Functional
Ecology 1998
The significance
12,185-194
photosynthetic
for return
of differences
acclimation
on investment
in the mechanisms
to light,
nitrogen
of
and CO2
in leaves
D. A. SIMS,*t
J. R. SEEMANNtand
Y. LUG*
* Desert Research Institute, Reno, NV, and t Department of Biochemistry, University of Nevada, Reno, NV 89506,
USA
Summary
1. We report changes in photosynthetic capacity of leaves developed in varying
photon flux density (PFD), nitrogen supply and CQ2 concentration. We determined the
relative effect of these environmental factors on photosynthetic capacity per unit leaf
volume as well as the volume of tissue per unit leaf area. We calculated resource-use
efficiencies from the photosynthetic capacities and measurements of leaf dry mass,
carbohydrates and nitrogen content.
2. There were clear differences between the mechanisms of photosynthetic acclimation to PFD, nitrogen supply and CQ2. PFD primarily affected volume of tissue per
unit area whereas nitrogen supply primarily affected photosynthetic capacity per unit
volume. CQ2 concentration affected both of these parameters and interacted strongly
with the PFD and nitrogen treatments.
3. Photosynthetic capacity per unit carbon invested in leaves increased in the low
PFD, high nitrogen and low CQ2 treatments. Photosynthetic capacity per unit nitrogen
was significantly affected only by nitrogen supply.
.
4. The responses to low PFD and low nitrogen appear to function to increase the
efficiency of utilization of the limiting resource. However, the responses to elevated
CQ2 in the high PFD and high nitrogen treatments suggest that high CQ2 can result in a
situation where growth is not limited by either carbon or nitrogen supply. Limitation of
growth at elevated CQ2 appears to result from internal plant factors that limit utilization of carbohydrates at sinks and/or transport of carbohydrates to sinks.
Key-words: Carbohydrates, A/Cj response, Glycine max, sink strength, water content
Functional
Ecology (1998) 12,185-194
Introduction
@ 1998
Ecological
British
Society
The photosynthetic capacities of leaves can change
dramatically in responseto photon flux density (PFD,
Boardman 1977; Bjorkman 1981), nitrogen supply
(Hunt, Weber & Gates 1985) or atmosphericCO2concentration (Gunderson & Wullschleger 1994; Griffin
& Seemann 1996). All of these responsesultimately
must result from changeseither in leaf thickness, and
thus the quantity of photosynthetic tissueper unit leaf
area,or the photosyntheticcapacity per unit volume of
that tissue. These two modes of responsemay have
quite different implications for resource allocation.
Reductions in leaf thickness reduce investment of all
resources equally per unit leaf area whereas reductions in photosynthetic enzyme concentrations,which
contain a large fraction of total leaf nitrogen (Evans
1989), may reduce nitrogen investment much more
than carbon investment.
Changes in photosynthetic capacity in responseto
light environment result primarily from changes in
leaf thickness and thus the quantity of photosynthetic
tissue per unit area (Ludlow & Wilson 1971;
Louwerse & Zweerde 1977; Patterson, Bunce &
Alberte 1977; Patterson, Duke & Hoagland 1978;
Sims & Pearcy 1992). This reduces resource investment per unit leaf area of shade leaves and allows
greater total leaf area production and thus increased
light capture and photosynthesisunder light-lirniting
conditions (Bjorkman 1981; Givnish 1988; Sims &
Pearcy 1994; Sims, Gebauer& Pearcy 1994).
Growth in elevated CO2 has been reported to
increaseleaf thicknessof soybean(Thomas & Harvey
1983; Leadley et al. 1987; Vu, Allen & Bowes 1989),
wheat (Robertson& Leech 1995),sweetgum and pine
(Thomas& Harvey 1983), ryegrass(Ferris et al. 1996)
and poplar (Radoglou & Jarvis 1990). However, these
increasesin leaf thicknessare generally lessthan 20%,
185
186
D. A. Sims
et a1
much less than the two- to threefold increases reported
for acclimation to high PFD. Photosynthetic capacities
per unit leaf area of plants grown at elevated CO2 often
decline even though leaf thickness is increased
(Gunderson & Wullschleger 1994). Consequently, in
contrast to PFD acclimation where photosynthetic
CO2. Within each pair of chambers four PFDs (ranging from 2.25 to 17.9 mol m-2 day-l) were established. The highest and lowest PFDs were in one
capacity per unit investment remains fairly constant
(Sims & Pearcy 1989), acclimation to elevated CO2
chamber and the two intennediate PFDs were in the
other chamber. Five nitrogen treatments (0-7.5 mM
appears to reduce photosynthetic capacity per unit carbon investment.
