Directory UMM :Data Elmu:jurnal:T:Tree Physiology:Vol16.1996:

Tree Physiology 16, 49--59
© 1996 Heron Publishing----Victoria, Canada

Growth and photosynthesis of loblolly pine ( Pinus taeda) after
exposure to elevated CO 2 for 19 months in the field
D. T. TISSUE, R. B. THOMAS and B. R. STRAIN
Duke University, Department of Botany, Durham, NC 27708-0340, USA

Received March 2, 1995

Summary To detect seasonal and long-term differences in
growth and photosynthesis of loblolly pine (Pinus taeda L.)
exposed to elevated CO2 under ambient conditions of precipitation, light, temperature and nutrient availability, seedlings
were planted in soil representative of an early, abandoned
agricultural field and maintained for 19 months in the field
either in open-top chambers providing one of three atmospheric CO2 partial pressures (ambient, ambient +15 Pa, and
ambient +30 Pa) or in unchambered control plots. An early and
positive response to elevated CO2 substantially increased total
plant biomass. Peak differences in relative biomass enhancement occurred after 11 months of CO2 treatment when biomass
of plants grown at +15 and +30 Pa CO2 was 111 and 233%
greater, respectively, than that of plants grown at ambient CO2.

After 19 months, there was no significant difference in biomass
between +15 Pa CO2-treated plants and ambient CO2-treated
plants, whereas biomass of +30 Pa CO2-treated plants was
111% greater than that of ambient CO2-treated plants. Enhanced rates of leaf-level photosynthesis were maintained in
plants in the elevated CO2 treatments throughout the 19-month
exposure period despite reductions in both leaf N concentration
and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity during the first 11 months of CO2 exposure.
Reductions in Rubisco activity indicated photosynthetic adjustment to elevated CO2, but Rubisco-mediated control of
photosynthesis was small. Seasonal shifts in sink strength
affected photosynthetic rates, greatly magnifying the positive
effects of elevated CO2 on photosynthesis during periods of
rapid plant growth. Greater carbon assimilation by the whole
plant accelerated plant development and thereby stimulated
new sinks for carbon through increased plant biomass, secondary branching and new leaf production. We conclude that
elevated CO2 will enhance photosynthesis and biomass accumulation in loblolly pine seedlings under high nutrient conditions; however, reductions over time in the relative biomass
response of plants to elevated CO2 complicate predictions of
the eventual magnitude of carbon storage in this species under
future CO2 conditions.
Keywords: biomass, carbon assimilation, carbon dioxide enrichment, leaf N concentration, Rubisco.

Introduction
Forest trees account for 65--70% of terrestrial net primary
production (Woodwell et al. 1978) and approximately 70% of
terrestrial atmospheric carbon fixation (Waring and Schlesinger 1985). Trees are a substantial sink for CO2, despite a
reduction in the global forested area as a result of tropical
deforestation (Hall and Uhlig 1991), and tree response to
rapidly increasing atmospheric CO2 may ultimately affect the
rate of change in CO2 partial pressure in the atmosphere (Harmon et al. 1990, Vitousek 1991). Thus, we need to determine
the long-term effects of elevated CO2 on growth and photosynthesis in forest trees both to predict future global carbon
budgets accurately and to manage forests for silviculture and
conservation.
Under nonlimiting resource conditions, growth of tree seedlings exposed to elevated CO2 is generally enhanced as a result
of increased carbon fixation; however, the enhancement is
often reduced when resources are limiting (see reviews by
Eamus and Jarvis 1989, Musselman and Fox 1991, Mousseau
and Saugier 1992, Ceulemans and Mousseau 1994). In shortterm (< 6 months) studies of elevated CO2 and varying resource availability, whole-plant biomass increased 38% for
conifers (12 species) and 63% for deciduous trees (52 species)
with increases in photosynthesis of 40% for conifers and 61%
for deciduous trees (Ceulemans and Mousseau 1994). Shortterm studies of loblolly pine (Pinus taeda L.) seedlings grown
in greenhouses under nonlimiting nutrient conditions and exposed to twice ambient CO2 showed either no change in

biomass (Tolley and Strain 1984a, 1984b) or approximately a
40% increase in biomass compared to plants grown in ambient
CO2 (Sionit et al. 1985, Griffin et al. 1993); nutrient limitation
significantly reduced the growth response to elevated CO2
(Griffin et al. 1993). Photosynthetic capacity of loblolly pine
seedlings exposed to elevated CO2 was enhanced in plants
provided nonlimiting water and nutrients, but there was no
enhancement of photosynthesis in response to CO2 enrichment
under limiting nutrient conditions (Thomas et al. 1994, Lewis
et al. 1994).
Enhancement of photosynthetic capacity at elevated CO2
often decreases with time (Gunderson and Wullschleger 1994,
Sage 1994), thereby potentially limiting the availability of
carbon for growth. Reductions in photosynthetic capacity and

