

of certain unicellular green algae in that they exhibit reduced photores-

piration but no C4 acid metabolism (Salvucci and Bowes, 1981). Reduced

photorespiration in the algae is probably due to the operation of an

electrogenic C02-concentrating mechanism which elevates the level of in-

ternal inorganic carbon (cf. Badger et al., 1980; Beardall and Raven, 1981).

A similar type of C02-concentrating mechanism has been postulated as a

mechanism for which to explain the low photorespiration state in non-C4

acid forming submersed macrophytes (Salvucci and Bowes, 1981, 1982: Raven

et al., 1982), based on the observation that their photorespiratory activity









is low when measured by gas exchange analysis. However, more recent data

show that a reduced fraction of the label fixed in photosynthesis is

shunted through photorespiratory intermediates (CHAPTER III), and this is

more direct evidence that a true reduction in photorespiratory activity,

as opposed to an enhancement of refixation, occurs in these plants. This

reduction is repressed when carbonic anhydrase activity is inhibited;

thus, as in algae, carbonic anhydrase is an important component of the

photorespiration-reducing mechanism in Myriophyllum (CHAPTER III). An

identical repression of the low photorespiration state is observed when

carbonic anhydrase is inhibited in Hydrilla; a possible indication that

aquatic plants require both elevated inorganic carbon and carbonic anhy-

drase in order to realize a change in the photorespiration state, regard-

less of the mechanism for carbon accumulation.


Concluding Statement

Although a renewed interest in the photosynthesis of submersed aquatic

macrophytes has occurred in the last few years, available information per-

taining to the physiological and biochemical details of photosynthetic

carbon metabolism in these plants lags far behind our present state of

knowledge for terrestrial plants. The variable nature of photorespiratory

activity in submersed macrophytes is unique among higher plants, and offers

many avenues for challenging future research, directed at investigating

biochemical and environmental regulation of photosynthesis and photores-

piration.













CHAPTER II
ETHOXYZOLAMIDE REPRESSION OF THE LOW
PHOTORESPIRATION STATE IN SUBMERSED ANGIOSPERMS


Introduction


Nonsucculent terrestrial plants can be divided into categories based

on their photosynthetic gas exchange characteristics. For C3, C4, and

C3-C4 intermediate species, the ratio of photosynthesis/photorespiration

is relatively constant within each category under a given set of measure-

ment conditions (Chollet and Ogren, 1975; Rathnam and Chollet, 1980),

except for minor changes during ontogeny (Ticha and Catsky, 1981). In

contrast, submersed freshwater angiosperms have been shown to exhibit

very variable photosynthetic/photorespiratory activities dependent upon

the conditions used for growth of the individual plants (Holaday and

Bowes, 1980; Salvucci and Bowes, 1981; Holaday et al., 1982; Salvucci

and Bowes, 1982). Submersed angiosperms incubated under a 30C/14 h day

exhibit high photosynthesis/photorespiration ratios as evidenced by low

CO2 compensation points, little inhibition of net photosynthesis by 21%

02, increased net photosynthetic rates, and low rates of CO2 evolution

in the light (Salvucci and Bowes, 1981; Holaday et al., 1982; Salvucci

and Bowes, 1982). Under a 12C/10 h day, a high photorespiration state

is induced as gas exchange characteristics of plants incubated under

these conditions indicate a measurable level of photorespiratory activity

(Salvucci and Bowes, 1981; Holaday et al., 1982; Salvucci and Bowes, 1982).








The induction of a continuum of high, through intermediate, to low photo-

respiration states distinguishes submersed angiosperms from nonsucculent

terrestrial angiosperms which appear to be "locked" into a particular

photorespiration state (Chollet and Ogren, 1975; Rathnam and Chollet, 1980);

either high (C3), low (C4), or intermediate (C3-C4).

The inducibility of low or high photorespiration states for submersed

angiosperms resembles a similar phenomenon reported for green algae

(Graham et al., 1971; Badger et al., 1980; Beardall and Raven, 1981;

Shelp and Canvin, 1981) and cyanobacteria (Badger et al., 1978). For

these organisms, certain features of the photosynthetic process can be
altered by the level of CO2 used during growth. Growth under air-levels

of CO2, as opposed to 1-5% C02, results in an increase in the apparent

affinity of photosynthesis for CO2 and decreases in the degree of 02 in-
hibition of photosynthesis and the rate of CO2 evolution in the light

(Badger et al., 1978; Tsuzuki and Miyachi, 1979; Beardall and Raven, 1981;

Shelp and Canvin, 1981). These changes in gas exchange parameters have

been attributed to the active accumulation of high levels of inorganic

carbon by these organisms (Badger et al., 1978; Beardall and Raven, 1981),

as evidenced by measurements of internal inorganic carbon concentrations

approaching 1 mM for the green algae Chlamydomonas and Chlorella (Badger

et al., 1980; Beardall and Raven, 1981) and over 4 mM for the cyanobac-

teria Anabaena and Coccochloris in the low-CO2 grown state (Badger et al.,

1978; Coleman and Colman, 1981). As these organisms are known to fix CO2
primarily via ribulose bisphosphate (RuBP) carboxylase-oxygenase (Graham

et al., 1971; Coleman and Colman, 1981) increasing the C02/02 ratio in-

side the cell could account for the observed gas exchange characteristics
indicative of reduced photorespiration.








The enzyme carbonic anhydrase, which facilitates the interconver-

sion of CO2 and HCO3 ,has been implicated in the inorganic carbon accu-

mulation mechanism. Thus, levels of carbonic anhydrase activity are

higher in algae and cyanobacteria grown at air-levels of CO2 than when

these organisms are grown at high CO2 (Graham et al., 1971; Dohler, 1974;

Hogetsu and Miyachi, 1979). Furthermore, treatment of air-grown algae

with acetazolamide (Diamox) or ethoxyzolamide, inhibitors of carbonic

anhydrase, suppresses the low photorespiration state, and cells thus

treated physiologically resemble cells grown under high CO2 (Graham et

al., 1971; Badger et al., 1978; Tsuzuki and Miyachi, 1979). It was the

purpose of this study to evaluate the involvement of carbonic anhydrase

in the low photorespiration state of submersed angiosperms by examining

the effect of ethoxyzolamide on various photosynthetic and photorespira-

tory activities. Two species were selected for comparison, Myriophyllum

spicatum and Hydrilla verticillata, because previous work has indicated

they exhibit biochemical differences in the low photorespiration state

(Salvucci and Bowes, 1981).


Materials and Methods

Plant Species

Myriophyllum spicatum L. was collected from either Crystal River or

the Suwanee River, Florida, and Hydrilla verticillata (L.F.) Royal was

collected from Orange Lake near Cross Creek, Florida. Apical segments

were grown in a solution containing 5% (v/v) Hoaglands solution under the

conditions described by Holaday and Bowes (1980) to induce a high or low

photorespiration state. Sorghum bicolor (L.) Moench, Nicotiana tabacum L.

and Moricandia arvensis (L.) DC. were greenhouse grown.








Infrared Gas Analysis

For the submersed macrophytes, photosynthetic and dark respiratory

activities were determined by measuring CO2 uptake of evolution in a closed

system as described previously (Salvucci and Bowes, 1981). For the ter-

restrial species, photosynthetic rates of detached leaves were calculated

from the decrease in CO2 concentration monitored in an opened system.

Carbon dioxide compensation point values were used to verify the photo-

respiration state of each plant used in an experiment, and were deter-

mined as described by Van et al. (1976). All gas exchange measurements

were performed at 30C using a saturating quantum flux density of 1,000

pE/m2.s. Gas exchange data presented in Figures 1-3 represent mean

values from at least three separate determinations.

Ethoxyzolamide Treatments

For studies involving ethoxyzolamide, the aquatic plants were incu-

bated and measured at 30*C in the light in a solution containing 10 mM

2-[N-morpholino] ethanesulfonic acid (Mes)-NaOH, 5% (v/v) Hoaglands solu-

tion, and 100 UM ethoxyzolamide at pH 5.5. Leaves from the terrestrial

plants were detached under water and incubated with their cut end sub-

merged in the ethoxyzolamide-containing solution. The incubation time

was generally 4 hours, since longer incubation periods (24 hours) did

not increase the ethoxyzolamide-stimulated effects.