nitrate) were applied to the plants in the highest PFD
treatment while the lower PFD plants received only
two different nitrogen treatments (0.75 and 7.5 mM
Photosynthetic acclimation to elevated CO2 may
instead improve nitrogen-use efficiency. Elevated
CO2 increases the efficiency of Rubisco, the primary
carboxylating enzyme of photosynthesis, and thus
may allow a decrease in the investment in Rubisco relative
to other
photosynthetic
components
(Sage
1990). Because Rubisco accounts for up to 30% of
total leaf nitrogen (Evans 1989; Evans & Seemann
1989), reductions in this enzyme could result in substantial savings in nitrogen investment. Nitrogen concentrations of elevated CO2 grown plants almost
always decline (Luo, Field & Mooney 1994) but
whether this results in increased photosynthetic nitrogen-use efficiency depends on the relative magnitude
of changes in nitrogen content and photosynthetic
capacity. It has also been suggested that elevated CO2
limits nitrogen uptake (Jackson & Reynolds 1996)
and thus might result in a response similar to the
response to limited nitrogen supply.
Our objective in this study was to determine how
the different mechanisms of photosynthetic acclimation to PFD, nitrogen supply and CO2 affect resourceuse efficiency in soybean. We measured the effect of
these treatments on photosynthetic capacity per unit
tissue volume as well as the change in tissue volume
per unit area. Dry mass, non-structural carbohydrates
and nitrogen content were used as measures of
resource investment. The results are discussed in
texms of the possible functions of these responses.
Materials and methods
PLANT MATERIAL AND GROWTH CONDITIONS
Seeds of a non-nodulating 'Lee' variety of soybean,
Glycine max (Hartwig 1994), were planted in 8 1 pots
in a 50:50 vol/vol mixture of fine sand and sandy loam
topsoil. The pots were placed in naturally lit growth
chambers inside a greenhouse at the Desert Research
Institute in Reno, NV, USA. Temperatures were controlled to 28:t2 °C in the daytime and 20:t1 °C at
@ 1998
British
Ecological
Society,
Functional
Ecology,
12,
185-194
7.5 mM nitrate). In the second experiment four growth
chambers were used. Two were controlled to
350 p.p.m. CO2 and the other two were at 700 p.p.m.
night. Relative humidity at midday was 66:t7%.
Two experiments were conducted over consecutive
growing seasons. In the first experiment, five growth
chambers were used. Each growth chamber was controlled to a different CO2 concentration (280, 350,
525, 700 or 1000 p.p.m.). Within each chamber there
were two levels of PFD (2.32:!:0.24 or 22.9:t2.2 mol
m-2 day-l) and two levels of nitrogen supply (0.75 or
nitrate). There were four plants per treatment except
for the five nitrogen levels in the highest PFD where
there were only three plants.
CO2 concentration in each chamber was measured
.
once every 6 min by an infra-red gas analyser (model
6262, LICOR Inc, Lincoln, NE, USA). A datalogger
(model CRlO, Campbell Scientific, Logan, UT, USA)
collected the data and controlled the duration of CO2
injection into the chambers on a 30 s cycle to maintain
the CO2 setpoint. Because the ambient CO2 concentration within the greenhouse was quite variable, CO2
scrubbers were used in the 280 and 350 p.p.m. chambers to maintain a constant CO2 concentration. The
scrubber boxes measured 14 cm x 45 cm x 56 cm high
and were constructed from Plexiglas with two fans in
the top and an open grill covered by screening in the
bottom. In the flfS~ experiment, CO2 in the air flowing
through
the boxes was absorbed by cooler pads
('Coolpad'
brand,
Research
Products
Corp.,
Avondale, AZ, USA) dipped in a slurry of hydrated
lime (Chemical Lime Co., Scottsdale, AZ, USA) and
water. In the second experiment, CO2 was absorbed
by soda lime ('Sodasorb' brand, W.R. Grace & Co.,
Atlanta, GA, USA). These boxes were placed inside
the chambers and the fans were operated continuously. Control to the setpoints was achieved through
additions ofCO2.