50

TISSUE, THOMAS AND STRAIN

the activity and content of ribulose-1,5-bisphosphate (RuBP)

carboxylase/oxygenase (Rubisco), are common indicators of
photosynthetic ‘‘acclimation’’ (Long and Drake 1991, Sage
1994). Reduced photosynthetic capacity may be the result of a
low sink demand imposed by genetic or environmental limitations (Stitt 1991) and is regulated by the flux of photosynthate
from the chloroplast to sink regions of the plant (Herold 1980).
Plant sink strength is an important factor controlling photosynthetic response to elevated CO2 (Thomas and Strain 1991).
There have been few long-term studies of the response of
loblolly pine to elevated CO2 (Fetcher et al. 1988, Tissue et al.
1993), and none have been conducted on plants grown in soil
under field conditions. Loblolly pine is an early successional
species that colonizes old fields, particularly following agricultural abandonment, and may grow on a wide variety of soils
of differing fertility (Pritchett and Smith 1975). Nutrient availability is often greatest in the early stages of colonization and
declines thereafter (Allen et al. 1990). In this study, loblolly
pine seedlings were planted in the field in soil representative
of an early abandoned agricultural field and maintained in
ambient atmospheric CO2, ambient +15 Pa CO2 (+15 Pa CO2),
or ambient +30 Pa CO2 (+30 Pa CO2) partial pressures for 19
months. The objective of the study was to detect seasonal and
long-term differences in growth and photosynthesis of loblolly
pine exposed to elevated CO2 under ambient conditions of

precipitation, light, temperature and nutrient availability.
Changes in leaf-level physiology were determined by measuring photosynthesis and Rubisco content and activity every
2 months.

+30 Pa CO2 (66.6 ± 0.3 Pa). Tree seedlings were grown under
ambient conditions of precipitation, light, temperature and
nutrient availability. Air temperature was measured at 1 m
above ground in the center of the plots with shielded thermocouples (one per plot) and photosynthetic photon flux density
(PPFD) was measured with one quantum sensor (Li-Cor Inc.,
Lincoln, NE) placed horizontally at 2.5 m in an open site. In
addition to removing trees during harvest periods, an additional four trees were removed from each plot in July 1993 to
thin the stands.
Mean monthly CO2 partial pressures fluctuated on a seasonal basis up to 4% from the yearly mean within a CO2
treatment with lower values occurring in the summer during
periods of high photosynthetic activity (Figure 1). Precipitation occurred year-round with an unusually dry period in June

Methods
Growth conditions
Loblolly pine seeds were germinated in April 1992, and seedlings were grown for 1 month in one of three greenhouses in
the Duke University Phytotron with CO2 partial pressures that

were automatically monitored and controlled at ambient CO2,
+15 or +30 Pa CO2 (Hellmers and Giles 1979). After germination, seedlings were inoculated with Pisolithus tinctorius
(Pers.) Coker and Couch (Mycorr Tech Inc., Pittsburgh, PA),
an ectomycorrhizal fungus commonly associated with loblolly
pine (Marx 1977). In May 1992, 24 seedlings were transplanted to each 3 m diameter × 3 m tall cylindrical open-top
chamber (Rogers et al. 1983) in Duke Forest. There were three
chambers for each of the three CO2 treatments and three
unchambered plots (NC). The native soil in each plot was
excavated to 1 m depth and replaced with a 1/1/1 (v/v) mix of
native clay soil, topsoil and sand mixture representative of soil
in a recently abandoned agricultural field. Soil mineral N
concentrations at the beginning of the experiment were 8.44 ±
3.55 µg N g −1 soil (mean ± SE, n = 12), with 80% of N as NO3
and 20% as NH4 (Reinhard and Richter, unpublished data).
The CO2 treatments were applied 24 h per day for the entire
experimental period. Mean CO2 partial pressures (± SE computed from monthly means, n = 18) during periods of photosynthetic activity (0800--1700 h) were: NC (36.6 ± 0.2 Pa),
ambient CO2 (36.6 ± 0.2 Pa), +15 Pa CO2 (51.6 ± 0.2 Pa) and

Figure 1. Environmental conditions in the open-top chamber and
unchambered (NC) sites include CO2 partial pressure calculated as a

monthly mean based on daily CO2 averages from 0800--1700 h,
monthly precipitation, mean total daily light in an NC site, and minimum and maximum daily air temperatures in chambered (ambient
CO2) and NC sites. Values are presented as means ± SE for 28--31
observations per month. Note for the CO2 graph that NC data are
hidden by the ambient CO2 data and SEs are not larger than the
symbols.

GROWTH AND PHOTOSYNTHESIS OF PINE IN ELEVATED CO2

1993. Daily PPFD in an unchambered plot was nearly four
times greater in the summer than in the winter; PPFD was not
measured in chambered plots. Maximum air temperature was
always higher in chambered plots than in unchambered plots,
but the difference was usually less than 1.5 °C (Figure 1). The
difference was greatest in summer with the maximum difference averaged over the month (2.9 °C) occurring in June 1993.
Growth measurements
Two plants from each chamber (six plants per CO2 treatment)
were selected at random at each of five harvests for determination of biomass accumulation and other growth parameters.
Aboveground biomass was harvested and separated into needles and stems. Because entire root systems could not be excavated without damaging the remaining trees, belowground
biomass was estimated by removing 15.7 dm3 of soil with a