Carbonic Anhydrase

Carbonic anhydrase (E.C. 4.2.1.1) assays were performed using 20 mM

veronal buffer by the method of Hogetsu and Miyachi (1979). Plant ex-

tracts were prepared at 4C by grinding 1.0 g of tissue in a Ten Broeck

homogenizer with 5 ml of 25 mM tris(hydroxymethyl) aminomethane (tris)-HC1,






























o d)
r 3 >
4) 0 r-
*i- -
*0- C( ( -
C 4 a) a)
E(0 4-3 0 0 06
C o +,o o a
=3 (A = S-
*Er- E o ,


+ r- 4-3 E
C -- C 4- -

0) 0 0o -

Q.r- S- to Q. fo
S .,- )- 0
w -r- *- C0-
= XN oI E S-
4-) 0).i- (1





r-
4- S 4PR M CA S

o S.. 0 0
C C'J. (V *1
004- 3 4S) U(-


r0 -- 01 *- *r-


U CUO *4 -
a V) -0 S- -
u r U o
C th M *r- 4- S-.
0 *r- O -0
o (A 3 a) = >1
a) 0 -C 0.:
N "C r- 4-)
0 C .CC
C W) >) 0 0
'4- (A 4- 0 -= 4.-
00 4-) (
4-) 0 r- .C *r-
U .C Q. 0 O.
0) Q. t0 1
4- r- 4- 3 )
4- 4- r- O 0 S-
) < *r- C r- o
a S.- 4-)
4 .. IV S- 0
.C4- >- .C 0
1- 0 = F- 4- C.







C)
*-
01.






















0 0I


* a-




0 0








I4 0
0 T
*- *^
*
>* > >I
nsi xi


I I
0 o -


AS SIS3HINASOOHd
13N JO NOllIBIHNI


O
I(n
O L
0

i0 o

00
_0
N
- 0

4
0 -


00


I-
z
w

0

o o
-o
o)


4.-


I I
10 o0
r- (D

(y~o %Iz

































*i- I
th .C 0
(0)4-) 4.
4 o .C


0 4
If .C



S.0) 0
4) _c


0 0 a).

S4.C- --
C 0... C>
O 4-In
Sr-4-) 0 0
4 Q) C 4- V



U r-- D to
0 C r- 0 -
4.O E.,c
SC0
Sr- O 4-) n



O -0 *

C r 0
U0 *r- 4-)
( .C 0: >




C U 4) 4*- "
4-* r- nto C
o 5-
4. 0 C 4-.S
40) C 4- 0 0




0) Ln 0
.C C 0-0 0)








*r-
0 a a)








cr>










-----INHIBITION OF NET PHOTOSYNTHESIS (%)
o
o in o 0i
-- I, -N O


\I I I I
II o0
E E 0
o 4 0

\ I I I
N N


\ 0
(iL 0
S I 1 W)
\ 3: I o a
II O




/ I I j


0I o
SI II-
11 z
o0 I
I I 0


-0
N I -
O
/o I I o


0 w cm
Om m 11-


(4q 1493 buJ/OO |IOWu 0 ) SIS3HINASOIOHd 13N-



































*r- 0 4-
(n .C cfl 01

C- U) CJ
0t C0 C







4- S'-!
C >*-- 0







o on t






0, r- OJ
- *r- *r-

0..C #U to
4-, C 0 4-








U) >4- U
CnOL S
0 .C 0
C 40. -
00 Id

C 0. 0E
0 *-
















+-> 1 US
4 O5- *r-
CE) .C U)



Y-r
U 4. CU




UC 43


U O+1
.- 5.-i U


C 3=r -





0 S C rd
43C0 >C





-- -a NV 0C







U)

*r-
.3-
U-









--- INHIBITION OF NET PHOTOSYNTHESIS (%)---
0 in 0 io 0
o r- i) CN

I o


O


0-
LO


+ E I
emo a
\0 0
0- a_ OT


_\ _.- 0)
ao 0
O o0
I -




I 0




0I 0
So/


-0
O





0 0
.o-0 -
0O


O /


7o If)



0 0 0 0

- (q4I14 bw/cO0 IOW//)SIS3HiNASO.LOHd 1.3N









5 mM dithiothreitol (DTT), 0.1 mM ethylenediamine-tetraacetate (EDTA),

and 2% (w/v) polyvinyl pyrrolidone (PVP)-40, pH 8.5. Extracts were fil-

tered rapidly through 4 layers of cheesecloth and immediately assayed.

Boiled extracts were used to determine the nonenzymatic rate of hydration.

Preliminary experiments revealed the presence of an endogeous, heat-stable

inhibitor of both the enzymatic and nonenzymatic activity in extracts from

the aquatic plants. Filtration of the extracts through Sephadex G-25

failed to remove the inhibitor, but the inclusion of 2% (w/v) PVP-40 to

the extraction and assay media eliminated inhibition. Enzyme activity is

expressed as enzyme units (EU), where EU = 10(Te/Tb-1) and Te and Tb are

the rates of enzymatic and nonenzymatic hydration, respectively.

PEP Carboxylase

Phosphoenolpyruvate (PEP) carboxylase (E.C. 4.1.1.31) was extracted

and assayed as described previously by Van et al. (1976) except that 50

mM [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate] (Hepes)-KOH, pH 7.3

was used in the extraction medium. After extraction, the centrifuged ex-

tracts were freed of low molecular weight components by rapid filtration

through Sephadex G-25 equilibrated with 25 mM Hepes-KOH and 10 mM MgCl2,

pH 7.2. As the eluants emerged from the columns, they were immediately

supplemented with DTT to a final concentration of 10 mM and assayed.

Chlorophyll concentration was determined according to the method of

Arnon (1949). Soluble protein was determined using the Coomassie Bril-

liant Blue dye method (Spector, 1978) with bovine serum albumin as a
standard. The results presented for all the enzyme activities represent

the mean values of triplicate determinations.








Results

The net photosynthetic rates for Myriophyllum and Hydrilla in the

high and low photorespiration states were measured at 21 and 1% 02 (gas
phase), as a function of CO2. The data are expressed as the percent inhi-
bition due to the decrease in photosynthesis when measured at 21% 02 as
compared to 1% 02 (Fig. 1). At air-saturated levels of CO2 (330 v1/1,
gas phase), the percent inhibition due to 21% 02 was less for Myriophyllum
and Hydrilla plants in the low photorespiration state (15.1 and 5.1%,

respectively) than for those same plants in the high photorespiration
state (22.6 and 23.8%, respectively). For the low photorespiration plants,

changing the CO2 level had no effect on the degree of 02 inhibition (Fig.

1). In contrast, the 02 inhibition in high photorespiration plants in-

creased as the CO2 concentration decreased (Fig. 1). Thus, at a CO2

level of 106 l C002/1, 02 inhibition of photosynthesis was increased to

49 and 74%, respectively, for Myriophyllum and Hydrilla in the high photo-

respiration state, as compared with 8 and 11%, respectively, at 2,500 pl
C002/ (Fig. 1).
An increase in carbonic anhydrase activity was associated with the

low photorespiratory state of both Myriophyllum and Hydrilla. The activ-
ity of carbonic anhydrase was up to three-fold greater in the low photo-
respiration plants (Table 2). Hydrilla exhibited a substantially greater
carbonic anhydrase activity than Myriophyllum when expressed on either a
chlorophyll or protein basis (Table 2). The activities of this enzyme
were considerably less in the submersed angiosperms, regardless of their
photorespiration state, than in the terrestrial C3 plant, tobacco (Table 2).
An inhibitor of carbonic anhydrase, ethoxyzolamide, at a concentra-

tion of 100 uM caused similar increases in the CO2 compensation points of















Table 2. Carbonic anhydrase activity in Myriophyllum
and Hydrilla in the low and high photorespir-
ation (PR) states, and in Nicotiana.


Plant and Carbonic anhydrase activity
Plant and
PR state (EU/mg Chl) (EU/mg protein)

Myriophyl um
Low PR 101 10
High PR 70 5

Hydrilla
Low PR 916 39
High PR 342 18

Nicotiana 3408 118








Myriophyllum and Hydrilla in the low and intermediate photorespiration

states (Table 3). An increase in the CO2 compensation point was evident

when measurements were made with 21 or 1% 02 in the gas phase (Table 3).

However, the increase was far greater for the CO2 compensation points

measured at 21% 02. The effect was reversible upon removal of the

ethoxyzolamide, and occurred, though to a lesser extent, with ethoxyzol-

amide concentrations as low as 20 1M (data not shown). The CO2 compen-

sation points of plants in the high photorespiration state, however,

were unaffected by the ethoxyzolamide treatment (Table 3). Similarly,

the CO2 compensation points determined for the detached leaves of three

terrestrial plants representing three distinct photosynthetic categories

(C4, C3-C4 intermediate, and C3) were unaltered by ethoxyzolamide (Table
3). No effect on the compensation points were observable even after 12

hours incubation at twice the concentration used for the aquatic plants

(200 vM). Apparently the inhibitor was taken up by these leaves, because

after only a 4 hour exposure to 100 vM ethoxyzolamide the net photosyn-

thetic rates were decreased by 3 to 11%.

When measured at 330 ul C02/1 in the gas phase, the net photosynthe-

tic rates of Myriophyllum and Hydrilla plants in the low photorespiration

state were inhibited greatly by ethoxyzolamide (Table 4). Also, at this

CO2 concentration, the 02 inhibition of photosynthesis was increased two-

to four-fold for the low photorespiration plants by the presence of the
inhibitor (Table 4). In contrast, for Myriophyllum and Hydrilla plants

in the high photorespiration state, net photosynthesis and 02 inhibition

were far less affected by ethoxyzolamide (Table 4). The rates of dark

CO2 evolution (respiration) for the low photorespiration plants were not

altered by the inhibitor (Table 4).















