Limnol. Oceanogr., 30(2), 1985, 322-334
0 1985, by the American Society of Limnology and Oceanography, Inc.
Photosynthesis, photorespiration, and productivity in
Lemna minor L.l
Gerald J. Filbin and R. Anton Hough
Department
of Biological
Sciences, Wayne State University,
Detroit,
Michigan
48202
Abstract
The roles of aqueous vs. atmospheric CO, fixation, photorespiration,
dark respiration, and
organic release in the primary productivity
of Lemna minor L. were investigated experimentally
in field populations and in the laboratory. The mean rate of net photosynthesis through the growth
season was 2.48 mg C g-l h-l during which an average of 86% of carbon fixed was from aqueous
inorganic carbon. Seasonal variations in photosynthesis correlated principally with light intensity
and temperature. Presence of photorcspiration
was suggested by early afternoon depressions of
photosynthesis, by experimental enhancement and inhibition of photosynthesis with low and high
oxygen, respectively, and by enhancement of 1ight:dark respiration ratios with high oxygen. However, under natural conditions L:D ratios were below unity most of the time, and CO, loss in the
light was never >4% of photosynthate fixed per hour. Experimental enhancement of photorespiration with oxygen was never as great as the inhibition of photosynthesis at comparable oxygen
concentrations.
Although plants exhibited a low CO2 compensation point and high light and
temperature optima, photosynthesis enzymology and fixation products indicated that C, photosynthesis was not a significant factor in maintenance of the low photorespiration
rates.
The duckweeds (Lemnaceae) have long
been known for their exceptionahy high rates
of productivity
(Hillman 196 1). Literature
on duckweed production ecology and physiology is extensive, the latter because of the
relative ease of maintaining axenic laboratory cultures under rigorously controlled
conditions. Wohler (1966) used 14C techniques to identify the respective roles of atmospheric and aqueous carbon uptakes as
well as the role of organic carbon in the
productivity
of natural populations. Very
little has been done, however, on the role
of photorespiration
and organic carbon loss
as factors which influence the productivity
of the duckweeds.
Photorespiration
has been defined broadly as the sum of all metabolism resulting in
the use of 0, and production of CO, in plants
it-, the light (Chollet and Ogren 1975). While
mitochondrial
respiration
generally remains relatively low in the light, glycolate
synthesis and oxidation can be a major loss
of photosynthetically
fixed carbon in some
plants, especially at high levels of light, temperature, oxygen, and pH, and low CO2
* Supported by National Science Foundation Grants
GB 40311 and DEB 76-04502 to R. A. Hough. Contribution
408, Department
of Biological
Sciences,
Wayne State University.
(Zelitch 197 1). Virtually all aquatic plants
examined in this context (mostly submersed
species) have shown characteristics associated with the presence of photorespiration
(Brown et al. 1974; Hough and Wetzel 1977;
Lloyd et al. 1977; Sarndergaard and Wetzel
1980; Van et al. 1976). Although evaluation
of true rates of photorespiration
is technically difficult,
particularly
in submersed
plants, rates appear to be somewhat lower
than in terrestrial C, plants in comparable
climates. The low rates may be due in general to less extreme conditions, particularly
internally, than those which develop from
stomata1 closure in terrestrial plants. Extreme conditions of light, temperature, pH,
dissolved oxygen, and low CO2 can occur
in dense macrophyte populations, however,
and for some species which are highly competitive under these conditions, some involvement of C4 photosynthesis has been
suggested (DeGroote and Kennedy 1977;
Bowes et al. 1978; Holaday and Bowes 1980;
Hough 1979; Hough and Wetzel 1977); this
probably has more relevance in subtropical
and tropical climates than in the temperate
zone (Beer and Wetzel 1982).
Floating macrophytes can be subject to
high light intensities, high pH and water
temperatures, and high dissolved and atmospheric oxygen levels (Dale and Gillespie 1976). On the other hand, high water
322
Productivity in Lemna minor
availability
minimizes need for stomata1
closure, and maintenance of rapid atmospheric gas exchange may minimize photorespiration. Our study was designed to examine the roles of photorespiration,
dark
respiration, and organic carbon release in
the primary productivity
of Lemna minor
in a productive lake under varying environmental conditions and on a seasonal basis,
and in axenic laboratory experiments.
Technical assistance was provided by D.
A. Putt, J. B. Bowen, A. F. Jarocha, P. Demmings, J. Alderink, and J. D. Reichkoff.
Axenic cultures of L. minor were provided
by W. S. Hillman. Advice on culture media
and techniques was provided by B. A. Manny. The assistance of these individuals and
the advice of R. Bowker, C. Cheney, R. G.
Wetzel, S. Izawa, D. C. Freeman, M. Adams,
and an anonymous reviewer are acknowledged.
Materials and methods
In situ studies--In situ investigations
were
made during two growing seasons in the littoral zone in Shoe Lake, a small (1.68 ha),
hard-water (alkalinity ca. 3.5 meq liter-l)
dimictic lake in southeastern Michigan. High
inorganic and organic nutrient loading from
an inflowing stream and surrounding wetlands supports high phytoplankton
productivity and abundant littoral macrophyte and
filamentous
algal growth (Cheney and
Hough 1983; Hough unpubl.).