cylindrical root corer (20 cm diameter × 50 cm deep). The root
cores included all of the tap root and varying amounts of lateral
roots. Complete excavation of an entire root system after 19
months growth (November 1993) indicated that the root core
technique captured 40--50% of total root biomass, including
the entire tap root (John King, unpublished data). A greater
percentage of total root biomass was probably obtained by the
root core technique when plants were smaller and the lateral
root system was less extensive. Root data presented in this
study represent actual recovered root biomass. Needles, stems
and roots were oven dried at 70 °C for 2 weeks before biomass
was determined. Biomass allocation between plant parts and
root to shoot ratios (RSR) were determined at each harvest.
Total needle surface area per plant was determined by measuring projected needle area with an LI-3100 leaf area meter
(Li-Cor Inc.) and calculating needle surface area (Thomas et
al. 1994). Leaf area ratio (LAR) was calculated as the ratio of
leaf area to total plant biomass (Kvet et al. 1971). Instantaneous relative growth rate (RGR: change in biomass per unit
biomass per unit time) and net assimilation rate (NAR: change
in biomass per unit leaf area per unit time) were estimated by
the regression method of Hunt (1990) that allows 95% confidence intervals to be determined. Tree height and number of

branches were measured during harvest periods, and the number of leaf flushes was measured by monitoring leaf production
nondestructively every 2 weeks. These parameters were used
to assess changes in plant morphology and phenology caused
by the elevated CO2 treatments.

51

which most needles experience for only a short period during
the day because of shading effects, these rates generally reflect
the maximum photosynthetic rate of needles at growth CO2
partial pressure rather than ambient net photosynthesis.
In addition, photosynthesis and Rubisco were measured
concurrently every 2 months on needles from three plants in
each chamber (nine plants per CO2 treatment) in each of two
CO2 treatments (ambient and +30 Pa CO2). Initial Rubisco
activity and total (fully activated) Rubisco activity were determined spectrophotometrically (Tissue et al. 1993). The activation state of Rubisco was calculated as the ratio of initial
activity to total activity. Rubisco content was determined by a
14
C-carboxyarabinitol bisphosphate binding assay (Sharkey et
al. 1986). Rubisco N was calculated from the amount of

Rubisco protein assuming 16.67% of Rubisco is N (Ridley et
al. 1967, Steer et al. 1968).
Needle properties
Specific leaf mass (SLM) was calculated as the ratio of needle
dry weight to needle surface area. Chlorophyll content was
determined by grinding needles in liquid N, extracting needles
twice with 80% acetone, centrifuging for 1 min, and measuring
absorbance of the supernatant at 646.6 and 663.6 nm (Porra et
al. 1989). Nitrogen was determined on needles dried at 70 °C,
ground in a Wiley mill, digested according to a micro-Kjeldahl
technique, and measured with a Technicon Traacs 800
autoanalyzer (Lowther 1980). Soluble sugar and starch content
of needles were determined as described by Tissue and Wright
(1995). Total nonstructural carbohydrate (TNC) was calculated as the sum of soluble sugar and starch.
Statistical analyses
Data were tested for normality and were natural log transformed where necessary to normalize variances among CO2
treatments. Main effects of CO2 on measured parameters were
tested by analysis of variance (ANOVA) models with the
random chamber block term nested within CO2 treatment
(Steel and Torrie 1980). Scheffe tests were used for mean

separation of the dependent variables due to planned comparisons among CO2 treatments (Data Desk Inc., Ithaca, NY).
Treatment effects were considered significant if P < 0.05.
Values of RGR and NAR were considered significantly different if 95% confidence intervals, generated by the Hunt (1990)
model, of compared values did not overlap.

Photosynthesis and Rubisco measurements
Photosynthesis was measured periodically on needles of current-year shoots of three plants in each chamber (nine plants
per CO2 treatment) in each of the four CO2 treatments with an
LI-6200 portable photosynthesis system (Li-Cor Inc.). All
photosynthesis measurements were conducted on clear days in
the open-top chambers under nearly saturating conditions of
PPFD (at least 1000 µmol m −2 s −1; Edwards 1989) and ambient
temperature. Photosynthesis was measured at the growth CO2
partial pressure of each plant and photosynthesis was expressed on a needle surface area basis. Because photosynthetic
rates of needles were measured under nearly optimal PPFD,

Results
Growth
Total plant biomass increased in response to the elevated CO2
treatments (P = 0.032, Figure 2). Differences in biomass between elevated CO2-treated plants and ambient CO2-treated
plants increased during the first 15 months of exposure to CO2
with maximum differences in relative biomass response occurring after 11 months of CO2 treatment when biomass of plants
grown at +15 and +30 Pa CO2 was 111 and 233% greater,
respectively, than that of plants grown at ambient CO2 (Fig-

52

TISSUE, THOMAS AND STRAIN

Figure 2. Total biomass for plants in the four CO2 treatments at
different harvest periods. Values are means ± SE for individual plants
in three chambers in each CO2 treatment.

ure 3). After 19 months of exposure to elevated CO2, biomass
of +30 Pa CO2-treated plants was 111% greater than that of
ambient CO2-treated plants (Figure 3), whereas there were no
significant differences in biomass between +15 Pa CO2-treated
and ambient CO2-treated plants (P = 0.152) although +15 Pa
CO2-treated plants were still, on average, 111 gDW larger than
ambient CO2-treated plants (Figure 3). There were no significant effects of chamber on leaf, stem, root or total biomass
during the five harvest periods (Table 1, Figure 2).
Biomass allocation between plant parts was similar for trees
in all CO2 treatments at all harvests (Figure 4). In all CO2
treatments, the proportion of root biomass declined and stem
biomass increased after 11 months. The RSR was greatest
during the first 11 months and declined thereafter, but did not

Figure 3. The absolute difference in biomass (gDW) and the relative
differences (% change) in biomass, photosynthetic rate and Rubisco
activity in plants grown and measured at elevated CO2 compared with
plants grown and measured at ambient CO2. Values are means for
individual plants in three chambers in each CO2 treatment.