Each chamber was divided into high and low PFD
halves. The front (south) half received full sun or
slightly reduced PFD under neutral density black plastic shade cloth (40% grade). The back (north) half
received lower PFDs under thicker shade cloth (60
and 90% ). PFD was measured continuously throughout the experiments with one gallium arsenide photodiode (model Gll18 Hamamatsu Corp., Bridgewater,
NJ, USA) in each PFD treatment connected to a datalogger (model CRlO, Campbell Scientific, Logan, UT,
USA). These sensors were previously calibrated
against a quantum sensor (model 190 s, LI-COR Inc.,
Lincoln, NE, USA). The mean daily PFD over the
4-6 week growing period of the plants was used in the
data analysis.
Nutrient treatments were randomly arranged within
the light and CO2 treatments. Nutrient treatments
were begun 3 days following seedling emergence. All
plants received either a half-strength Hoagland
solution (7.5 mM NO3, 0.5 mM PO4, 3 mM K, 2.5 mM
Ca, 1 mM Mg, 1 mM SO4' 0.067 mM Fe-EDTA, plus
187
Acclimation
to
light,
CO2
N and
micronutrients)
or a similar solution modified to
reduce nitrate conce.ntrations while keeping all other
experiments we found diurnal fluctuations of 10% or
more in leaf dry mass per unit area but less than 2%
ions constant, except for S04 and Cl which were
changes in leaf water content. Central leaflets were
used in equal portions to maintain charge balance.
detached from the plant and the midrib excised. One
half of the leaflet was weighed as quickly as possible
GAS-EXCHANGE
{less than 1 min) after detachment from the plant to
MEASUREMENTS
Photosynthesis of one leaf per plant was measured
after 4-6 weeks growth. Measurements were made on
fully
expanded leaves two nodes down from
the
youngest expanding leaf greater than 1 cm long. This
leaf was found to have the highest photosynthetic
rates in preliminary measurements of all leaves on
determine fresh mass. Leaf area was then measured
with an area meter {model 3000, LI-COR Inc.,
Lincoln, NE, USA) and the samples were dried for
48 h at 60 °C prior to determination of dry mass and
total nitrogen content {model 2400 CHN analyser,
PerkinElmer, Norwalk, CT, USA). The other half of
the leaflet was frozen in liquid nitrogen and stored in a
three high- and three low-C02 grown plants (data not
-80
shown). Because we wanted to compare plants at
similar developmental stages, measurements were
made on the higher PFD plants before the lower PFD
fructose, sucrose and starch using the technique of
Hendrix {1'993).
plants, which grew more slowly.
Photosynthesis was measured in an open flow gasexchange system (model 6400, LICOR Inc., Lincoln,
NE, USA). C02 and O2 concentrations in the air supplied to the system were controlled by mixing pure O2
and N2 with C02 in N2 using mass flow controllers
(model 825, E!iwards High Vacuum International,
Wilmington, MA, USA). An O2 concentration of 21 %
was used for all measurements. Dew-point of the air
was controlled by a dew-point humidifier (model
DPH02, Armstrong Enterprises, Palo Alto, CA, USA).
The light source for all measurements was a tungsten
halogen projector lamp (model ENH, 120 V-250 W,
Radiac Inc, Japan) reflected off a 45 ° cold mirror.
The response of assimilation
to intercellular
C02
concentration was measured at PFDs exceeding light
saturation for the leaf. Measurement PFDs for high
°C freezer until they were analysed for glucose,
Results
The effect of growth CO2 on photosynthesis (measured at light saturation and the growth CO2 concentration) depended on the PFD and nitrogen treatments
(Fig. 1 and Table 1; note the significant three-way
interaction
in
Table
1). Photosynthesis
of
high
PFD/high-nitrogen
plants increased strongly with
CO2 up to 700 p.p.m. and then declined at 1000 p.p.m.