We evaluated the relative contributions
of atmosphere and water to the photosynthetic CO, uptake of L. minor several times
during the growth season using 14C02 and
NaH14C0, in a technique devised by Wohler (1966). An open-ended Plexiglas chamber was placed open-side down in a pan of
filtered lake water with plants floating on
the surface, half of them under the chamber.
The atmosphere in the canopy was partially
evacuated with a cannula through a serumstoppered sampling port. The atmosphere
within the canopy was labeled by adding
1O-60 PCi of aqueous NaH14C02 by syringe
into a receptacle, acidifying it, and purging
the resulting 14C0, from the receptacle with
the cannula into the chamber air, in the process restoring equal air pressure and water
level inside and outside the chamber. This
323
assembly was incubated in the Shoe Lake
littoral zone or in the laboratory for 1.251.50 h with frequent stirring. The concentration of 14C0, in the water absorbed from
the chamber atmosphere was determined
three times during each incubation by liquid
scintillation
radioassay of 1-ml samples.
Available 14C0, in the chamber air was calculated by subtracting the total cpm in the
water and the cpm remaining in the receptacle from the total initial inoculated activity. We assumed that loss from the system
by diffusion out of the water outside the
canopy was minimal, as the pH was high
(ca. 8) and the temperature did not exceed
29°C. The concentration of 12C0, in the air
was assumed to be 335 ppm. The increase
in CO2 concentration
in the chamber air
caused by the addition of 14C02 (< 5%) was
accounted for in the calculations.
At the end of incubations, plants were
rinsed, freeze-dried, weighed, and radioassayed by dry combustion and liquid scintillation counting of ethanolamine-trapped
14C02 (Burnison and Perez 1974). Oxidation efficiency was measured by combustion
of 14C-labeled organic standards; counting
efficiency was computed with quench curves
and external standard efficiency. Photosynthetic carbon fixation from the water was
computed on the basis of the measured 14C
availability and 12Cavailability determined
from alkalinity,
temperature,
and pH
(Saunders et al. 1962) with calculations described in Vollenweider
(1969). Plants incubated outside the chamber were used to
compute 14C02 uptake from the water alone.
The rate of atmospheric uptake was computed by subtracting the aqueous 14C fixation rate of plants outside the chamber from
the total 14C fixation rate of plants in the
chamber.
Detailed biweekly and diurnal trends in
photosynthesis were monitored on the basis
of the aqueous carbon uptake with triplicate
125-ml glass bottles containing 50 ml of
filtered (0.45~pm membrane) lake water and
20-30 fronds of L. minor. Bottles were incubated with [14C]bicarbonate at 0.5 PCi
ml-* for 1.25-l .50 h in the littoral zone with
frequent swirling, after which the plants were
collected and radioassayed as described
above.
324
Filbin and Hough
Rates of photorespiration,
dark respiration, and organic carbon release were assayed by the 14C method of Zelitch (1968)
as used with L. minor by Filbin and Hough
(1979). Plants from the lake were washed
with filtered lake water from a squeeze bottle to remove loosely attached epiphytes and
detrital material. Photosynthetic
carbon
pathways in the plants were 14C-saturated
by incubation in sterile-filtered lake water
containing 1 PCi ml-l [ 14C]bicarbonate for
1 h. Plants then were rinsed and placed in
unlabeled filtered lake water in duplicate gas
flow-through
assay flasks. A continuous
stream of CO,-free air or oxygen was bubbled through the flasks at 65 ml min-l into
ethanolamine CO, traps to collect released
14C02. The traps were subsampled at 5-min
intervals and radioassayed by liquid scintillation. Duplicate samples of water in the
flasks were drawn by syringe at 5-min intervals and radioassayed by liquid scintillation to determine the amount of labeled
organic and inorganic carbon released into
the water by the plants (Filbin and Hough
1979). Incubations were run for 30 min in
the light and 25 min with the chambers
darkened, after which plants were rinsed,
lyophilized, and radioassayed as above. 14C
loss was expressed as a percentage of initial
plant radioactivity
(Hough and Wetzel
1972); rates were determined by regression
of 14C loss over time expressed as percent
loss per hour. Mean rates were determined
by averaging the slopes of duplicate assays.
The influence of manipulated dissolved
oxygen concentration on photosynthesis was
measured on a diurnal
basis biweekly
through the growth season. A container of
lake water was sparged with oxygen to give
25-35 mg liter-l dissolved 02, another was
sparged with nitrogen for the same duration
to give 0.5 mg liter-‘, and a third was kept
at the ambient O2 level (6.8-l 2.4 mg liter-l)
as a control. Water of each oxygen level was
placed in triplicate 300-ml glass bottles. A
fourth set contained water with high oxygen content to which sodium hydroxymethanesulfonate
(HMS: sodium formaldehyde bisulfite) was added as a potential
inhibitor
of glycolate oxidation and photorespiration
( 10d4 M, see Zelitch 1968).