Table 3. Effect of ethoxyzolamide on the C02 compensation
points of Myriophyllum, Hydrilla, and Proserpinaca
in different photorespiration (PR) states, and of
Sorghum, Moricandia, and Nicotiana.


C02 compensation point (pl C02/1)
Plant type
and PR state Control Ethoxyzolamide

21% 02 1% 02 21% 02 1% 02


Myriophyl um
Low PR 14 7 47 18
Intermediate PR 37 15 64
High PR 81 55 81 55

Hydrilla
Low PR 14 7 41 25
Intermediate PR 48 29 70
High PR 91 60 98 65

Proserpinacaa
Low PR 7 7 45 26

Sorghum
C4 species 3 4 -

Moricandia
C3-C4 species 22 2 21 1

Nicotiana
C3 species 60 10 61 8


aEmergent form incubated and measured under water.









47













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As shown in Figure 2, for Myriophyllum plants in either photorespir-

ation state, net photosynthesis could be increased by raising the CO2

concentration, but saturation was not achieved even at 5,000 pl C02/1.

Treatment of the low photorespiration Myriophyllum plants with 100 UM

ethoxyzolamide substantially inhibited photosynthetic CO2 fixation (Fig.

2 inset), as previously indicated in Table 4. However, the percent inhi-

bition of photosynthesis caused by the ethoxyzolamide treatment could be

decreased by raising the CO2 concentration (Fig. 2). Myriophyllum plants

in the contrasting high photorespiration state exhibited only a 5% inhi-

bition of photosynthetic CO2 fixation as a result of the ethoxyzolamide

treatment, and this small degree of inhibition was independent of the CO2

concentration (Fig. 2). In a related experiment, the photosynthetic rates

of detached leaflets of low photorespiration Myriophyllum were determined

in terms of 02 evolution. The data obtained were similar to that shown

in Figure 2, in that at pH 8, photosynthetic 02 evolution was decreased

by ethoxyzolamide at sub-saturating, but not at saturating, HC03 levels

(data not shown).

The data in Figures 2 and 3 produced linear responses, when trans-

formed into double reciprocal plots. From an evaluation of these data

in terms of Michaelis-Menton kinetics, estimates of the apparent Km(CO2)

values for photosynthesis were calculated using the statistical methods

of Wilkinson (1961). This provided an approximation of the apparent

affinity of the plants for CO2. For high and low photorespiration Myrio-

phyllum, the apparent Km(C02) values for photosynthesis were calculated

as 175 and 64 VM, respectively. When low photorespiration Myriophyllum

plants were measured in the presence of 100 pM ethoxyzolamide, their

apparent photosynthetic affinity for CO2 decreased to a level approaching









that of high photorespiration plants, as indicated by a rise in the ap-

parent Km(C02) to 123 yM. No increase in the apparent Km(CO2) for photo-

synthesis occurred for high photorespiration Myriophyllum plants treated

with ethoxyzolamide.

Net photosynthesis for low photorespiration Hydrilla increased with

increasing CO2 concentration, but as with Myriophyllum, 5,000 ul C02/1

did not saturate photosynthesis (Fig. 3). An inhibitory effect of 100

vM ethoxyzolamide on net photosynthesis similar to that observed for low

photorespiration Myriophyllum was evident for Hydrilla plants in the low

photorespiration state, and this inhibition could similarly be relieved

by increasing the CO2 concentration (Fig. 3). Calculation of the apparent

Km(C02) value for photosynthesis in low photorespiration Hydrilla indi-

cated it increased from 74.7 to 90.2 yM in the presence of ethoxyzolamide.

Because Hydrilla plants in the low photorespiration state utilize

PEP carboxylase as a major carboxylation enzyme (Holaday and Bowes, 1980),

the effect of ethoxyzolamide on the in vitro activity of this enzyme from

Hydrilla was examined. The activity of PEP carboxylase was 120 pmol/mg

Chl-h, at saturating substrate concentrations, and 100 vM ethoxyzolamide

had no inhibitory effect on this rate. Similarly, 100 vM ethoxyzolamide

had no effect on the enzyme rate when the enzyme was assayed under limit-

ing substrate levels of 0.4 mM HCO3- or 0.5 mM PEP.


Discussion


It has been well documented for terrestrial C3 plants that the 02

inhibition of net photosynthesis is increased at low CO2 concentrations

(Chollet and Ogren, 1975). Substantial evidence indicates that this








phenomenon is due to the competitive interactions between CO2 and 02 at

the active site of RuBP carboxylase-oxygenase (Chollet and Ogren, 1975).

The degree of 02 inhibition of net photosynthesis and its sensitivity to

CO2 suggests that gas exchange measurements with Myriophyllum and Hydrilla

in the high photorespiration state also reflect the properties of RuBP

carboxylase-oxygenase. Thus in the high photorespiration state, these

aquatic angiosperms, like terrestrial C3 plants, apparently lack an

effective mechanism to insulate the initial events of photosynthetic and

photorespiratory carbon metabolism from external CO2 and 02 concentra-

tions. In contrast, the low level of 02 inhibition and its insensitivity

to CO2 observed with Myriophyllum and Hydrilla in the low photorespira-

tion state imply that some type of photorespiration-reducing mechanism

is induced in these plants (Salvucci and Bowes, 1981; Holaday et al.,

1982; Salvucci and Bowes, 1982).

Several photosynthetic characteristics found in Myriophyllum and

Hydrilla upon induction of the low photorespiration state resemble those

reported for unicellular green algae. For example, the induction of the

low photorespiration state in Myriophyllum and Hydrilla results in in-

creased carbonic anhydrase activity; a similar phenomenon has been re-

ported for algal cells transferred from high (5%) CO2 to low (air-levels)

CO2 (Graham et al., 1971; Badger et al., 1978; Hogetsu and Miyachi, 1979).

Furthermore, ethoxyzolamide treatment decreases the net photosynthetic

rate but increases the apparent Km(C02) for photosynthesis, the 02 inhi-

bition of photosynthesis, and the CO2 compensation points of low photo-

respiration Myriophyllum and Hydrilla. Similar results have been reported

for low C02-grown Chlorella and Chlamydomonas cells (Tsuzuki and Miyachi,

1979; Badger et al., 1980). The competitive nature of increasing CO2 levels








decreasing the ethoxyzolamide inhibition of photosynthesis also appears

to be common to these aquatic angiosperms and unicellular green algae

(Tsuzuki and Miyachi, 1979; Badger et al., 1980). As in the green algae,

all of these results point to the involvement of carbonic anhydrase in

the photorespiration-reducing mechanism of both Myriophyllum and Hydrilla.

Rather than the sole factor, carbonic anhydrase in Myriophyllum and

Hydrilla appears to be but one component of the photorespiration-reducing

mechanism, in that the low C02 compensation points of plants in the low

photorespiration state were increased to the level of those in the inter-

mediate state but not to those of the high photorespiration plants.

Although carbonic anhydrase has been implicated in the green algae,

the observed photosynthetic characteristics of low C02-grown algal cells

appear to be due to an elevated level of inorganic carbon at the site of

fixation by RuBP carboxylase-oxygenase, rather than to carbonic anhydrase

activity per se. Thus, by measuring the internal inorganic carbon concen-

trations in Chlamydomonas, Badger et al. (1980) showed that ethoxyzolamide

decreased the rate but not the final concentration of inorganic carbon

accumulated. Recently, Spalding et al. (1982) have isolated high CO2-

requiring mutants of Chlamydomonas, one deficient in carbonic anhydrase

activity but capable of accumulating high levels of inorganic carbon, and

the other unable to accumulate inorganic carbon despite normal levels of

carbonic anhydrase. The physiological phenotypes of both mutants resemble

those of high C02-grown wild type Chlamydomonas cells indicating that
inorganic carbon transport/accumulation and carbonic anhydrase activity

are two separate components, both of which are required for the low

photorespiration (low C02-grown) characteristics. Membrane hyperpolariza-

tion studies with the green algae Chlorella (Beardall, 1981; Beardall and









Raven, 1981) and the cyanobacterium Anabaena (Kaplan et al., 1982), using

the lipophilic cationic probe tetraphenyl phosphonium (TPP ), have charac-

terized the inorganic carbon accumulation component of the photorespiration-

reducing mechanism as an electrogenic HCO3- pump located either at the

plasmalemma (Kaplan et al., 1982) or possibly on the chloroplast membrane

(Beardall, 1981; Beardall and Raven, 1981).

To date, all higher plants and algae with reduced photorespiratory

capacity utilize mechanisms that increase the CO2 concentration at the

site of RuBP carboxylase-oxygenase (Chollet and Ogren, 1975; Rathnam and

Chollet, 1980; Beardall and Raven, 1981). For cyanobacteria, there is evi-

dence to suggest that the very high levels of internal inorganic carbon

(4 mM) can be utilized for photosynthesis without increased carbonic

anhydrase activity (Badger et al., 1978). In the green algae, however,

it appears that carbonic anhydrase is needed to facilitate the rapid

equilibration of the inorganic carbon forms in order for these organisms

to effectively use the elevated internal levels of inorganic carbon as a

concentrated source of free CO2 (Badger et al., 1980). An equilibration

function for carbonic anhydrase may also be required for Myriophyllum and

Hydrilla to achieve the low photorespiration state.