In contrast, photosynthesis of the high PFD/1ow-nitrogen plants changed very little across the growth CO2
gradient. For low PFD plants, photosynthesis
increased with CO2 in both high and low nitrogen
treatments.
Photosynthesis at the growth CO2 was a function of
both direct effects of CO2 on photosynthetic rates and
indirect effects of light, nitrogen and CO2 on photo-
photosynthetic
capacity sun grown leaves were
1200-1400 ~mol m-2 S-I but this was reduced to
around 800 ~mol m-2 S-I for shade leaves to avoid
problems with photoinhibition.
28 °C and water-vapour
30:t2 mmol mol-l.
40
Leaf temperature was
concentration
was
Leaves were initially
35
allowed to
equilibrate for 30 min at 350 p.p.m. C02, then the C02
concentration was reduced to ..80 p.p.m. and subsequently increased in eight steps to ..1000 p.p.m.
allawing 6-10 min for equilibration at each C02 concentration. The measured responses of assimilation to
intercellular C02 concentration (A/Cj curves) were fit
to a photosynthesis model (Farquhar, von Caemmerer
& Berry 1980) for estimation of carboxylation capac-
~
30
25
'iln
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a
20
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ity (V cmax).
LEAF CHARACTERISTICS
Leaf samples were collected from the same leaves
used in the gas-exchange measurements. Prior to collection of the leaves, the plants were returned to the
@ 1998 British
Ecological Society,
Functional Ecology,
12,185-194
growth chambers for 1 day and then leaves were collected in the mid-aftemoon. Collection at a consistent
time of day reduced variation owing to diurnal
changes in carbohydrate contents. In preliminary
0
IIi
0
I
200
400
Growth
600
GO?
III
800
1000
1200
(ppm)
Fig. 1. Photosynthesis measured at the growth CO2 concentration and light saturation (Agrow) for plants grown in a
range of CO2 concentrations and PFDs of 18 mol (photon)
m-2 day-l (open symbols) or 2.3 mol (photon) m-2 day-l
(filled symbols) and nitrogen supplies of 7.5 rnM nitrate (circles) or 0.9 rnM nitrate (triangles).
188
D. A. Sims
et a1.
Table I. Significance levels for the effect of light, nitrogen and CO2 treatments on photosynthesis measured at light saturation
and the growth CO2 concentration (Agrow), carboxylation capacity [V cmax'calculated from the response of assimilation to intercellular CO2 concentration and expressed per unit leaf area (area), leaf nitrogen (N), leaf dry mass (DM) or leaf water content
(w)], leaf dry mass per unit area (h), TNC free leaf dry mass per unit area (hs)' nitrogen as a percentage of leaf dry mass (nm)'
nitrogen as a percentage ofTNC free leaf dry mass (ns)' nitrogen per unit leaf area (n.), leaf water content per unit area (w, leaf
fresh mass minus dry mass), and total non-structural carbohydrates as percentage of leaf dry mass (TNC), Individual carbohydrates; glucose (glu), fructose (fru), sucrose (suc) and starch (st), each expressed per unit leaf area (area) or per unit leaf
water content (w) were only measured in the second experiment, Interaction terms for which there were no significant effects
are not listed. Significance levels: *** p < 0.001; ** p < 0.01; * p < 0.05
v cmax
1sI experiment
Light (L)
Nitrogen (N)
CO2 (C)
LxN
LxC
NxC
LxNxC
AglOw
area
N
DM
w
h
hs
nm
ns
na
w
TNC
~w~
~w~
~w
~w*
***
***
***
***
*¥*
~¥*
*¥~
***
N
DM
w
h
hs
nm
ns
n.
w
TNC
v cmax
2nd experiment
area
Light
Nitrogen
CQ2
LxN
LxC
NxC
synthetic capacities. Carboxylation capacity per unit
leaf area (V cmax)was significantly affected by all the
treatments although the magnitude of these effects
varied between the two experiments (Table I). Vcmax
increased with increasing PFD (Fig. 2a) and increasing nitrogen supply (Fig. 2b) although in both cases
Vcmaxtended to saturate at the highest resource levels.