Table 1. Biomass of loblolly pine at different harvest periods when grown at four CO2 conditions: no chamber (NC), ambient CO2 (Amb), ambient
plus 15 Pa CO2 (+15), and ambient plus 30 Pa CO2 (+30). Plants were exposed to elevated CO2 beginning April 1992. Duration of CO2 exposure
(months) is indicated below harvest dates. Values are means (standard errors) for individual plants in each of three chambers per CO2 treatment.
Different letters within a column and measurement indicate statistically different values at P < 0.05.
Measurement

CO2 (Pa)

5/92
(1)

11/92
(7)

3/93
(11)

5/93
(13)

7/93
(15)

11/93
(19)

Leaf biomass
(gDW)

NC
Amb
+15
+30

0.036 (0.001) A
0.036 (0.001) A
0.037 (0.001) A
0.036 (0.001) A

9 (2) A
10 (1) A
17 (3) B
21 (5) B

13 (2) A
10 (1) A
23 (3) B
34 (7) B

23 (4) A
33 (6) A
48 (7) B
80 (16) C

79 (12) A
88 (11) A
146 (20) B
181 (22) B

325 (76) A
267 (64) A
298 (30) A
703 (179) B

Stem biomass
(gDW)

NC
Amb
+15
+30

0.009 (0.002) A
0.009 (0.002) A
0.009 (0.003) A
0.008 (0.001) A

5 (1) A
5 (1) A
10 (2) B
12 (3) B

10 (2) A
7 (1) A
16 (2) B
27 (6) C

19 (4) A
28 (6) AB
46 (5) BC
62 (11) C

53 (7) A
63 (8) A
118 (18) B
153 (20) B

195 (25) A
322 (76) A
375 (34) AB
592 (154) B

Root biomass
(gDW)

NC
Amb
+15
+30

0.017 (0.006) A
0.017 (0.006) A
0.021 (0.008) A
0.019 (0.006) A

4 (1) A
5 (1) A
10 (1) B
11 (2) B

12 (2) A
10 (1) A
18 (1) B
29 (5) C

19 (7) AB
11 (1) A
24 (3) B
43 (9) C

17 (2) A
21 (3) A
27 (1) B
50 (8) C

70 (13) A
80 (8) AB
108 (11) AB
116 (23) B

Root/shoot ratio

NC
Amb
+15
+30

0.32 (0.05) A
0.32 (0.05) A
0.39 (0.07) A
0.40 (0.05) A

0.35 (0.03) A
0.38 (0.02) A
0.38 (0.04) A
0.36 (0.03) A

0.51 (0.02) A
0.57 (0.03) A
0.49 (0.07) A
0.49 (0.03) A

0.51 (0.20) A
0.19 (0.02) B
0.26 (0.02) AB
0.31 (0.05) AB

0.14 (0.01) A
0.14 (0.01) A
0.11 (0.01) A
0.15 (0.01) A

0.15 (0.02) A
0.16 (0.03) A
0.16 (0.01) A
0.10 (0.03) A

GROWTH AND PHOTOSYNTHESIS OF PINE IN ELEVATED CO2

Figure 4. Biomass allocation between leaves, stems and roots for
plants in the four CO2 treatments at different harvest periods. Values
are means for individual plants in three chambers in each CO2 treatment.

differ among treatments except in May 1993 in the NC and
ambient CO2-treated plants (Table 1). Reductions in RSR associated with duration of CO2 exposure partly reflected a lower
percentage of lateral roots recovered by the root core technique
as plants became larger.
Both RGR and NAR were highest during the first 7 months
in all CO2 treatments and were higher for plants in the elevated

53

CO2 treatments than for plants in the ambient CO2 treatment
during the first 11 months and were generally lower or similar
thereafter (Table 2). The LAR was similar for plants in all CO2
treatments within each harvest date, except for the harvest at
15 months (July 1993) when LAR was significantly higher in
NC plants than in plants in the chamber treatments (Table 2).
In all CO2 treatments, LAR declined as plants aged.
Total leaf area per plant was increased by elevated CO2 (P =
0.028), but differences between +15 Pa CO2-treated and ambient CO2-treated plants disappeared after 19 months, at which
time only +30 Pa CO2-treated plants had higher leaf areas than
ambient CO2-treated plants (Table 3). Trees were taller when
grown in elevated CO2 (P = 0.047) compared with ambient
CO2, although +15 Pa CO2-treated and +30 Pa CO2-treated
plants were generally not significantly different from each
other (Table 3). In response to elevated CO2, trees produced
more secondary (P = 0.012) and total branches (P = 0.023), but
not more primary branches (P = 0.088); however, there were
no significant differences in total number of branches between
+15 Pa CO2-treated and +30 Pa CO2-treated trees (Table 3).
Elevated CO2 increased the number of leaf flushes (P = 0.035)
after 203 days of treatment and this difference was maintained
throughout the experiment (Figure 5). Ambient CO2-treated
and NC plants had similar height, number of branches, total
leaf area, and number of leaf flushes until the final harvest,
when height (P = 0.002) and number of primary branches (P =
0.042) were significantly greater in ambient CO2-treated plants
than in NC plants (Table 3, Figure 5).
Photosynthesis and Rubisco
Plants grown in elevated CO2 had substantially higher photosynthetic rates (P < 0.001) than plants grown in ambient CO2,
with the greatest differences occurring during the active
growth period (May--October, Figure 6). In general, +30 Pa
CO2-treated plants had higher photosynthetic rates than +15 Pa
CO-treated plants, and ambient CO2-treated and NC plants had