The biochemical basis for the reduced photorespiration state of

submersed aquatic macrophytes involves at least two different mechanisms.

For Hydrilla, low photorespiration is associated with the induction of

increased C4 acid metabolism (Holaday and Bowes, 1980; Salvucci and Bowes,
1981), and it now appears likely that decarboxylation of the malate pool

results in elevated internal inorganic carbon levels analogous to the

situation in terrestrial C4 plants (CHAPTER III). Despite the likely use

by Hydrilla of a C4 system to elevate the CO2, the data in this study









indicate that carbonic anhydrase is still a component required to effec-

tively maintain the low photorespiration state. In the high photorespira-

tion state of Hydrilla, the function of carbonic anhydrase in photosyn-

thesis is far less apparent.

In contrast to Hydrilla, Myriophyllum in the low photorespiration

state exhibits little phosphoenolpyruvate carboxylase activity and C4

acids account for less than 10% of the initial photosynthetic products

(Salvucci and Bowes, 1981; CHAPTER III). Thus, a C4 acid system for ele-

vating internal CO2 levels cannot be operative in Myriophyllum. Whether

an algal-like electrogenic HCO3- pump together with the elevated activity

of carbonic anhydrase function as components of the Myriophyllum system

is presently unclear. Evidence for active HCO3- utilization mechanisms

have been presented for a number of submersed freshwater macrophytes in-

cluding Myriophyllum and Hydrilla (Lucas, 1980; Walker, 1980; Prins et al.,

1982a) Consequently, although free CO2 generally appears to be the pre-

ferred form for the photosynthesis of submersed angiosperms (Van et al.,

1976), the internal accumulation of significant amounts of inorganic

carbon by an electrogenic HC3- pump, which in Myriophyllum would be in-

duced or increased in the low photorespiration state, cannot be ruled out.
The lack of an ethoxyzolamide-stimulated effect on the CO2 compensa-

tion point of Sorghum suggests that in terrestrial C4 plants, the utiliza-

tion of elevated inorganic carbon levels to overcome photorespiration in

the bundle sheath does not depend on the activity of carbonic anhydrase.

In a similar manner, the CO2 compensation point was not increased in the

C3-C4 intermediate Moricandia or in the C3 plant tobacco, a possible indi-
cation that the involvement of carbonic anhydrase in maintaining a particu-

lar photosynthesis/photorespiration ratio, at least at levels of CO2 near

the CO2 compensation point, may be restricted to aquatic plants.













CHAPTER III
TWO PHOTOSYNTHETIC MECHANISMS MEDIATING THE
LOW PHOTORESPIRATION STATE IN
SUBMERSED AQUATIC ANGIOSPERMS


Introduction

It has become a common practice to differentiate among plants on the

basis of their photosynthetic and photorespiratory carbon metabolism.

Thus, terrestrial C3 and C4 plants can be distinguished on the biochemi-

cal basis of their first products of carbon fixation. However, they

concomitantly exhibit characteristic physiological and ecological differ-

ences, especially in regard to gas exchange (Chollet and Ogren, 1975).

Consequently, the terms C3 and C4 take on a much broader meaning when

they are used to describe the complete syndrome of biochemical and physio-

logical characteristics associated with each group. Recently some inves-

tigators have made use of this more descriptive interpretation in applying

the term C3-C4 intermediate species to terrestrial plants which exhibit

gas exchange characteristics, anatomical features, and, for some species,

levels of C4 acid metabolism, which are intermediate between C3 and C4

plants (Rathnam and Chollet, 1980; Holaday et al., 1982). In the same

vein, the term CAM (Crassulacean Acid Metabolism) has come to refer to

a cadre of photosynthetic characteristics, found largely in succulent

plants, and not just to the biochemical mechanisms involved (Kluge and

Ting, 1978).








Aquatic autotrophs pose a problem in regard to categorization. Cer-

tain unicellular green algae and cyanobacteria, such as Chlorella (Bear-

dall and Raven, 1981; Shelp and Canvin, 1981), Chlamydomonas (Badger et

al., 1978), and Anabaena (Badger et al., 1978), exhibit changeable photo-

synthetic and photorespiratory characteristics depending upon the CO2

concentration at which they are grown. Thus on the basis of their gas

exchange features, these algae do not fit into either the C3 or C4 cate-

gory. Based on their first product of carbon fixation they have been

referred to as "C3 plants" (Beardall and Raven, 1981), however, this

designation becomes confusing for low C02-grown algae which physiologi-

cally are C4-like, and thus could be described as C4-like C3 plants

(Beardall and Raven, 1981). Unicellular green algae and cyanobacteria

also differ from C3-C4 intermediates in that the algae appear to exhibit

a continuum of photorespiration states and not a fixed intermediate level

as found in terrestrial C3-C4 intermediate species (Badger et al., 1980).

Submersed freshwater angiosperms also exhibit a continuum of photo-

respiratory states, ranging from high to low, and in this regard are

similar to unicellular green algae (Bowes et al., 1978; Salvucci and

Bowes, 1981). However, the difficulties encountered in assigning green

algae to the C3 or C4 categories are complicated further in submersed

angiosperms by evidence that C4 acids can be major products of photo-

synthesis in some, but not all, aquatic macrophytes (Stanley and Naylor,
1972; Holaday and Bowes, 1980; Beer and Wetzel, 1982b) even in those with

considerable photorespiratory activity (Browse et al., 1980). In addition,

features such as dark 14C fixation, net fixation of CO2 in the dark, and

diurnal fluctuations in the level of titratable acidity, which are gen-

erally associated with CAM plants (Kluge and Ting, 1978), have been









observed in certain submersed aquatic macrophytes (Holaday and Bowes,

1980; Beer and Wetzel, 1981; Keeley, 1981; Keeley and Bowes, 1982; Hola-

day et al., 1982). Thus, photosynthetic carbon metabolism in submersed

aquatic macrophytes can involve a combination of features, each generally

thought to be characteristic of a particular photosynthetic category

(i.e., C3, C4, C3-C4 intermediate, or CAM).

Part of the difficulty in categorizing submersed angiosperms is due

to the fact that it is not completely understood how the various pathways

for carbon assimilation relate to the level of photorespiratory activity

or to its variable nature (Browse et al., 1980; Holaday and Bowes, 1980).

A 14C labeling study using the submersed angiosperm Myriophyllum indicates

that carbon assimilation in this plant is via the Calvin cycle (Stanley

and Naylor, 1972), however, the photorespiration state of the plants

used in the study was not determined. Reportedly low levels of PEP car-

boxylase activity provide further evidence that CO2 fixation in Myriophyl-

lum is predominately via the Calvin cycle, even when the plant is in the

low photorespiration state (Salvucci and Bowes, 1981, 1982). In contrast,

from previous studies with Hydrilla, it appears that C4 acids are major

early products of photosynthesis and that the induction of the low photo-

respiration state in Hydrilla is associated with an increase in the

activities of several enzymes involved in C4 acid metabolism (Holaday

and Bowes, 1980; Salvucci and Bowes, 1981). The formation of C4 acids

in photosynthesis has been reported for several other submersed macro-

phyte species but,as in Hydrilla, their role in photosynthetic carbon

metabolism is unclear (Brown et al., 1974; De Groote and Kennedy, 1977;

Beer and Wetzel, 1982b). Pulse-chase labeling experiments have not re-

solved this question in that the turnover of label fixed in malate is









reported either not to occur (Browse et al., 1980), or to turn over at a

slower rate during the chase period than in terrestrial C4 species (De

Groote and Kennedy, 1977; Holaday and Bowes, 1980).

In order to clarify some of these problems, the relationship between

C4 acid metabolism and the variable photorespiration states in Hydrilla

was examined especially with regard to the fate of malate produced in

photosynthesis. By way of comparison, the intermediates of photosynthetic

carbon metabolism in Myriophyllum were also investigated. Because a low

photorespiration state is inducible in Myriophyllum, as well as in Hydrilla,

an attempt was made to further elucidate the mechanisms) responsible for

this state. This was accomplished by examining the interactive effects

of 02; C02; ethoxyzolamide, which is a repressor of the low phororespir-

ation state (CHAPTER II); and isonicotinic acid hydrazide, which inhibits

carbon flow through the photorespiratory pathway (Servaites and Ogren,

1977);on the gas exchange characteristics and on the relative fluxes

of carbon through the photorespiratory pathways of these plants.


Materials and Methods

Plant Material

Myriophyllum spicatum L. and Hydrilla verticillata (L.F.) Royal

were collected and incubated as described in CHAPTER II.

Infrared Gas Analysis

Net photosynthetic rates were determined by measuring CO2 uptake in

a closed system as described previously (Salvucci and Bowes, 1982).