In the CO2 gradient experiment, Vcmax was significantly affected by CO2 concentration only in the high
PFD/1ow-nitrogen treatment (Fig. 2c). There was a
tendency for greater depression of Vcmaxby elevated
CO2 in the intermediate light and nitrogen treatments
(Fig. 2a,b). Responses of the photosynthetic rate at
light and CO2 saturation were qualitatively similar to
the results for Vcmax(results not shown).
Vcmax per unit nitrogen invested in the leaf
@ 1998 British
Ecological Society,
Functional Ecology.
12,185-194
(V cmax/N), a measure of nitrogen-use efficiency, was
substantially
increased at low nitrogen supply
(Fig. 2e). High PFD increased Vcmax/Nbut only at low
N supply (Fig. 2d,f). CO2 concentration had no significant effects on Vcmax/N (Table I). Vcmaxper unit leaf
dry mass (V cmax/DM), a measure of the return on carbon investment, was increased at the lowestPFDs but
otherwise was little affected by PFD (Fig. 2g).
Vcmax/DM increased strongly with increasing nitrogen
supply (Fig. 2h) and decreased with increasing CO2
concentration (Fig. 2i).
Changes in leaf non-structural carbohydrate content
(TNC) can dramatically affect leaf dry mass without
having any effect on actual leaf thickness and volume.
Expressing Vcmaxper unit leaf water content (V cmax/w)
eliminates the effect of TNCs and provides a measure
of the average concentration of photosynthetic components in the cell solution. On this basis, PFD had
very little effect on Vcmax(Fig. 2j). At 350 p.p.m. CO2,
Vcmax/w increased strongly with increasing nitrogen
supply up to 4 mM nitrate and then declined (Fig. 2k).
At 700 p.p.m. CO2, Vcmax/Wwas relatively constant
except for a drop at the lowest nitrogen supply.
Consequently, the CO2 effect on Vcmax/Wwas much
greater at intermediate nitrogen supplies. This appears
to account for the lack of a significant CO2 effect on
189
100
Acclimation
to
light,
CO2
N and
(b)
(a)
(c)
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Growth
(mol
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PFD
I
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Nitrogen supply
(mM nitrate)
m-2day-,>
I
I
,~ .
I t,
200
400
It
600
I
800
,
1000 1200
Growth CO2 (ppm)
Fig. 2. Carboxylation capacity (Vcmax'estimated from the response of assimilation to intercellular CO2 concentration)
expressedper unit leaf area, per unit leaf nitrogen (N), per unit leaf dry mass (DM) or per unit leaf water content for plants
grown in a range of photon flux densities (PFD), nitrogen supplies and CO2 concentrations. Symbols for PFD gradient:
700 p.p.m. CO2 (open symbols), 350 p.p.m. CO2 (filled symbols), 7.5 rnM nitrate (circles) and 0.9 rnM nitrate (triangles).
Symbols for nitrogen gradient: 700 p.p.m. CO2 (open symbols) and 350 p.p.m. CO2 (filled symbols). Symbols for CO2 gradient: 18 mol (photon) m-2 day-l (open symbols) and 2.3 mol (photon) m-2 day-l (filled symbols), 7.5 rnM nitrate (circles) and
0.9 rnM nitrate (triangles). Arrows indicate resourcelevels usedin the other gradients.
v cmax/W in the CO2 gradient experiment (Fig. 21)
where only the high and low nitrogen treatments were
used.
Leaf dry mass per unit area (LMA)
<91998 British
Ecological
Society,
Functional
Ecology,
12, 185-194
is the sum of
non-structural carbohydrates (TNC) and TNC free dry
mass, where the latter should be related to the structural investment in the leaf. Increasing PFD increased
LMA (Fig. 3a) as a result of increases in both TNC
(Fig. 3d) and TNC free dry mass (Fig. 3g). The
increasein TNC was particularly large from the lowest to the second lowest PFD for the high CO2 plants
(Fig. 3d). Increasing nitrogen supply decreasedTNCs
(Fig. 3e) but increasedTNC free dry mass (Fig. 3h)
resulting in no change in LMA (Fig. 3b). Elevated
CO2 resulted in increased TNC (Fig. 3f) and thus
LMA (Fig. 3c) but had little effect on TNC free dry
mass (Fig. 3i). The CO2 effects were much greaterat
high than at low PFD. Responsesof leaf water content
190
D. A. Sims
70
(b)
(a)
et al.