Table 2. Relative growth rate (RGR), net assimilation rate (NAR), and leaf area ratio (LAR) of loblolly pine at different harvest periods when
grown at four CO2 conditions: no chamber (NC), ambient CO2 (Amb), ambient plus 15 Pa CO2 (+15), and ambient plus 30 Pa CO2 (+30). Duration
of CO2 exposure (months) is indicated below harvest dates. Values are means (standard errors for LAR, and 95% confidence intervals for RGR
and NAR) for individual plants in each of three chambers per CO2 treatment. Different letters within a column and measurement indicate
statistically different values at P < 0.05.
Measurement

CO2 (Pa)

11/92
(7)

3/93
(11)

5/93
(13)

7/93
(15)

11/93
(19)

RGR
(g g −1 day −1)

NC
Amb
+15
+30

0.030 (0.001) A
0.031 (0.001) A
0.034 (0.001) B
0.035 (0.001) B

0.003 (0.001) A
0.003 (0.001) A
0.006 (0.001) B
0.007 (0.001) B

0.010 (0.003) A
0.016 (0.002) B
0.013 (0.001) AB
0.013 (0.002) AB

0.015 (0.002) A
0.014 (0.002) A
0.014 (0.001) A
0.012 (0.002) A

0.012 (0.001) A
0.012 (0.001) A
0.008 (0.002) B
0.010 (0.001) AB

NAR
(g m −2 day −1)

NC
Amb
+15
+30

2.92 (0.25) A
2.97 (0.18) A
3.44 (0.20) B
3.61 (0.30) B

0.37 (0.05) A
0.27 (0.08) A
0.65 (0.07) B
0.77 (0.10) B

1.14 (0.15) A
1.94 (0.12) B
1.52 (0.10) C
1.69 (0.09) C

1.12 (0.10) A
1.16 (0.12) A
1.20 (0.11) A
1.08 (0.09) A

0.90 (0.08) A
1.14 (0.20) A
0.81 (0.09) A
0.93 (0.10) A

LAR
(dm2 g −1)

NC
Amb
+15
+30

1.21 (0.05) A
1.36 (0.12) A
1.28 (0.14) A
1.22 (0.13) A

0.82 (0.19) A
0.67 (0.03) A
0.69 (0.05) A
0.63 (0.05) A

1.17 (0.16) A
1.17 (0.03) A
1.05 (0.07) A
1.07 (0.08) A

1.66 (0.21) A
1.32 (0.07) B
1.39 (0.06) B
1.20 (0.05) B

0.87 (0.18) A
0.89 (0.12) A
0.85 (0.10) A
0.74 (0.11) A

54

TISSUE, THOMAS AND STRAIN

Table 3. Growth characteristics of loblolly pine at different harvest periods when grown atfour CO2 conditions: no chamber (NC), ambient CO2
(Amb), ambient plus 15 Pa CO2 (+15), and ambient plus 30 Pa CO2 (+30). Duration of CO2 exposure (months) is indicated below harvest dates.
Values are means (standard errors) for individual plants in each of three chambers per CO2 treatment. Different letters within a column and
measurement indicate statistically different values at P < 0.05.
Measurement

CO2 (Pa)

11/92
(7)

Total leaf area
(m2)

NC
Amb
+15
+30

Tree height
(cm)

NC
Amb
+15
+30

27 (3) A
32 (3) A
42 (3) B
43 (5) B

34 (3) A
36 (3) A
43 (2) B
52 (7) B

35 (2) A
43 (6) AB
46 (3) B
57 (2) C

Number of branches

NC
Amb
+15
+30

12.0 (4.0) A
9.0 (2.3) A
11.8 (1.5) A
11.3 (1.8) A

15.2 (3.2) A
13.0 (1.8) A
21.7 (4.6) B
33.7 (7.7) B

25.2 (8.3) A
22.0 (4.6) A
32.2 (3.8) AB
44.0 (6.8) B

32.5 (4.1) A
27.2 (4.3) A
56.0 (8.5) B
49.3 (7.8) B

63.5 (5.7) A
69.2 (4.2) A
81.2 (2.4) B
100.0 (17.0) B

Number of primary
branches

NC
Amb
+15
+30

9.2 (1.7) A
8.8 (2.1) A
11.7 (1.5) A
10.8 (1.7) A

11.7 (1.6) A
12.3 (1.7) A
14.0 (2.0) A
15.7 (1.8) A

18.2 (3.4) A
14.5 (1.8) A
17.8 (1.3) A
20.8 (2.2) A

19.2 (1.7) A
18.7 (2.1) A
25.0 (2.3) B
24.8 (2.9) B

24.5 (1.6) A
32.5 (2.4) B
38.3 (1.4) C
28.3 (2.6) AB

Number of secondary
branches

NC
Amb
+15
+30

2.8 (2.6) A
0.2 (0.2) A
0.2 (0.2) A
0.5 (0.3) A

3.5 (1.7) A
0.7 (0.7) A
7.7 (2.9) A
18.0 (6.1) B

7.0 (5.3) A
7.5 (2.9) A
14.3 (3.3) A
23.2 (5.2) B

13.3 (3.5) A
8.5 (2.9) A
31.0 (6.7) B
24.0 (5.0) B

38.0 (5.5) A
36.3 (2.4) A
42.8 (9.3) A
69.0 (13.1) B

0.22 (0.04) A
0.18 (0.04) A
0.40 (0.09) B
0.56 (0.16) B

3/93
(11)