Carbon dioxide compensation point values were used to verify the photo-

respiration state of each plant used in an experiment, and were determined









as described by Van et al. (1976). The CO2 compensation point has been

shown to be a reliable indicator of the photorespiration state (Salvucci

and Bowes, 1981). All gas exchange measurements were performed at 30C
2
using a saturating quantum flux density of 1,000 vE/m 2s. Gas exchange
data represent mean values from at least three separate determinations.

Initial Product and Pulse-Chase Experiments

Carbon fixation experiments were performed at 300C in the light

under a saturating quantum flux density of 900 yE/m2*s (400-700 nm).

After a 1-2 h equilibration period, apical segments of Myriophyllum and

Hydrilla, 5 to 10 cm in length, were transferred from an aerated (317 pl

C02/1 and 21% 02, gas phase) solution of 10 mM Mes-NaOH and 5% Hoaglands

solution at pH 5.5 (Mes-Hoaglands solution) and pulsed for 20 s in 200 ml
of Mes-Hoaglands solution containing 20 yM NaH14C03 (50 vCi/pmol). Prior

to the addition of H14CO3-, the pulse solution was purged for 1 h with

CO2-free air. In pulse-chase experiments, the labeled plants were trans-

ferred to a second aerating solution to complete the duration of the

chase. Aeration established a free CO2 concentration of 9.0 yM for the

chase portion of the experiment. Carbon fixation was stopped by rapidly

plunging the plants into liquid N2.

Experiments involving ethoxyzolamide employed the above procedures

except that ethoxyzolamide at 100 vM was included in all solutions, and

the equilibration period was extended to 4 h (CHAPTER II).

Glycine Accumulation

Carbon fixation experiments were performed in a shaking water bath

using detached leaves of Myriophyllum and Hydrilla. The light and tem-

perature conditions were as described above. Entire apical segments were








incubated at 25"C in the dark for 1 h in Mes-Hoaglands solution contain-

ing various isonicotinic acid hydrazide (INH) concentrations. After 1 h,

2 leaves (approximately 50 ug Chl) were detached and added to 25 ml re-

action flasks with 10 ml of the INH-containing solution, which had been

previously purged for 20 min with a C02-free N2/02 mixture. Purging was

continued for 7 min in the light to equilibrate the leaves, after which

time the flasks were rapidly sealed and fixation initiated by the addi-

tion of 0.1 ml NaH14C03 (50 uCi/umol) through a serum cap. After 15 min,

carbon fixation was stopped by rapidly plunging the leaves into liquid

N2. Experiments were run in duplicate and, for each experiment, the

pooled contents from two replicate vials were analyzed.

Extraction of Labeled Compounds

The plant material frozen in liquid N2 was ground in a Ten Broeck

homogenizer containing 80% (v/v) acetone at 4C. The resulting homogenate

was quantitatively transferred to a 15 ml centrifuge tube and aliquots

were taken to determine chlorophyll content (Arnon, 1949) and total 14C

fixed. For the latter, aliquots were added to scintillation vials con-

taining 0.5 ml H20 acidified with 0.1 ml of a 6 N HC1 solution saturated

with 2,4-dinitrophenylhydrazine (DNPH). Triplicate determinations for

each sample were taken to dryness and 14C dpm were determined by liquid

scintillation spectrometry. Eighty-five percent ethanol containing 0.12%

(w/v) DNPHwas added to the remainder of the homogenate and the resulting
mixture was warmed to room temperature. After 1 h, the solution was

centrifuged for 20 min at 10,000 g and the pellet was extracted sequen-

tially with boiling solutions of ethanol (85%, 40%, and 20%), distilled

H20 (twice) and 0.5 N formic acid, each for 10 min. The supernatants

from each extraction were pooled and taken to dryness overnight at 40C








under a stream of air. The amount of 14C label remaining in the pellet

was determined and represented the insoluble fraction. The supernatant

fraction was reconstituted with H20 and then extracted twice with chloro-

form to remove lipids and unreacted DNPH, and finally lyophilized. The
1C in the chloroform fraction was generally less than 1.0% of the total

C fixed. Determinations of radioactivity in the chloroform, insoluble

and supernatant fractions indicated that greater than 93% of the total
1C fixed was recovered at this stage.


Separation of Labeled Compounds

Following the reconstitution of the lyophilate in H20, the extract

was quantitatively fractionated by ion-exchange chromatography (Atkins

and Canvin, 1971). Basic compounds were eluted from an 8.0 x 60.0 mm

column of Dowex 50X8-400 with 2 N NH40H. Nonbasic compounds were sepa-

rated into neutral, acid 1 and acid 2 fractions by chromatography on

Dowex 1X8-400, eluted with H20, 3 N formic acid, and 4 N HC1, respective-

ly. These elution conditions, as confirmed by the separation of authen-

tically labeled standards (3H-glucose, 14C-malate, 14C-P-glycerate, 14C-

fructose 1,6 bisP), provided virtually complete separation of malate

from P-glycerate. The 14C dpm were determined for each fraction and re-

covery of 14C from the ion-exchange columns was nearly 100% as has been

reported by other workers (Atkins and Canvin, 1971). Each fraction from

the columns was lyophilized and then reconstituted by the addition of

100 Pl of 10% ethanol, with the exception of the neutral fraction to

which 100 pl of H20 was added.








Thin-Layer Chromatography

Individual compounds in each ion-exchange fraction were separated

by one-dimensional chromatography on plastic backed thin-layer plates

(MN-Cellulose 300, Brinkmann Instruments, Westbury, New York, U.S.A.).

The plates were developed twice in the appropriate solvent system after

overspotting five 2 pl aliquots of each sample using a capillary pipet.

Aliquots of each sample were also added directly to scintillation vials

with the 2 p1 capillary pipet to determine the total 14C added to the

plate prior to separation. Recovery of 14C from the thin-layer plates,

after separation, exceeded 95%. In the basic fraction, amino acids were

separated in n-butanol-acetone-water-diethylamine (20:20:15:3). Compounds

in the acid 1 fraction were separated in ethanol-ammonium hydroxide-water

(6:1:1.6, equilibrated for 24 h) and in sec-butanol-formic acid-water

(6:1:2). This latter solvent system was also used for the acid 2 fraction.

The neutral fraction was separated by chromatography in n-butanol-acetic

acid-water (12:3:5). Individual compounds were localized by co-

chromatography with authentic standards, and areas corresponding to

these markers were cut out and their 14C dpm determined. Authentic amino

acids, chromatographed in adjacent lanes, were visualized by spraying

these marker lanes with ninhydrin (Touchstone and Dobbins, 1978). Cold

carrier standards were co-chromatographed with the acid 1 fraction and

visualized with bromocresol purple and ammonium hydroxide (Touchstone

and Dobbins, 1978). In a similar manner, phosphorylated compounds in

the acid 2 fractions were identified using ammonium molybdate (Hanes

and Isherwood, 1949).
In order to check the accuracy of the procedures outlined previously,

in some experiments individual compounds were also separated by 2-dimensional








thin-layer chromatography of samples taken prior to ion-exchange chroma-

tography. The results were in agreement with those from the procedures

outlined above with regard to the identity of, and percent 14C incorpor-

ated in, individual compounds. Solvent systems used were sec-butanol-

formic acid-water (6:1:2), ethanol-ammonium hydroxide-water (6:1:1.6)

and the phenol-water and butanol-propionic acid-water system of Pratt

and Rand (1979).


Results


For Hydrilla in both the low and high photorespiration states, greater

than 50% of the label that was incorporated after 20 s photosynthesis in

20 PM H14CO3 was recovered in the C4 acids malate and aspartate (Fig.

4). During the ensuring chase period in unlabeled CO2, the low photo-

respiration Hydrilla plants showed a rapid decline in the percent 14C in

both malate and aspartate, after a 1 min lag period (Fig. 4). This de-

cline continued for 60 min. In contrast, in the high photorespiration

plants the malate did not decline during the chase, but instead exhibited

an increase in percent 14C incorporation (Fig. 4). Unlike malate, the

aspartate labeling of high photorespiration plants declined steadily,

dropping from 28 to 5% during the 2 h chase (Fig. 4). This decline in

aspartate coincided with the increase in malate labeling (Fig. 4), and

also with increases in the amino acids glutamate and alanine (data not

shown).