About 30-40 freshly collected fronds were
added to each bottle and allowed to preincubate for 10 min to allow absorption of the
HMS. Photosynthesis was measured by 14C
uptake as above.
The photosynthetic
light optimum was
determined for the natural population of L.
minor by 14Cuptake in bottles placed in the
littoral zone under different light intensities
ranging from zero to full sunlight using foliage cover, cheesecloth shades, or dark bottles to reduce ambient sunlight. Temperatures remained constant at 28.4” + l.O”C.
In all experiments, we measured light with
a Weston meter and with a LiCor radiometer obtained during later portions of the
study, temperature with a mercury thermometer, pH with a Corning model 7 meter, dissolved oxygen by unmodified Winkler titration, and total alkalinity by standard
sulfuric acid titration.
Laboratory studies- Laboratory experiments were done with axenic cultures of L.
minor using Hutner’s (1953) medium supplemented with 1.O g liter-’ glucose, 20 mg
liter-’ trypticase-peptone,
and 50 mg liter-l
EDTA as a chelator. Cultures were grown
at 24°C pH 6.8, in a 14-h photoperiod at
120 PEinst m-2 s-l yielding doubling times
of 4-5 days. In all experiments (except senescence experiments) we used 20-day-old
cultures with fresh sterile medium.
Rates of net photosynthesis in the cultures were determined as above using the
14C bottle method and the Plexiglas chamber to measure atmospheric CO, uptake.
Experiments testing effects of manipulated
dissolved oxygen were performed under 100
fluorescent light
PEinst m-2 s- l “cool-white”
at 22°C.
The CO2 compensation point was determined in closed flasks from aqueous CO2
uptake in duplicate long term incubations.
Medium containing NaH14C0, (0.06 &i
ml-l) was equilibrated with the air in the
sealed flasks overnight with constant stirring, after which about 500 Lemna fronds
were quickly added to each flask and the
flasks resealed within 2 s. Flasks were incubated at 475 PEinst rnd2 sp1and 24°C with
continuous stirring; duplicate 1-ml water
samples were withdrawn through a serum
stopper port initially and at 10 intervals over
16 h and radioassayed as above for 14C re-
Productivity in Lemna minor
10.0
I
0
6 JUN
7 JUL
21 AUG
4 SEP
325
I
I
I
I
I
1976
170CT
Fig. 1. Inorganic carbon uptake from the air and
from the water in situ during the 1977 growth season
and in carbonate-free medium (axenic culture).
maining in the water. The CO2 in the water
was assumed to be at equilibrium
with the
air in the flasks initially,
and subsequent
CO2 concentrations were calculated from the
percent 14C cpm remaining at each sample
interval (corrected for volume and counting
efficiency), multiplied
by the equilibrium
concentration of CO, in water at pH 6.8 and
24°C (Hutchinson 1957). The CO, compensation point was determined as the lowest
constant concentration
remaining in the
flasks.
Environmental effects on respiration were
tested with the 14Cmethod described above.
Control assays were done on 20-day-old
cultures in Hutner’s medium at pH 6.8,
24”C, 7.40 mg liter-l 02, under 120 PEinst
m-2 s-l. Experimental variables (tested separately) included increase of light to 740
PEinst rnd2 s-l, increase of oxygen to 28.5
mg liter-l, increase of pH to 8.2 (halfstrength Hutner’s medium was necessary for
this variable to avoid precipitation; controls
were done with half-strength medium at pH
6.8). The performance of senescent plants
relative to controls was tested with plants
from 5 8-day-old cultures.
Two- and ten-second 14C fixation products of photosynthesis in the Lemna plants
were examined for any contribution
of C4
photosynthesis with 2-way descending paper chromatography
of ethanol extracts in
butanol : propionic acid : water and phenol :
water, and autoradiography
with 14C-labeled malate and aspartate as standards
(Benson et al. 1950). The activity of phosphoenolpyruvate
carboxylase, which catalyzes the initial step of C4 photosynthesis,
0
MAY
JUN
JU L
AUG
SEP
OCT
NOV
Fig. 2. Mean net photosynthesis (mean & SD) during the 1976 and 1977 growth seasons.
was assayed by the 14Cmethod of Slack and
Hatch (1967), in which incorporation
of
[ 14C]bicarbonate into acid-stable organic
compounds is measured in the presence of
buffered, reduced plant tissue extracts and
phosphoenolpyruvate.
Leaves of corn and
spinach (C, and C3 plants, respectively) were
subjected to the same assay for comparison.
Enzyme activity in terms of CO, fixation
was calculated as moles mg-l Chl and determined as percent of activity in the corn
extracts.
Differences between control and experimental rates in both laboratory and in situ
experiments were tested statistically by the
“Student’s”
t-test or ANCOVA
(Woolf
1968). Analysis of covariance was used to
test differences (0.95 C.I.) in rates of photorespiration, dark respiration, and organic
excretion. Regression analyses and coefficients of correlation were used to identify
the relationship between in situ metabolic
rates and ambient conditions of light, temperature, O2 concentration,
and pH. Photosynthetic rates are expressed on the basis
of dry weight.