5/93
(13)

0.28 (0.05) A
0.28 (0.02) A
0.49 (0.05) B
0.57 (0.11) B

similar photosynthetic rates, indicating no chamber effect on
photosynthesis.
When photosynthesis and Rubisco were measured concurrently on plants grown in the ambient CO2 and +30 Pa CO2
treatments, photosynthesis was always higher in +30 Pa CO2treated plants, especially during periods of high light and
temperature (Table 4, Figure 3). Rubisco activity was generally
reduced in +30 Pa CO2-treated plants in the first year of CO2
treatment, but activities were similar to those of ambient CO2
plants thereafter (Table 4, Figure 3). Rubisco activation state
(P = 0.082) and Rubisco content (area basis, P = 0.645) were
not affected by elevated CO2 (Table 4). Elevated CO2 had no
effect on Rubisco content expressed on a chlorophyll basis (P
= 0.218), used to estimate shifts in allocation between Rubisco
and thylakoid components (Evans and Terashima 1987), or on
Rubisco content expressed on an N basis (P = 0.086), used to
determine N allocation to Rubisco (Table 4).
Needle properties
Chlorophyll content was unchanged by elevated CO2 (P =
0.428). Leaf N concentration (mg g −1) was reduced by elevated
CO2 (P < 0.001), but leaf N content (mg m −2) was not significantly affected by elevated CO2 (P = 0.839, Table 5). Reduced
leaf N concentration in plants exposed to elevated CO2 may be
a consequence of N dilution by greater starch (P = 0.003) and
TNC (P = 0.002) concentrations in leaves resulting in leaves
with greater SLM (P < 0.001). Soluble sugar (P = 0.079) was
not significantly affected by elevated CO2 (Table 5).

0.67 (0.12) A
0.83 (0.13) A
1.25 (0.17) B
1.96 (0.35) C

7/93
(15)
2.55 (0.60) A
2.23 (0.23) A
4.04 (0.60) B
4.51 (0.43) B
75 (3) A
92 (5) A
110 (9) B
123 (4) B

11/93
(19)
5.39 (0.87) A
5.98 (1.34) A
6.75 (0.90) A
10.79 (3.38) B
120 (7) A
174 (6) B
203 (9) C
200 (8) C

Discussion
Loblolly pine seedlings exhibited an early and positive
biomass response to elevated CO2 that resulted in a rapid
increase in total plant biomass. However, the relative biomass
response peaked after 11 months of CO2 exposure, such that
after 19 months there was no significant difference in biomass
between +15 Pa CO2-treated and ambient CO2-treated plants.
For +30 Pa CO2-treated plants, the relative enhancement of
biomass compared with ambient CO2-treated plants declined
from 233% at 11 months to 111% at 19 months. In studies with
container-grown loblolly pine supplied with nonlimiting nutrients, the relative enhancement of biomass for plants in +30 Pa
CO2 declined from 59% after 5 months of CO2 treatment
(Thomas et al. 1994) to 33% after 17 months (Strain and
Thomas 1992). Many studies on trees have demonstrated a
large initial increase in biomass that was reduced after extended exposure to CO2 (see Ceulemans and Mousseau 1994).
However, some trees such as sour orange and Pinus eldarica
L. attain maximal relative biomass enhancement after 16--18
months of elevated CO2 treatment and do not exhibit a subsequent reduction in the relative rate of growth (Idso and
Kimball 1992, 1994). For loblolly pine, exposure to elevated
CO2 increased plant biomass and subsequently increased carbon storage. However, over time, reductions in the relative
biomass response of plants subjected to elevated CO2 complicate predictions of the eventual magnitude of carbon storage in
this species.
Partitioning of biomass between plant parts was similar for

GROWTH AND PHOTOSYNTHESIS OF PINE IN ELEVATED CO2

55

Figure 6. Needle photosynthetic rates at the growth CO2 condition in
the field under nearly saturating conditions of PPFD (at least 1000
µmol m −2 s −1) and ambient temperature for plants in the four CO2
treatments. Values are means ± SE for individual plants in three
chambers in each CO2 treatment.

Figure 5. Percentage of trees producing the indicated number of needle
flushing events in the four CO2 treatments after 203 (December 1992),
359 (May 1993) and 526 days (November 1993) of CO2 treatment.
Values are means for three chambers in each CO2 treatment.

trees in all CO2 treatments regardless of differences in total
biomass. Generally, plants grown in nutrient-poor soils and
elevated CO2 allocate more carbon to belowground tissues
than plants grown in ambient CO2, resulting in increased root
mass and, in turn, increased uptake of nutrients (Norby et al.