The data in Figure 5 demonstrate that for the low photorespiration

Hydrilla plants, the decline in malate labeling during the chase period

was relatively rapid, with a 50% decrease occurring within 180 s. During

this time period, there was a steady and concomitant increase in 14C





























*0 I
Oy 0
t( tA-4 4-3
tI Cn 3- .C
0 *- Q. tO J Q.
S 4-O 0 4-)
0 1o C. tn 0 +1 .C
0)O C0 '-
0) ECN ) *-
4-C i- .CN C
tO 0 4 tC 4- -4 "0
4- *,- C"
S- 4 0 t04- 0) to
Sto S- CO S-
C. S- a. "- a) 3
n -r- N 3 .4- 3 0
rd OC r0 -
ln 0 )- ) (A
'3 0) J- U- U 0 S-
a S-L.-- O S-4 *
4- Q. r-
a00 )* .C 0 )
4-'. 4-In 0 C >
(a OC Cr a 0 r- r-
r- a) .= 1 -f 4
0 .C EU (04-. U


0 C Q ) -C '- E t
C -0 I)X Cl S-
i- C 0 4- CU L.) 0
to -r 5 OL *
3: (A 4-3X X i- I
I-- t= to S E EO-
4|- L 4-) 0 0 $-
0 0) I *- O
C 4- IV) r C
0 f- ,- O 0 -4
*- C 4- *r- 0) C
4- r -4-) +1 0
5- O O S-R- 4-,
0 "- )00 t
CL oi 0c. m LC 5-
5- r- C ) S- 5-. r-
U 'O 5- 0 L UC -n
c >3 = w c c c
CA OCCCO
-I 'aO S- ( *- to S-




0)
5,-

*U-
Lu
























0




0

00
Ob

o
0 2



c ;
D -


0 0 0


(%) Ga31VIOdiOONI 3tl
























Distribution of 14C among photosynthetic inter-
mediates of Hydrilla in the low photorespiration
state during the initial period of the pulse-
chase experiment. The zero time point represents
the start of the chase following a 20 s pulse.
Incorporation is expressed as a percent of the
total 4C incorporated and the 4C fixation rates
were as in Figure 4.


Figure 5.

















60
Malate +
Aspartate
V Phosphorylated
Compounds
48 0 Insoluble +
Neutral
A Glycine +
Serine








12-
a
w 36 -



0
0
24





12






0 60 120 180
CHASE TIME ( S )








entering the neutral and insoluble fractions, composed largely of sucrose

and starch, respectively (Fig. 5). Glycine and serine labeling also rose

during the chase period. Concomitant with the rise in sucrose and starch,

the percent label in total phosphorylated compounds showed a slight ten-

dency to decline from an initial value of 32% (Fig. 5).

Figure 6 shows in more detail the labeling patterns for Hydrilla

during the extended 2 h chase period. Both high and low photorespiration

plants initially exhibited similar percentages of 14C incorporation into

Calvin cycle intermediates. During the chase period, for both photores-

piration types, the phosphorylated intermediates gradually decreased as

a percentage of the total labeled products, coinciding with an increased

labeling of the neutral (sucrose) and insoluble (starch) fractions (Fig.

6). However, after a 2 h chase, neutral and insoluble compounds accounted

for more than 60% of the total 14C incorporated by low photorespiration

Hydrilla, as compared to less than 23% for the plants in the high photo-

respiration state (Fig. 6). At all times during the chase, the high

photorespiration plants incorporated a greater fraction of the label into

the photorespiratory intermediates glycine and serine than did the low

photorespiration plants (Fig. 6). For example, after 2 h of chase,

glycine and serine comprised 18% of the labeled products of the plants

in the high photorespiration state as compared with only 7% in the low

photorespiration state (Fig. 6).

For both the high and low phororespiration plants, total dpm incor-

porated during the pulse showed only a slight decrease during the chase

period, even after a 2 h chase. This indicates that 14C loss due to the

excretion of labeled compounds such as glycolate must have been negligible,

and that the percent changes in the various compounds reflect actual dpm

changes.



























1 I
v( 0
0U 4- I -0
4- 0 0)
O1 = S- a

EmX QL
.,- 4- X W
- X~ ) 0 0a) X
S- ( O4-) (A
tn )

t- W 0o -
C 4- 0 -a -i 'D






0 U. 10 CS
U I4- CB

4-) r- 0 0M
C O C 4
C.C OS
V) rI4 (D
o 0) QCS-
0: -C : V : 0 S
O -0 Q CO
.- Q S- L
,-- QO -
- *r- E 0 -'O
4 -0 ro 0 n tin-I LL



to 4-4 -. 0
0 u 5 0) 0 I


D-r- (n *-) -) )

4-( to 0 0)4-
o 4-4P-CO
n a r- 0o
O CL O (
*0- 01 03 C 5-
4C) *r .C U0
S-- I- 0- 0 S- C

X. C Sr- CU 0

S0 0 0 .C tW *r-
O rs- U 04





L-
*r
1 ( 1 0 0
0 L -COV
C a n a <
CO NS4- -
0 C<



















0 'Z
g.-cI
*, c
a2o
~P Q[ 0
w= a a~ ~~

*o,+t~


0 0 0 0 0 0 0
- r0 O C -


(%) a03"IVOd8O3NI


ot,3


0
c0
d
(O

O
0




o-
0
cV)
o

0










co
cJ
0 2







d








In contrast to Hydrilla, carbon assimilation in low photorespiration

state Myriophyllum was almost exclusively via the Calvin cycle as C4 acids

were less than 10% of the initial labeled products while sugar-P and P-

glycerate comprised 77% (Fig. 7). The fraction of 14C incorporated into

C4 acids did not increase at low inorganic carbon concentrations, even

when the equilibration and fixation were carried out at only 1 pM HCO3

(data not shown). After a 20 s fixation period at 20 pM H14C03 label

in phosphorylated compounds decreased steadily during the chase period;

this decrease coincided with a substantial increase in labeling of com-

pounds in the insoluble and neutral fractions (Fig. 7).

No major differences in C4 acid and sugar-P labeling patterns were

evident when the low photorespiration Myriophyllum plants were treated

with ethoxyzolamide to repress the low photorespiration state (Table 5).

However, Myriophyllum plants treated with ethoxyzolamide did exhibit an

increased labeling of glycolate, glycine, and serine after a 20 s fixa-

tion period (Table 5) and through 10 min of a subsequent chase (data not

shown). Low photorespiration Hydrilla plants, in contrast, showed no

affect of ethoxyzolamide on the labeling pattern of these photorespira-

tory intermediates or on the C4 acids malate and aspartate (Table 5).

Sugar monophosphates did increase somewhat, apparently at the expense of

P-glycerate and triose-P. For both Hydrilla and Myriophyllum, ethoxyzol-

amide treatment reduced the total 14C incorporated.

Isonicotinic acid hydrazide, an inhibitor of the glycine to serine

conversion in photorespiration, can be used to halt the flow of label

through the photorespiratory pathway by inhibiting the further metabolism

of glycine (Servaites and Ogren, 1977). When detached leaves of low photo-

respiration Myriophyllum were treated with increasing concentrations of INH,




























U)
0) 4-) L-

d CC O- O
0 3 Q. -

01) E 0 r-4
4+J 4.- .- 4-)
r (0 4-) C0 +1
CS.. O J +1
U0 a *
4-) 0 N CO 4-




-J~
. *r- C O

C S- 0 3
>, r- a)
tU Q. U
0 U) 0 -)
+4 0) 4J 4- () t(
0 S- C S-
Q.4- E W 0 Cr



3 X l) -0 x
Cr- 4-U W 4-
0 a) tW
E 0)n 4 0) C-)
eto 0 *---
4-) .C -4
U) U4- X
C I S- Q) 0)




C i- .CO0 -C
0 > + *r- ) *
4- Q.C" X <0 4 0 -C
3 0 C C S- S- u
*r- .1 tr Q. Q. O
S-. X S- S-E
4- : a) 5s- o 0 *-
t1 Q. U u CNJ
*r- 4- C ) C C0
0 0 1 *r- '-



*r-
s-
3~cco
01L ,PPT
u UL


















0
N

a

0 'C
CL
.0
:a)

S, + +


+ 1 o


a--
0 O

C








U)
--o



0
) + I \ -







*o,


CD N c o D N 0c
1- N -

(%) 031VHOdHODNI o














Table 5. Effect of ethoxyzolamide (100 uM) on the distribution of 14C
among the photosynthetic intermediates of Myriophyllum and
Hydrilla in the low photorespiration state.



C Incorporated Myriophyllum Hydrilla
Control Ethoxyzol- Control Ethoxyzol-
amide amide


Total 14C Fixed 13.8 10.0 13.6 9.1
(pmol C02/mg Chl.h)

Fraction
(% of 14C incorporated)

BASIC 6.7 9.2 30.3 23.8
Aspartate 1.4 0.6 24.6 18.8
Alanine 0.4 0.6 1.0 0.7
Glycine + Serine 2.8 7.1 1.0 1.1

ACID 1 7.0 9.8 33.5 41.1
Malate 4.0 4.5 27.9 31.8
Glycolate 1.1 4.8 0.4 0.4
ACID 2 84.1 81.0 35.4 32.2
Sugar-P 35.8 23.2 6.3 14.5
Sugar-P2 19.1 24.8 6.6 7.3
P-glycerate + Triose-P 27.3 25.2 18.9 5.8

INSOLUBLE 1.8 1.8 0.8 2.8


Plants were pulsed for 20 s at 20 yM H14CO 3


and 21% 02 (gas phase).








glycine represented an increasingly larger percentage of the 1C incorpor-

ated (Fig. 8). At a saturating concentration of 10 mM INH, 30% of the
14C incorporated by low photorespiration state Myriophyllum was recovered

in glycine as compared with less than 15% in the control (Fig. 8). As has

been reported for terrestrial plants (Kumarasinghe et al., 1977; Servaites

and Ogren, 1977), total 14C fixation decreased in plants treated with 10

mM INH to 70% of the control rate (Fig. 8). Unlike in terrestrial plants,

however, serine did not decrease as a percentage of the 1C incorporated.