Results
In axenic plants grown in the laboratory
in carbonate-free Hutner’s medium, 63.2%
of the carbon used in photosynthesis was
from the atmosphere under saturating illumination
(Fig. 1). However, for plants
growing in Shoe Lake, the mean percentage
of carbon obtained from the air throughout
the season was only 14.7 + 3.4, with most
of the carbon taken from the biocarbonaterich water. The relative portion of carbon
326
Filbin and Hough’
Table 1. Diurnal patterns of photosynthesis
1977 growth season.
(measured as carbon) and environmental
Net photosynthesis
(mg g-’ h- I)
Water temp
(“Cl
Light
Wux)
Oxygen
(mg liter-l)
21 May
iEon
0.50
0.38
14.2
16.0
16.0
10.9
6.80
7.11
P.M.
7 Jun
A.M.
0.22
0.52
0.42
0.15
1.04
1.00
0.73
0.92
1.02
0.70
3.00
2.35
1.75
3.80
3.50
2.61
8.72
8.05
6.90
6.74
6.46
5.49
3.40
3.15
2.20
2.62
2.52
2.18
2.89
2.58
1.18
0.32
0.28
0.08
18.0
19.0
20.8
21.4
21.0
21.8
23.1
25.4
26.1
28.0
25.9
26.6
29.1
27.0
28.8
29.4
24.2
26.8
27.8
19.0
20.8
20.2
13.0
13.2
13.8
10.8
11.4
11.9
2.2
2.2
2.0
0.9
1.8
2.4
8.4
68.0
102.0
116.0
7.97
5.41
8.40
12.20
6.04
8.02
10.94
6.11
8.58
10.29
8.40
10.10
12.90
7.90
10.40
11.30
10.10
12.81
14.00
5.20
8.60
10.20
10.10
12.20
14.00
8.80
10.00
10.20
7.60
8.40
8.00
7.90
8.40
8.40
Noon
21 Jun
P.M.
A.M.
Noon
P.M.
1 Jul
A.M.
Noon
P.M.
15 Jul
A.M.
Noon
P.M.
1 Aug
A.M.
Noon
P.M.
14 Aug
A.M.
Noon
P.M.
27 Aug
A.M.
Noon
P.M.
12 Sep
A.M.
Noon
P.M.
30 Sep
A.M.
Noon
P.M.
20 Ott
A.M.
Noon
P.M.
16 Nov
A.M.
Noon
P.M.
taken from the air was minimal in July at
9.8% and maximal in September at 20.5%.
In the more detailed seasonal analyses of
photosynthetic
uptake of inorganic carbon
from the water, rates were low during spring,
never exceeding 1 mg g-l h-l until the first
of July (Fig. 2). A seasonal maximum of
7.84 occurred in mid-August, with subsequent decline through late summer and fall.
Plants in the fall appeared to be senescent
and displayed the lowest rates of photosynthesis. Data from the last half of the 1976
growth season display a trend similar to that
in 1977. Rates ofphotosynthesis for the 1977
64.8
104.0
128.0
66.0
124.0
118.0
74.0
136.0
101.0
48.8
108.0
118.0
91.0
101.0
76.0
74.0
86.0
71.0
61.0
72.0
78.0
48.0
42.0
17.0
21.2
20.0
17.2
conditions
during the
Alkalinity
(meq liter-l)
3.42
3.04
3.04
3.61
3.63
3.88
3.82
3.61
3.40
3.80
3.90
3.56
3.81
3.44
3.30
3.66
3.55
3.02
3.61
3.22
3.08
3.80
3.72
3.40
3.40
3.60
3.40
3.88
3.82
3.49
3.73
3.64
3.60
3.66
3.61
3.62
season correlated best to temperature (r =
0.86) and to light (r = 0.74).
Photosynthesis was light-saturated at 45
klux (ca. 440 PEinst rnw2 s-l) when tested in
situ. While light intensities at Shoe Lake
usually were at or above this well after noon,
the rate of photosynthesis was maximal in
the late morning or at midday throughout
the season (Table 1). The afternoon depressions of photosynthesis averaged 36% below maximal photosynthesis for the day; the
maximum afternoon depression (60%) occurred in late October. The photosynthesis
depressions invariably correlated with daily
327
Productivity in Lemna minor
Table 2. Respiration in the light and in the dark
during the 1976 and 1977 growth seasons.