(c)
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Nitrogen supply
(molm-2day-1)
(mM nitrate)
6
7
0
200
,tI
400
600
I
800 1000
Growth CO2 (ppm)
Fig. 3. Leaf dry mass per unit area. total non-structural carbohydrates (TNC) as a percentage of leaf dry mass. TNC free leaf
dry mass per unit area and leaf water content (fresh mass minus dry mass) per unit area for plants grown in a range of photon
flux densities (PFD), nitrogen supplies and CO2 concentrations. Symbols are as in Fig. 2.
per unit area (w, an estimate of leaf thickness,
Fig. 3j-l) were qualitatively similar to responses of
TNC free leaf dry mass (Fig. 3g-i) except for a greater
CO2 effect on w at intermediate
@ 1998 British
Ecological Society,
Functional Ecology,
12, 185-194
nitrogen
supply
(Fig. 3k).
Starch accounted for more than 90% of the TNC
content (Fig. 4) and consequently the responses of
starch content to PFD, nitrogen and CO2 were similar
to those observed for TNC. The responses of soluble
carbohydrates were quite different from those of
starch. When expressed per unit area, glucose
increased with increasing PFD (Table 1, Fig. 4a) and
was higher at elevated COz concentration (Fig. 4a,b).
There was an interaction between nitrogen supply and
COz concentration such that the soluble carbohydrate
contents were greatest at high COz and internlediate
nitrogen (Fig. 4b,d,f). For fructose and sucrose there
was an interaction between PFD and COz such that
the COz effect was greater at higher PFD (Table 1,
Fig.4c,e).
The PFD effect on soluble sugar contents was
largely a result of increased leaf thickness. PFD had
no significant effect on glucose and fructose concentration expressed per unit leaf water (i.e. average
concentration in aqueous solution) (Table I, data not
191
Acclimation
to
light, N and CO2
shown). Sucrose per unit water content did, however,
increase with PFD (data not shown). Because nitrogen
supply and CO2 concentration had little effect on
Similarly, increasing nitrogen supply resulted in a
large increase in na at high PFD (Fig. Sb) but had little
effect at low PFD (Fig. Sa). At low PFD, CO2 had little effect on na (Fig. Sc) but, at high PFD, na decreased
water content per unit leaf area, the effects of these
treatments on sugars were similar regardless of the
slightly with increasing CO2 concentration.
unit of expression.
There was a strong interaction between PFD and
nitrogen supply on leaf nitrogen per unit area (nJ
percentage of dry mass regardless of whether it was
expressed per unit total dry mass (Fig. Sd) or LNC
free dry mass (Fig. Sg), suggesting that the increase in
(Fig. Sa). Nitrogen per unit area increased strongly
with PFD at high nitrogen supply but was little
affected by PFD when nitrogen was limiting.
na with high PFD resulted primarily from increases in
LMA. In contrast, increasing nitrogen supply resulted
in proportional increases in na (Fig. Sb) and nm
Increasing PFD had little effect on leaf nitrogen as
(Fig. Se) suggesting that the increase in na resulted
entirely from increases in nm, rather than LMA.
Elevated, CO2 concentration decreased nitrogen concentration across all treatments when nitrogen concentration was expressed per unit total dry mass
(Fig. Sd-f). Expressing percentage nitrogen on a TNC
free dry mass basis eliminated the CO2 effect in many
but not all cases (Fig. Sg-i).
Discussion
There were clear differences between the mechanisms
of photosynthetic acclimation to PFD, nitrogen supply
and CO2. Acclimation to PFD resulted largely from
changes in leaf thickness and thus the quantity of pho0)
rn
O
u
2
11.
tosynthetic tissue per unit area rather than changes in
photosynthetic capacity per unit volume of tissue. In
contrast, limited nitrogen supply had little effect on
leaf thickness but substantially reduced photosynthetic capacity per unit volume of tissue. Elevated
CO2 concentration also decreased photosynthetic
capacity per unit tissue volume and this effect was
most pronounced at intermediate nitrogen supply.