1986, Bazzaz 1990, Rogers et al. 1992). The RSR of loblolly
pine was not altered by the elevated CO2 treatments suggesting
that investment of additional photosynthate into root growth
for improved acquisition of nutrients is not necessary for
elevated CO2-treated plants growing in nutrient-rich soil. Similar results have been observed in Betula pendula Roth. (Pettersson et al. 1993) and six co-occurring trees in a northern
temperate forest, including Pinus strobus L. (Bazzaz et al.
1990).
Differences in total plant biomass among CO2 treatments
can generally be attributed to the effect of CO2 on NAR, which
is an integrated value of whole-plant photosynthesis and respiration and reflects the efficiency with which leaves produce
plant biomass. Higher RGR in elevated CO2-treated plants in
the first 11 months and similar RGR in the last 8 months
compared with ambient CO2-treated plants was a result of

Table 4. Net photosynthesis and properties of Rubisco for needles of loblolly pine grown at ambient CO2 (Amb) or ambient plus 30 Pa CO2 (+30).
Duration of CO2 exposure (months) is indicated below measurement dates. Values are means (standard errors) for individual plants in each of three
chambers per CO2 treatment. Different letters within a column and measurement indicate statistically different values at P < 0.05.
Measurement

CO2 (Pa)

9/92
(5)

11/92
(7)

1/93
(9)

3/93
(11)

5/93
(13)

7/93
(15)

9/93
(17)

11/93
(19)

Photosynthesis
(µmol m −2 s −1)

Amb
+30

4.0 (0.2) A
8.8 (0.4) B

3.7 (0.2) A
4.7 (0.2) B

2.8 (0.1) A
3.2 (0.2) B

3.3 (0.2) A
4.4 (0.3) B

4.0 (0.3) A
9.0 (0.5) B

3.3 (0.2) A
7.0 (0.4) B

5.0 (0.5) A
8.0 (0.2) B

4.4 (0.4) A
8.4 (0.5) B

Rubisco activity
(µmol m −2 s −1)

Amb
+30

20.3 (0.9) A
17.5 (0.7) B

15.5 (2.1) A
16.7 (1.2) A

22.5 (1.3) A
15.0 (1.5) B

24.2 (1.4) A
17.9 (1.6) B

19.0 (1.4) A
16.6 (2.5) A

12.9 (1.6) A
10.1 (1.3) A

21.0 (1.7) A
19.1 (2.0) A

11.8 (0.9) A
12.8 (1.0) A

Activation state
(%)

Amb
+30

80.5 (2.5) A
80.7 (2.8) A

70.8 (4.6) A
89.6 (6.0) B

86.1 (4.0) A
91.8 (3.0) A

80.8 (5.2) A
88.9 (3.3) A

72.8 (2.8) A
75.6 (4.4) A

82.2 (5.8) A
83.9 (6.1) A

87.7 (2.5) A
70.8 (4.5) B

73.4 (2.2) A
68.8 (2.8) A

Rubisco content
(mg m −2)

Amb
+30

465 (24) A
422 (35) A

388 (37) A
477 (59) A

621 (53) A
414 (46) B

614 (55) A
487 (49) B

512 (69) A
472 (52) A

365 (22) A
300 (47) A

514 (42) A
522 (39) A

290 (12) A
322 (11) A

Rubisco content
(g mmol chl −1)

Amb
+30

3.8 (0.4) A
3.5 (0.6) A

3.1 (0.3) A
3.1 (0.2) A

3.7 (0.8) A
2.3 (0.5) B

2.7 (0.3) A
2.6 (0.2) A

2.5 (0.4) A
2.1 (0.4) A

1.5 (0.1) A
1.2 (0.2) A

2.1 (0.2) A
2.3 (0.2) A

1.4 (0.1) A
1.5 (0.1) A

Rubisco content
(% of leaf N)

Amb
+30

11.8 (0.8) A
7.8 (0.6) B

5.2 (0.7) A
6.5 (0.8) A

7.9 (1.2) A
5.6 (1.3) A

5.8 (0.5) A
4.8 (0.6) A

9.6 (1.8) A
7.1 (1.1) A

7.4 (0.7) A
6.5 (1.2) A

10.3 (0.8) A
10.4 (1.4) A

4.3 (0.3) A
5.1 (0.3) A

56

TISSUE, THOMAS AND STRAIN

Table 5. Leaf characteristics of loblolly pine measured during photosynthesis and Rubiscomeasurements for plants grown at ambient CO2 (Amb)
or ambient plus 30 Pa CO2 (+30). Duration of CO2 exposure (months) is indicated below measurement dates. Values are means (standard errors)
for individual plants in each of three chambers per CO2 treatment. Different letters within a column and measurement indicate statistically different
values at P < 0.05.
Measurement

CO2 (Pa)

9/92
(5)

11/92
(7)

1/93
(9)

3/93
(11)

5/93
(13)

7/93
(15)

9/93
(17)

11/93
(19)

Chlorophyll
(µmol m −2)

Amb
+30

122 (10) A
130 (12) A

125 (5) A
158 (7) B

165 (19) A
184 (14) A

229 (25) A
189 (18) A

202 (8) A
229 (19) A

238 (10) A
255 (14) A

255 (15) A
230 (10) A

198 (16) A
224 (14) A

Nitrogen
(mg g −1)