(o/o
i”i,?&&~,c
MAY
JUN
JUL
AUf3
SEP
OCT
Light
Dark
LED
Aug
Sep
Sep
Ott
76
76
76
76
0.82
3.82
1.83
1.22
1.44
2.60
1.63
1.25
0.57
1.47
1.12
0.98
19 May
7 Jun
18 Jun
10 Jul
25 Jul
13 Aug
26 Aug
11 Sep
22 Sep
1 Ott
18 Nov
77
77
77
77
77
77
77
77
77
77
77
0.56
0.82
0.70
1.08
1.16
3.15
3.60
3.04
1.62
1.50
0.58
0.84
0.96
0.90
1.22
1.30
2.74
3.05
2.80
1.50
1.75
1.70
0.68
0.85
0.77
0.88
0.89
1.15
1.18
1.09
1.08
0.86
0.34
18
4
15
5
0
h-,)
NOV
Fig. 3. Seasonal patterns of photosynthesis and the
effect of high and low oxygen and 10M4 M hydroxymethanesulfonate
during the 1977 growth season.
ambient dissolved oxygen and
maximum
temperature, and daily minimum alkalinity
(total dissolved inorganic carbon concentration).
Manipulation
of oxygen influenced the
rate of photosynthesis throughout the day
and throughout the 1977 season. High oxygen concentration inhibited photosynthesis,
maximally by 50% early in August during
the afternoon (Fig. 3), and by 60% in late
June, also in the afternoon. Oxygen inhibitions of significantly lower magnitude occurred during the morning (Fig. 3). Enhancement of photosynthesis by low (below
ambient) oxygen concentration was significant only in the afternoon measurements
and only between early July and late October. The addition of hydroxymethanesulfonate (HMS) was drastically inhibitory
to photosynthesis in all cases. Daily patterns
of photosynthesis were closely correlated to
the ambientconcentrations
of oxygen in the
water (r = 0.81).
Midday assays of photorespiration
conducted during the last half of the 1976 season and during the entire 1977 season revealed similar patterns (Table 2). The
seasonal maximum rates in both seasons
occurred late in the growth season (late Au-
gust 1977 and early September 1976). The
lowest rates measured during the 1977 season occurred early (in the last week of May)
and very late in the season (during the second week of November). Rates of photorespiration were most closely correlated to
water temperature (r = 0.91) and dissolved
oxygen (r = 0.88) during the 1977 growth
season. The rates of dark respiration were
low early in the season, increased through
summer, and decreased somewhat in the
fall. The seasonal change in dark respiration
was most closely correlated to water temperature. The 1ight:dark ratio of respiratory
rates was low in May and rose to a seasonal
maximum
in late August. The seasonal
minimum L:D ratio was in mid-November
under ice cover.
On both occasions when 14C in situ photorespiration was measured simultaneously
with photosynthesis
and related environmental factors (Fig. 4), the daily maximum
rate of photorespiration
correlated inversely
(weakly) to the daily minimum rate of photosynthesis (r = -0.68 on both dates). Photorespiration correlated positively to light
(r = 0.87 in June, 0.99 in August), dissolved
oxygen (r = 0.97 in June, 0.98 in August),
water temperature (r = 0.93 in June, 0.90
in August), and air temperature (r = 0.99 in
June, 0.59 in August). According to the ANCOVA, rates determined from the three as-
328
Filbin and Hough
7 JUNE
14
AUGUST
I
20 -0
10 II
20 -
a
a -k
II
II
a
0
I
I
I
I
I
I
1
I
I
I
I
I
2.0
1.0
1.0
0.5
0
0600
1200
TIME
Fig. 4. Diurnal patterns of photosynthesis,
and 14 August 1977.
0600
1600
OF
photorespiration,
says done on 7 June are all significantly different from each other. On 14 August the
morning rate is different from the two afternoon rates, while the latter are not statistically different from each other. The effect
of experimentally
increased dissolved oxygen concentration on the rates of photorespiration was significant in three midday assays (Table 3). Increased dissolved oxygen
concentration raised the rate of photorespiration by 28% in mid-June, 24% in early
August, and 20% in early September. Rates
1200
I
1600
DAY
and related environmental
factors on 7 June
of dark respiration also were increased by
elevated dissolved oxygen concentration by
18% in June, 12% in August, and 11% in
September.
Seasonal rates of organic carbon release
in the light (Table 4) were minimal in midJuly, somewhat higher earlier in the season,
and maximal in late August. Loss in the
light was virtually undetectable in November. The four assays from late summer and
fall 1976 demonstrated a higher and earlier
peak of organic loss. Rates of organic loss
329
Productivity in Lemna minor
Table 3. The effect of elevated oxygen concentration on respiration in the light and in the dark during
the 1977 growth season.
Respiration
(% initial internal ‘T h-0
7 Jun
14 Aug
30 Sep
Table 4. Organic carbon release in the light and in
the dark during the 1976 and 1977 growth seasons.
Organic carbon release
(O/oinitial internal 14Ch-l)
.
Oxygen level
Light
Dark
L:D
Ambient
High
Ambient
High
Ambient
High
0.82
1.25
2.76
3.72
1.65
1.98
0.96
1.13
2.49
2.78
1.83
2.03
0.85
1.11
1.11
1.34
0.90
0.96
in the dark were lower than those in the
light through most of the season, except for
one assay in September and one in October.
The overall seasonal pattern of dark release
was similar to the pattern of organic release
in the light.
Diurnal rates of organic carbon release in
the light varied considerably with season.