At 5 mM INH, the concentration used in subsequent experiments, excretion

of organic 1C by Myriophyllum was less than 3% of the total 14C fixed,

which was the same rate as the control (data not shown).

For Myriophyllum in both the high and the low photorespiration state,

5 mM INH caused label to accumulate in glycine, apparently at the expense

of neutral and acid 2 compounds (Table 6). In the presence of INH, glycine

represented a much greater proportion of the photosynthetically labeled

products in high than in low photorespiration plants; the percent values

were 48 and 25, respectively (Table 6). In the absence of INH, little

difference in glycine formation between the two photorespiratory states

was evident (Table 6). The INH had a similar effect on 14C incorporation

into glycine in Hydrilla (Table 6).

When glycine accumulation was examined at 20 vM HC03 with respect

to the 02 concentration, at 02 concentrations greater than 1 or 2% (gas

phase), glycine comprised a greater fraction of the 14C labeled products

in the high than in the low photorespiration state of Myriophyllum (Fig. 9).

At 80% 02, glycine constituted only 25% of the total 14C incorporated by

the low photorespiration plants compared with greater than 45% in the high

photorespiration state (Fig. 9). In both photorespiration states, formation





























I I
S- 4-) d) r- "0
4-o U O Lth ) *
c"a- .:: a)-4 Q..C: C f- S. -
U 0. 0 4-- -.U
C 4- 3 U C
U i-- =Iq U E
co cnz0-0
-- 0 --'' C- J
: *r- -Cr- 0C
4- 4-3 4J 0'|J= 10 C) L)

0w ( U 4-) 0

(D CL C- 9( 0 : (V
0 S -. f ) e- l
o U .o J- a.E



- .= C)4->- Cr W
4. 0 E C- 4. -

-3 0 0 0 C -




4- 0 C 4-3 M-
L C-- C S-
to(0 tr to C (- c
S_ Q101. a)
U 0r S U) U
0 r- 3- OC4-'
Sr S. 4 O







C *0) i 03

4-) M C0 C% J C04
0 *Xr- O n c4-'
0- 0) (0 34)-
4-.0 0 IA
*Vt -0 4>- r t4 0-)








0 to a) 0 (1)4-) C
u4-E C C a 0








U 4-' U -- 0 *p- C 4->
a) >>4-) S- g-o
4- Cr- to C CEX
4r o a M o *CL *
r- 0 CL 0 4-c -






C 0 r- n U O
c *- c0 < c n cU
..C C O) th








4- +-) r .I .- 4- To








u-
0 L L e- *--










----14C FIXED (% OF CONTROL)---
o o o
0) (D r')


i
/
/
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/
I
I
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00)

o0 CM *i- >r( S- E0
Or c- u *e LEo
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4- -0 X (0 4- ) a) CD 0 -U
(1 r 4-) 0) (L. a) 4-', L M- -A M:
S- to c e u a) .- t 0 to
o >-(A4-3 r- r4-) (U4-^
-. 3 -- o XS- 0 C-Cl
5- 0 o U o 0C
U 0 0 C .-0 0 m
- C- C 4-o (0 + 4-' 4- 4--
4- tIo S.- to C tO
r_ E S- 0 01 4-j sL 0) L.- 2~
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on c L +'4C- 0

4-) E S- -- .-. r CM4 O.
S- 0 (A S- CD

- *- 0 0 0 0 c
C i- 0U (U + U 4C-S







0 .0 > C a U+4-4- 0O 0
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4-+ aS.- CL) 0 Q C4-) U
u 0 U 01 CO (a-c
s-r- -0 C- u o z:us.-o0
4- -' 0 = -- 0 0-0- 4-) C
E 4-' (A 4- 4-) 4-) 1- '- C 93
O aa: O to "M W S-
u *-) Ec 4 4- S- a E

0- U O C C-4-)

C~(U r r_ Q1) U *i "4-+
OC 0 04- 4- C a CD t
4r--(A 0 V) O S-
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r- .- ClJ aU-)-C O cl o0 U
4- C U () +J Cf 4 I -- C I 4- U



(U t- o GcU410 0 a Z IC,)
4J> 3: M U W r-- 4-J M
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I-,4 mCX J fo ui" S- CCA. 4) U( =



















_0

C C
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of labeled glycine at 20 VM HC03 approached saturation at 02 concentra-

tions greater than 50%. When measured under a constant 02 concentration,

glycine labeling in low photorespiration Myriophyllum was independent of

the HCO3 concentration and, therefore, was not a function of the 02/C02

ratio (Fig. 9, inset).
A similar response pattern to that observed for glycine labeling was

recorded for 02 inhibition of net photosynthesis in low but not high photo-

respiration state plants. Thus, for plants in the low photorespiration

state, the percent inhibition of net photosynthesis by 21% 02 was inde-

pendent of the CO2 concentration and therefore inhibition of net photo-

synthesis by 02 was not a function of the external 02/C02 ratio (Fig. 10).

However, inhibition of net photosynthesis was affected by the 02 concen-

tration when measured under a constant CO2 concentration (330 ul C02/1).

In contrast to these results with low photorespiration plants under

either constant 02 or constant C02, the percent inhibition of net photo-

synthesis by 02 in high photorespiration state Myriophyllum responded to

changes in both 02 and CO2 and was thus a function of the external 02/C02

ratio (Fig. 10). The percent inhibition of net photosynthesis by 02 was

considerably less in the low photorespiration plants than in the high
photorespiration plants whether measured under conditions of either

constant 02 or constant CO2 (Fig. 10).


Discussion

Evidence for C4 acid metabolism has been presented for a number of

submersed angiosperm species based either on direct data from labeling

studies (Brown et al., 1974; De Groote and Kennedy, 1977; Browse et al.,

1980; Holaday and Bowes, 1980; Beer and Wetzel, 1982; Helder and van Harmelen,





























s C
S- *r- .XO C
O O L r ( A
0.0 S- E S 3 Ct
ed CAr- Ot s- 0




*r- 0 1 0 -
1 0 T n to < tn
SO- 0t 5C ( CU

a = o 4 C = CD
- ) 4-~~C 4J CJ
C> Q) U 0 E0 0)0)oth
UCL) CD


uO r (- Et
( a) 4- Co1 ) C4
0 4- -C (0 M W )
S-U 0 4 I 4-) 0
C4- 0 E: CC E
rO- 0-0C E S




r- ,-0. O 0 00

.- f 0 )COu -

.gM) 0 *- 3 E


C r- r-L O --
4 =r- 4- C .00


S0 C *r- 4- CA W



U (v 0- S0 >j -
4- C 0 0 0-




4-0) U)Cl (X: C S- 0
cL 0. (v m eto o

4) 4-. 0 0. E 4
C) r- .0 EU En ) OJ0E

>-j4 (V M C- r < .- S
l C r- r- MU (0 4-
0 > r-E CC C1






I- ) r U o( E0 L..
. uE > Me
U)- -0 + U 0 1n i- eu To






0
'-1


0C

*r-
.-


































































0 0
N


13N JO NOII1BIHNI


.-
Co
0
U)
0

NO


0
'n






CM








o



0


-0
0


- Cg
rg
Co

NO
Oo
00(-


(%) SIS3HI.NASO.OHd








1982) or indirectly by implication from measurements of relatively high

PEP carboxylase activities (Bowes et al., 1978; Salvucci and Bowes, 1981).

All of the angiosperms cited are monocotyledonous and five genera, Egeria,

Elodea, Hydrilla, Lagarosiphon, and Valisneria, belong to one family,

Hydrocharitaceae. Labeling studies with Hydrilla and Egeria have shown

that the proportion of label incorporated into C4 acids in the light in-

creases as the level of inorganic carbon is decreased (Browse et al.,

1979a;Holaday and Bowes, 1980). Thus the formation of C4 acids in photo-

synthesis by certain submersed angiosperm species may be an adaptation to

low external levels of inorganic carbon (Van et al., 1976) and appears to

be associated with the low photorespiration state which is induced under

these conditions (Holaday and Bowes, 1980; Salvucci and Bowes, 1981).