This interaction between nitrogen supply and CO2
highlights the complexity of CO2 responses and the
need for multi-factor experiments. The CO2 responses
in the PFD and CO2 gradients might have been substantially greater had an intermediate nitrogen supply
been used in addition to the high and low nitrogen
treatments. Elevated CO2 increased leaf thickness but
this effect was much smaller than the effect on leaf dry
mass per unit area. The greater effect on leaf dry mass
was the result of increased total non-structural carbohydrate content (TNC) in the elevated CO2 leaves.
These different mechanisms of acclimation have
important consequences for the return on investment
in leaves. Acclimation to high PFD decreased photosynthetic capacity per unit dry mass but had little
effect on photosynthetic capacity per unit nitrogen.
Low nitrogen supply increased photosynthetic capacity per unit nitrogen but decreased photosynthetic
capacity per unit dry mass. Elevated CO2 decreased
photosynthetic capacity on both dry mass and nitrogen bases.
The differences in return on investment provide
Fig. 4. Soluble carbohydrate and starch per unit leaf area for plants grown in a range
of photon flux densities (PFD), nitrogen supplies. Symbols are as in Fig. 2.
insight into whether these responses could function
to increase acquisition
of a limiting
resource.
2.5
192
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V. A. Sims et al.
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3 4
5
6
Nitrate supply
7
0
I
200
*
.~,
,
400 600 800 1000
Growth CO2 (ppm)
(mM nitrate)
Fig.5. Leaf nitrogen per unit area and as a percentage of leaf dry mass (DM) or percentage of dry mass minus total non-strucrural carbohydrate (TNC) for plants grown in a range of photon flux densities (PFD). nitrogen supplies and CQ2 concentrations. Symbols are as in Fig. 2.
@ 1998 British
Ecological Society,
Functional Ecology,
12,185-194
Conversely, they also suggest when growth is not
limited by resource supply. The increase in photosynthetic capacity per unit dry mass of shadeleaves
is consistent with a carbohydrate limitation of
growth in low PFD. Conversely, the reduction in
photosynthetic capacity per unit dry mass as PFD
increasessuggeststhat the increasein photosynthetic
capacity per unit area of sun leaves does not directly
function to increasecarbon gain. Whole plant carbon
gain as a percentage of dry mass (which is proportional to relative growth rate), for shade acclimated
plants measuredat PFDs sufficient to saturatephotosynthesis, has been found to be equal to or greater
than that of sun acclimated plants (Blackman &
Wilson 1954; Hughes 1966; Rice & Bazzaz 1989;
Sims & Pearcy 1994; Sims, Gebauer & Pearcy
1994). This is possible becausethe reduced photosynthetic capacity per unit leaf areaof shadeplants is
compensatedfor by increasedtotal leaf area. Instead
of directly increasing carbon gain, sun-leaf characteristics may function to reduce the susceptibility of
leaves to damage under stressful conditions. Sun
leaves
are
less
susceptible
to
photoinhibition
(Bjorkman
1981) but this may be function of
increases in xanthophyll cycle pigment"' contents
(Demmig-Adams et al. 1995; Koniger et al. 1995) as
well as increases in photosynthetic capacity.
Whereas PFD primarily affected photosynthetic
capacity per unit dry mass, changes in nitrogen supply
affected the return on investment for both carbon and
nitrogen. At low nitrogen supply, photosynthetic
capacity per unit nitrogen is increased while photosynthetic capacity per unit dry mass is decreased. As
nitrogen supply increases, photosynthetic capacity per
unit nitrogen decreases but photosynthetic capacity
per unit dry mass increases, suggesting a shift from
nitrogen to carbon limitation of growth.
If elevated CO2 resulted in increased nitrogen limitation as has been proposed (Jackson & Reynolds
1996), we would expect a shift to greater nitrogen-use
efficiency with increasing CO2. However, we found
no significant effect of elevated CO2 on photosynthetic capacity per unit nitrogen. Also, elevated CO2
resulted in increased w whereas limiting nitrogen
193
Acclimation
to
light.