The strongest correlations
with environmental conditions in June were with air
temperature (r = 0.99) and water temperature (7 = 0.92), in August with air temperature (r = 0.93) and light (r = 0.94), and in
September also with air temperature (r 7
0.97) and light (r = 0.4 1). When oxygen tension was manipulated, there was no significant difference in rates of organic loss in
the light in June or August, although there
was a significant elevation (17%) in September. There was no significant effect of high
oxygen on rate of organic loss in the dark
at any time.
In laboratory cultures, elevated oxygen
inhibited
photosynthesis
to 38% of ambient oxygen controls (Table 5). The 9% apparent increase in the rate of photosynthesis
in plants incubated in low oxygen was statistically insignificant. The addition of HMS
virtually stopped net carbon fixation.
Light saturation of photosynthesis in axenic L. minor occurred at about 350 PEinst
m-2 s-l; there was no evidence of photoinhibition
up to 700 PEinst me2 s-l. The
photosynthetic
temperature optimum was
relatively wide, in the range of 25”-35°C. At
45°C photosynthesis was greatly inhibited.
The CO, compensation
point in axenic
plants was attained after 4 h and was very
Light
Dark
LID
Aug
Sep
Sep
Ott
76
76
76
76
5.52
4.05
3.92
1.82
3.30
1.04
0.90
0.80
1.60
3.89
4.35
2.28
19 May
7 Jun
18 Jun
10 Jul
25 Jul
13 Aug
26 Aug
11 Sep
22 Sep
1 Ott
18Nov
77
77
77
77
77
77
77
77
77
77
77
1.55
1.48
1.09
0.82
1.18
2.34
4.40
4.28
4.10
1.04
0.18
2.20
2.00
2.03
1.81
1.70
2.60
3.66
5.11
3.44
1.12
0.38
0.70
0.74
0.54
0.44
0.69
0.90
1.20
0.83
1.19
0.92
0.47
18
4
15
5
low (Fig. 5); the initial concentration of inorganic carbon was reduced by 99.7% to 1.2
pg liter-l or the equivalent of 0.95 ppm in
air.
Rates of photorespiration
in the axenic
plants increased with experimental
increases in both oxygen and light (Table 6).
The rate of dark CO2 loss was also enhanced
by increased oxygen; increased light intensity before the dark period did not significantly influence rates of dark respiration.
The L:D ratios increased more than twofold
in both treatments. In a similar experiment
with senescent plants, rates in ambient oxygen were 11.7% h-l in the light and 23.5%
h-l in the dark (L:D = 0.50), while at high
oxygen they were 25.5% h-l in the light and
47.0% h-l in the dark (L:D = 0.54).
The rate of organic carbon release in 20day-old axenic plants was significantly increased by high dissolved oxygen concentration (Table 7). An apparent increase in
the rate of release in the dark was not statistically significant. Treatment of senescent
plants with high oxygen did not influence
the rate of organic carbon release in either
the light or dark.
Lemna minor incorporated 1.2% of fixed
14Cinto the C, acid aspartate during the first
10 s of photosynthesis.
No activity was
found in C, acids in the 2-s fixation products. There were no traces of malate. The
330
Filbin and Hough
Table 5. The effects of high and low oxygen and
10m4 M hydroxymethanesulfonate
on net photosynthesis measured as carbon in axenic Lemna minor (dissolved 0, levels in mg liter-l: ambient-8.48;
high21.20; low-0.6;
rates as mean f SD).
Treatment
16
Fig. 5.
CO, compensation
20
Ambient oxygen
High oxygen
High oxygen + HMS
Low oxygen
Net photosynthesis
(mg g-l h-l)
2.OlzkO.14
0.77kO.49
0.03+0.06
2.2OkO.22
point in axenic L. minor.
activity of the C, carboxylation
enzyme,
phosphoenolpyruvate
carboxylase, was 4.6%
of that found in corn leaves assayed at the
same time.
Discussion
The season-long predominance of photosynthetic uptake of inorganic carbon from
the lake water over that from the air by L.
minor at Shoe Lake was striking. Other than
a similar observation by Wohler (1966), this
phenomenon in L. minor evidently has not
been reported. One would expect the more
highly diffusable atmospheric CO, to be
more available than that in water; this was
the case in our laboratory experiment using
carbonate-free Hutner’s medium, and it has
been assumed generally that these plants use
primarily atmospheric CO2 (e.g. Bowker et
al. 1980). The lake water was high in bicarbonate, however, and this carbon appears to have been more available than generally thought for aquatic plants, especially
at pH near 8. The percentage of atmospheric
CO2 uptake, although always relatively small
at the lake, did increase through summer,
in correlation with a summer-long decline
in total bicarbonate content reported for the
same year there by Cheney and Hough
( 1983) as contributing to a decline in photosynthetic productivity
in the filamentous
alga Cladophora fracta. Thus, L. minor appears to be sensitive to the relative availabilities of carbon from the two sources. The
extent to which this occurs in other floating
species is unknown, but it is clear that measurements of photosynthesis in this plant in
water of moderate to high bicarbonate content cannot be made accurately from atmospheric CO, flux alone.