In Hydrilla, although C4 acids accounted for a similarly large per-

centage of the photosynthetically fixed carbon in both the low and the

high photorespiration states, differences in the fate of malate were

apparent between the respective photorespiration states. In the low

photorespiration state plants, as in terrestrial C4 plants, malate was

utilized to form photosynthetic end-products (sucrose and starch), prob-

ably as a result of the activities of C4-pathway enzymes which increase

with induction of the low photorespiration state (Salvucci and Bowes,

1981). This fixation and decarboxylation of C4 acids in the light and

the enzymatic capacity to regenerate PEP via pyruvate Pi dikinase are

features similar to the photosynthetic carbon metabolism in terrestrial

C4 plants (Chollet and Ogren, 1975), and in an analogous manner they may

also function as a C02-concentrating mechanism in low photorespiration

Hydrilla. The resulting increase in the concentration of CO2 at the site








of RuBP carboxylase-oxygenase could explain the C4-like photosynthesis/

photorespiration ratio which is characteristic of Hydrilla in the low

photorespiration state (Bowes et al., 1978; Salvucci and Bowes, 1981;

Holaday et al., in press).

In contrast, in the high photorespiration state of Hydrilla, label

was accumulated in malate throughout an extended chase period and did not

show any evidence of turnover. This lack of malate turnover during photo-

synthesis is probably the result of the low activities of C4 enzymes in

the high photorespiration plants (Salvucci and Bowes, 1981). It is none-

theless unusual, considering the large percentage of carbon that was in-

corporated into this compound. Similar results have been reported for

Egeria plants with high CO2 compensation points (Browse et al., 1980).

In these Egeria plants, the malate pool expands during the light period

and decreases in the dark, which suggests that malate formed in the high

photorespiration state is an end-product of photosynthesis, possibly

being used for some function such as ion (HC03 ) balance rather than as

a source of CO2 for RuBP carboxylase-oxygenase (Browse et al., 1980).

In the low photorespiration state, the available evidence with Hydrilla

indicates that the malate pool fluctuates with an opposite diel rhythm,

as higher levels of titratable acidity are measured after a dark than

after a light period (Holaday and Bowes, 1980). No measurements of malate

pool sizes were made in the present study, but it is likely that in the

low photorespiration state, under natural conditions, the malate pool

increases during the dark period due to the relatively high capacity for

dark fixation in the low photorespiration plants (Holaday and Bowes, 1980)

and then decreases in the light as malate is utilized in photosynthesis.








This scheme for malate metabolism in low, but not in high, photorespira-

tion plants is somewhat reminiscent of terrestrial CAM plants, and diur-

nal acid patterns similar to that reported by Holaday and Bowes (1980)

for Hydrilla have since been reported for the submersed lower vascular

plant Isoetes (Keeley, 1981) and the submersed angiosperm Scirpus (Beer

and Wetzel, 1981). Unlike the classical CAM situation, however, it ap-

pears that in Hydrilla some fixation into malate potentially can occur

in the light, as well as in the dark.

To date, at least three genera of aquatic angiosperms (Myriophyllum,

Ceratophyllum, and Ranunculus), all dicotyledonous, are known to lack

significant C4 acid metabolism based either on C3-type labeling patterns

(Stanley and Naylor, 1972) or on low levels of PEP carboxylase activity

(Van et al., 1976; Salvucci and Bowes, 1981; Valanne et al., 1982). The

pulse-chase labeling data presented here confirm the C3-type labeling

patterns for Myriophyllum. Two of the three groups, Myriophyllum and

Ceratophyllum, are known to exhibit variable photorespiration states

(Salvucci and Bowes, 1981; Holaday et al., 1982) like Hydrilla, despite

the lack of a C4 acid-type mechanism to achieve the low photorespiration

state. In this regard they seem to be similar photosynthetically to low

C02-grown unicellular green algae (Berry et al., 1976; Beardall and Raven,

1981). As with the algae the mechanism involved in reducing photorespir-

ation in these submersed angiosperms appears to depend, in part, on the

activity of carbonic anhydrase as demonstrated by the manner in which

the carbonic anhydrase inhibitor ethoxyzolamide altered the gas exchange

characteristics (CHAPTER II) and increased the labeling of photorespiratory

intermediates in low photorespiration Myriophyllum plants (Table 5).








Low C02-grown algae probably utilize an inorganic carbon accumulat-

ing system in conjunction with the carbonic anhydrase activity, in order

to concentrate CO2 at the active site of fixation by RuBP carboxylase-

oxygenase (Badger et al., 1978; Spalding et al., 1982). The labeling

data in the present study are consistent with the operation of a similar

system in Myriophyllum in the low photorespiration state. Firstly, the

increase in glycine labeling which occurred in the presence of INH suggests

that the low photorespiration state in Myriophyllum is not the result of

an alternate pathway that bypasses the reactions involved in the release

of photorespiratory CO2. Secondly, the reduced label in glycine of low,

as compared to high, photorespiration plants suggests that less carbon

passes through the photorespiratory pathway, as opposed to there being

an increase in refixation of photorespired CO2 in the low photorespiration

plants. Both of these lines of evidence support the contention that the

reduction of photorespiratory CO2 release in low photorespiration Myrio-

phyllum is due to elevated CO2 at the site of RuBP carboxylase-oxygenase.

Similar arguments can be made for low photorespiration Hydrilla from the

inhibitor and labeling data presented in this study (Fig. 6 and Table 6).

When evaluated as a function of changing 02 but constant inorganic

carbon concentrations, at any given [02]/[C02] ratio, the percentage of
14C incorporated into glycine in high photorespiration state Myriophyllum

was virtually identical to the percentage reported for leaf cells iso-

lated from the C3 plant, soybean, treated similarly with INH (Servaites

and Ogren, 1977). In contrast, glycine labeling in low photorespiration

Myriophyllum was considerably less than that reported for soybean cells,
but responded in a similar manner to 02 by increasing with increasing 02

concentration. From the results of the reverse experiment, in which the








02 was held constant and the inorganic carbon level was varied, it ap-
pears that at a given 02 concentration the percent of glycine labeling

and the percent of 02 inhibition of net photosynthesis in low photores-

piration state Myriophyllum, by being responsive only to changes in the

02 concentration, are not strictly a function of the [02]/[C02] ratio.
Shelp and Canvin (1980) have reported a similar response in low C02-grown

Chlorella cells. These authors suggested that the 02 inhibition of net

photosynthesis exhibited by Chlorella may not be photorespiratory in nature

(Shelp and Canvin, 1980). However, this cannot be the case for Myriophyl-

lum, because when the 02 concentration was raised, the increased percent
of 02 inhibition of net photosynthesis was accompanied by an increase in

the percent labeling of the photorespiratory intermediate glycine.

These gas exchange and labeling characteristics exhibited by Myrio-

phyllum plants in the low photorespiration state, in response to changes

in the external CO2 and 02, superficially appear to be inconsistent with

the properties of RuBP carboxylase-oxygenase. In vitro determinations

of the vo/Vc ratio of the enzyme from low photorespiration state Myrio-

phyllum using several CO2 and 02 concentrations indicated that the rela-

tive activity of the carboxylase and oxygenase is a linear function of

the [02]/[C02] ratio as is the case for RuBP carboxylase-oxygenase from

other sources (Jordan and Ogren, 1981b)including the high photorespira-

tion state Myriophyllum (APPENDIX A). Thus, for Myriophyllum in the low
photorespiration state, the observed physiological responses of photo-
synthesis and photorespiration, which are catalyzed by RuBP carboxylase

and oxygenase, respectively, are probably not due to any kinetic changes
in the two activities of this enzyme, but rather appear due to an un-

coupling of the enzyme from the external [02]/[C02] ratio. Since the








diffusion of CO2 and 02 into the active site of the RuBP carboxylase-

oxygenase is one of the factors determining the rates of photosynthesis

and photorespiration, it may be possible that carbonic anhydrase, in asso-

ciation with a HC03 accumulation system, modulates the internal pool of

inorganic carbon that exists in both the HCO3 and free CO2 form so as to

act as a "buffer" for CO2. This could,to some extentuncouple CO2 at the

site of fixation from the external CO2 concentration without affecting

the access of external 02 to the oxygenation site. The observed physio-

logical responses could then still be attributed to RuBP carboxylase-

oxygenase but in association with the accumulation/utilization mechanism

dependent on carbonic anhydrase.

Under certain conditions, carbonic anhydrase has been shown to en-

hance CO2 fixation by isolated RuBP carboxylase and this enhancement (as

a percent) increases with decreasing CO2 concentration (Bird et al., 1980,

1982). Tsuzuki et al. (1980) have observed a similar enhancement effect

of photosynthetic CO2 fixation by adding carbonic anhydrase exogenously

to Chlorella cells. These investigators have postulated that internally,

carbonic anhydrase facilitates the utilization of an elevated HCO3 pool

in the chloroplasts as an indirect source of CO2 which can enhance photo-

synthesis to the detriment of photorespiration (Tsuzuki et al., 1980).

Carbonic anhydrase may function in a somewhat similar way in the low

photorespiration state of submersed angiosperms as evidenced by the en-

hanced percent of inhibition of net photosynthesis caused by ethoxyzola-

mide at low CO2 concentrations for Myriophyllum and Hydrilla in the low,

but not the high, photorespiration state (CHAPTER II).

Thus, it appears likely that both C4 acid- and non-C4 acid-dependent

systems for concentrating CO2 occur among submersed aquatic angiosperms,