CO2
N and
@ 1998 British
Ecological
Society,
Functional
Ecology,
12, 185-194
generally decreasedw, suggesting that these are in
fact distinct responses.These results, combined with
the large accumulation of non-structural carbohydrates in elevated CO2 leaves, suggest that growth
under elevated CO2 is not limited by either carbon or
nitrogen supply.
Even in the absenceof a nitrogen limitation elevated CO2 increases the efficiency of Rubisco and
thus might allow reductions in the investment in
Rubisco relative to other photosynthetic components
(Sage 1990).BecauseRubisco accountsfor up to 30%
of total leaf nitrogen (Evans 1989; Evans & Seemann
1989) this could have a substantial effect on nitrogen
investment. However, our results suggest that any
increase in nitrogen-use efficiency is quite small and
thus is probably not the primary factor driving
responseto elevatedCO2.
An emphasison resource-useefficiency makes the
assumption that growth is primarily resource limited.
This is no doubt the case in low-PFD and low-nitrogen environments but may not be true for elevated
CO2 combined with high PFD and high nitrogen.
Instead of improving resource-useefficiency, photosynthetic acclimation to elevated CO2 may be a
responseto liniited plasticity in sink demand for carbon (Stitt 1991) and/or limited ability to move carbohydrates from the sites of photosynthesisto the sink
tissues.The large increasesin leafTNC content of elevated CO2 grown plants suggestsuch a limitation. We
have already argued that growth of elevated CO2
plants does not appear to be nitrogen limited. Sink
strengthmight be limited by other factors such as temperature (Hofstra & Hesketh 1975) or maximal rates
of cell division and expansion (Kinsman et at. 1996).
However, some studies suggest that the limitation
involves export of carbon and transportto sinks rather
than limited sink demand. In a study of carbohydrate
production and utilization in soybean,Cure, Rufty &
Israel ( 1991) concluded that rates of phloem loading
and/or sucrose synthesis, rather than sink demand,
limited carbohydrate export from source leaves. A
similar conclusion can be drawn from studies currently underway in our laboratory.Treatmentof single
soybean leaflets with high CO2, while the rest of the
plant remained at ambient CO2, resulted in carbohydrate accumulation in the treatedleaflet to levels similar to those of leaflets where the whole plant was
exposedto high CO2 (D. A. Sims, unpublisheddata).
Carbohydrateaccumulationin leavesis often correlated with reducedphotosyntheticcapacity of elevated
CO2 grown plants (Stitt 1991).This might result from
interactions between carbohydrates and hexokinase
leading to changesin the rate of synthesisof several
photosynthetic enzymes (Jang & Sheen 1994).
However, we found no overall correlation between
whole leaf sugar concentrations, or the change in
these sugar concentrations with growth in elevated
CO2, and changesin Vcmaxper unit volume. Also, in
the experimentsmentioned above, treatmentof single
leaflets with elevated CO2 substantially increased
sugar concentrations but had no effect on photosynthetic capacity. Further evidence that photosynthetic
responses are not a simple function of sugar concentrations comes from studies of plants transfonned to
express the movement protein from tobacco mosaic
virus. These plants accumulate carbohydrates in their
leaves without significant effects on photosynthesis
(Lucas et al. 1996).
These results suggest that photosynthetic responses
at the leaf level may be a function of the integrated
control of growth and development throughout the
plant. An understanding of these responses will
require an understanding of both the limitations to
growth and resource utilization. and the signals which
act to maintain a balance between resource uptake and
utilization. This may require an understanding of
internal limitations to carbohydrate utilization, such
as limitations on the rate of transport to sinks in addition to the effects of external environmental conditions. Future efforts to predict photosynthetic
acclimation to CO2 should focus on the factors limiting growth under natural conditions as well as the signals linking
a growth limitation
at sinks to
photosynthetic responses of sources.
Acknowledgemen~
We thank Carol Johnson for nitrogen analyses and
Gavin Chandler for carbohydrate analyses. This work
was supported by USDA NRICGP grant no.940136
to Y.L. and NSF grant no.IBN 1940709 to J.R.S.
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Received 5 February 1997; revised 26 May 1997; accepted
4 June 1997