The rates of photosynthesis in L. minor
in Shoe Lake were similar to estimates by
Wohler ( 1966) using similar methods and
approximated the productivity
represented
by biomass data reported elsewhere (Natl.
Acad. Sci. 1976). On an overall seasonal
basis the productivity
of L. minor was most
strongly influenced by positive relationships with temperature and light, a normal
circumstance for plants growing in a temperate lake of high nutrient content.
The afternoon depressions in photosynthesis, their daily correlation with afternoon
dissolved oxygen maxima and dissolved inorganic carbon minima, and their experimental enhancement by elevated oxygen and
partial alleviation
by lowered oxygen, all
suggested the presence of photorespiration.
The oxygen sensitivity
of photosynthesis
usually was demonstrable at all times of day
tested in the field, as well as in the laboratory
cultures. The presence of photorespiration
also was suggested by the enhancement of
1ight:dark respiration ratios by high oxygen
and high light in laboratory cultures, and by
high oxygen in the field where the effect was
demonstrable most times of day and most
of the season, but particularly in the afternoon late in summer correlating with maximal afternoon depressions in photosynthesis. Furthermore, the diurnal progression of
respiration in the light correlated inversely
with net photosynthesis and positively with
all the environmental parameters known to
enhance photorespiration
when all were
measured simultaneously;
similar relationships were observed in situ for the submersed angiosperm Najas flexilis (Hough
1974).
However, while our data suggest that
Productivity in Lemna minor
Table 6. Responses of light and dark respiration to
elevated oxygen and increased light in axenic Lemna
minor (% initial internal 14C h-l). Ambient O,-7.40
mg liter- *; elevated O2 - 28.5 mg liter-‘; low light- 120
PEinst mm2 s-l; high light-740
PEinst m-2 s-l.
331
Table 7. Organic 14C release in 20-day-old and senescent Lemna minor cultures under ambient and elevated dissolved oxygen concentrations (O/oinitial intemal 14C h-l).
Senescent
20-day-old
Respiration
Ambient
High 0,
Ambient
High 0,
Variable
Light
Dark
L:D
Light
Dark
Light
Dark
Light
Dark
Light
Dark
Ambient oxygen
High oxygen
Low light
High light
1.7
6.1
1.0
3.2
3.2
5.4
2.9
3.2
0.5
1.1
0.3
1.0
0.42
2.73
1.85
3.26
5.96
3.69
6.65
3.88
photorespiration
is demonstrable in Lemna, the rates appear to be relatively low. At
ambient oxygen concentrations the L:D ratios were below unity for much of the growth
season at Shoe Lake, similar to or lower
than L:D ratios found in several submersed
angiosperms (Hough 1974, 1979; Jana and
Choudhuri 1979; Sondergaard 1979), suggesting low photorespiration,
high internal
refixation of respired C02, or both. The L:D
ratios were slightly above unity late in summer when respiration in the light was maximal, but the rates of CO, loss in the light
were never > 4% of photosynthesis per hour.
In oxygen manipulation
experiments the
enhancement of photorespiration
was never
as great as the inhibition
of photosynthesis
at comparable O2 concentrations. Thus, the
oxygen inhibition of photosynthesis, shown
to be present naturally, particularly in the
afternoons, and experimentally
inducible
most of the time, probably involved more
than photorespiration,
as was found for
Cladophora fracta by Cheney and Hough
(1983). Oxygen inhibition
can also result
from oxidation of a component of the photosynthetic electron transport chain (Shelp
and Canvin 1980), hydrogen peroxide production (Gibbs 1970), and other mechanisms (Haugaard 1968). Our use of the glycolate oxidase inhibitor HMS provided no
information
regarding photorespiration,
as
it was totally inhibitory to photosynthesis.
The same results occurred with C. fracta in
a similar study (Cheney and Hough 1983);
others have reported that HMS can act as
a general inhibitor to photosynthesis (Smith
et al. 1976) and to other metabolic processes
(Lian and Sin 1974).
While we believe that oxygen inhibition
in general plays a role in L. minor productivity, the question remains why photorespiration itself was apparently so low when
conditions were sufficient to cause oxygen
inhibition and other correlates of photorespiration. C, plants have the most highly developed system for minimizing
photorespiration. Several aquatic plants exhibit some
attributes of the C4 system, notably relatively high levels of PEP carboxylase and
C4 acid production in photosynthesis (Beer
and Waisel 1979; Beer and Wetzel 1982;
Bowes et al. 1978; DeGroote and Kennedy
1977; Holaday and Bowes 1980), and in one
case both a Krantz-like
leaf structure and
somewhat elevated C4 biochemistry (Hough
and Wetzel 1977). The strongest evidence
that C4 biochemistry
(in the absence of
Krantz anatomy) actually reduces photorespiration in a submersed plant has been
reported by Bowes et al. (1978) and Holaday
and Bowes (1980) for Hydrilla verticillata,
which shows seasonal variation in the ratio
of C, and C3 metabolism, with a correlative
adjustment of photorespiration
and CO,
compensation point. In most cases, however, aquatic plants have been interpreted
as fundamentally
C3 plants with photorespiration controlled by factors other than C4
photosynthesis
(Hough and Wetzel 1977;
Beer and Wetzel 1982).
Although L. minor had been reported to
lack Krantz anatomy (Hough and Wetzel
1977), we examined our plants for possible
C, involvement.
Lemna minor did show a
high temperature optimum and a high lightsaturation capacity, found also in this plant
by Wedge and Burris (1982), which are
characteristic of C, plants. The low CO2
compensation point described here is also
characteristic of C, plants. However, we
332
Filbin and Hough
found that activity of the PEP carboxylase
and levels of the C4 acids in early fixation
products were very low and clearly in the
range normal for C3 plants. The low compensation point and apparent utilization of
bicarbonate by these plants are similar to the
observations of Salvucci and Bowes (1983)
for Myriophyllum spicatum and H. verticillata; their studies suggested that carbonic
anhydrase and a substantial bicarbonate accumulation-utilization
system control the
internal inorganic carbon pool, consequently uncoupling CO;! concentration at the carboxylation site from the external COz concentration. Similarly, the access of external
oxygen to the oxygenation/carboxylation
site
is not affected by the function of the carbonic anhydrase system; this is consistent
with the effects of experimentally
elevated
oxygen conditions in our study. On the basis
of the insignificant levels of C, biochemistry
and the lack of Krantz anatomy, we agree
with the recent conclusion of Wedge and
Burris (1982), and that of Hough and Wetzel (1977) earlier, that L. minor is not a C4
plant; thus the low photorespiration
must
be related to other factors.
Photorespiration
may be expressed in
some algae by glycolate excretion rather than
oxidation to CO* (Tolbert 1974). Although
specific excretion products were not analyzed here, total organic carbon release in
L. minor was influenced experimentally
somewhat by oxygen concentration and was
correlated with the overall results suggestive
of photosynthetic depression. However, the
rates of release were very low and there was
little to suggest that glycolate excretion was
significant or that organic release in general
was involved in more than a minor way in
the progression of productivity in this plant.
Low photorespiration and consequent low
CO1 compensation may be partly a result
of highly efficient CO2 refixation in the light,
even in the absence of the C4 system. There
are relatively abundant gas lacunae in the
basal tissues in which respired CO* may accumulate and be reabsorbed by the mesophyll adjacent to them, as hypothesized for
submersed plants by Hough and Wetzel
(1972) and Hough (1974) and supported by
the observations
of Sondergaard (1979).
Moreover, as Lemna plants have ready ac-
cess to water, there is little need for stomata1
closure in conditions of high temperature
and high light. Thus with continually
free
atmospheric gas exchange there would be
minimal internal O2 buildup and minimal
CO2 limitation
(especially if the water is a
ready carbon source as well). This may play
a major role in the low photorespiration,
low CO2 compensation point, high temperature optimum, and high light saturation
capacity, allowing the very high productivity well known in this plant. The major limiting factor, assuming adequate nutrients,
temperature, and light, would be the inorganic carbon content of the water.
The light optima of L. minor found here
and by Wedge and Burris ( 19 8 2) were higher
than those found by White (1937) and Zurzycki (1957); this plant probably is rather
adaptable in this regard. The results of both
White and Zurzycki were obtained with laboratory cultures. Our laboratory cultures had
lower light optima than did our field populations. Wedge and Burris used plants cultured from the field; those cultures may have
been close enough in number of generations
to the field population not to have lost their
high light optima.
Plant senescence was implicated in increased rates of photorespiration
and organic carbon loss in a field population of the
submersed angiosperm N. jlexilis (Hough
1974). Our senescent laboratory cultures of
Lemna exhibited higher rates of loss of CO,
and organic carbon both in the light and
dark, and a higher oxygen sensitivity of respiration, than did young cultures. Nevertheless, photorespiration
and organic release in the field population decreased in
the fall. The field population probably was
not senescent in the usual sense. Unlike
many rooted submersed macrophytes, L.
minor populations are not of uniform physiological age as the season progresses (Harper 1977; Lehman et al. 198 1). At any time
during the season, populations are comprised of plants at various physiological ages
because of continual vegetative reproduction from budding of new daughter fronds,
analogous to a multiaged microbial population in a chemostat and exhibiting an integrated physiological performance. Thus,
the observed decline of photorespiration late
333
Productivity in Lemna minor
aquatic macrophyte
(Hydrilla
verticillata).
Plant
in the growth season probably is more a
Physiol. 65: 331-335.
function of decreasing temperature and light,
HOUGH, R. A. 1974. Photorespiration
and productivity in submersed aquatic vascular plants. Limas well as increasing bicarbonate content of .
nol. Oceanogr. 19: 912-927.
the water after fall overturn, than of senes1979. Photosynthesis,
respiration, and or-.
cence.
ganic carbon release in Elodea canadensis Michx.
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Submitted: 7 March 1984
Accepted: 17 September 1984