Amb
+30

21.9 (0.5) A
22.6 (0.8) A

26.1 (0.5) A
21.6 (0.6) B

25.8 (0.6) A
21.3 (0.7) B

27.8 (0.6) A
23.5 (0.9) B

22.6 (0.5) A
20.3 (0.2) B

17.9 (0.5) A
13.9 (0.7) B

17.4 (0.8) A
14.2 (0.8) B

18.8 (0.6) A
16.5 (0.8) B

Nitrogen
(mg m −2)

Amb
+30

674 (32) A
796 (26) B

1282 (57) A
1278 (86) A

1427 (108) A 1641 (67) A
1321 (73) A 1434 (89) A

823 (32) A
985 (102) A

805 (44) A
725 (34) A

889 (48) A
855 (50) A

1036 (46) A
1209 (73) A

Specific leaf mass Amb
(g m −2)
+30

30.8 (1.4) A
35.5 (1.4) B

49.2 (2.3) A
58.9 (3.3) B

55.0 (3.1) A
61.8 (2.1) B

59.0 (1.7) A
60.7 (1.8) A

36.5 (1.3) A
47.8 (2.6) B

44.8 (1.5) A
52.3 (1.3) B

51.4 (2.5) A
60.8 (2.8) B

55.2 (1.6) A
73.4 (3.0) B

Soluble sugar
(% by DW)

Amb
+30

NA
NA

6.6 (0.3) A
6.9 (0.2) A

7.2 (0.5) A
6.2 (0.3) A

5.0 (0.2) A
5.0 (0.1) A

3.8 (0.5) A
4.0 (0.2) A

3.6 (0.3) A
4.3 (0.2) B

4.2 (0.1) A
4.9 (0.2) B

5.8 (0.3) A
6.5 (0.2) B

Starch
(% by DW)

Amb
+30

NA
NA

4.5 (0.1) A
4.6 (0.1) A

4.1 (0.2) A
3.7 (0.2) A

5.2 (0.3) A
4.6 (0.2) A

5.9 (0.4) A
7.3 (0.6) B

4.1 (0.5) A
7.6 (1.0) B

6.3 (0.5) A
9.5 (1.3) B

4.2 (0.1) A
4.6 (0.2) A

TNC
(% by DW)

Amb
+30

NA
NA

11.2 (0.3) A
11.5 (0.3) A

11.3 (0.5) A
9.9 (0.2) B

10.2 (0.5) A
9.6 (0.3) A

9.7 (0.6) A
11.3 (0.7) B

7.7 (0.8) A
12.0 (1.0) B

10.5 (0.5) A
14.4 (1.2) B

10.0 (0.3) A
11.1 (0.3) B

parallel changes in NAR. Despite maintenance of high leaf
photosynthetic capacity, reductions in the elevated CO2-induced enhancement of NAR during the last 6 months of the
CO2 treatment occurred. This suggests that whole-plant carbon
gain of elevated CO2-treated trees was reduced relative to that
of ambient CO2-treated trees during this time period, thereby
reducing the positive effect of elevated CO2 on biomass accumulation. Reductions in NAR may be due to increased wholeplant respiration as a result of more respiring, nonphotosynthetic woody tissue or reduced whole-plant photosynthesis
as a result of increased needle self-shading. In loblolly pine,
there was no difference in LAR among CO2 treatments indicating that there was no increase in the relative amount of woody
tissue. Therefore, it seems likely that reductions in NAR of
elevated CO2-treated plants were a result of reductions in
whole-plant photosynthesis induced by needle self-shading.
Norby and O’Neill (1991) found that Liriodendron tulipifera
L. increased NAR and decreased LAR when exposed to increasing CO2 partial pressures.
Changes in plant architecture were detected in plants grown
at +30 Pa CO2. In addition to increased tree height and greater
leaf area, plants grown at +30 Pa CO2 exhibited greater branch
production, especially of secondary branches. Increased leaf
area per unit tree height and increased branch number per unit
tree height changed the vertical structure of the canopy in +30
Pa CO2-treated plants, which may alter the red/far red ratio of
understory tree seedlings, thereby affecting their pattern of
growth (Arnone and Korner 1993). Leaf phenology was also
altered by CO2 partial pressure as loblolly pine grown in
elevated CO2 generally exhibited more flushes of new leaves,
as has been observed in Fagus sylvatica L. (El Kohen et al.
1993) and Quercus petraea L. ex Liebl. (Ceulemans and
Mousseau 1994). Greater carbon assimilation in response to

elevated CO2 often stimulates new sinks for carbon such as
increased secondary branching (Idso et al. 1991a) and new leaf
production.
The leaf-level photosynthetic response of loblolly pine seedlings to elevated CO2 indicated a small and transient acclimation response to long-term exposure to elevated CO2 partial
pressures. Photosynthetic acclimation often involves a reallocation of resources, particularly N, away from Rubisco to other
limiting photosynthetic processes or to nonphotosynthetic
processes resulting in optimal use of resources (Sage 1994).
Complete photosynthetic acclimation to elevated CO2 would
have occurred if loblolly pine grown and measured at elevated
CO2 had similar photosynthetic rates, reduced Rubisco content
and total Rubisco activity, and similar Rubisco activation
states as plants grown and measured at ambient CO2 (Sage et
al. 1989). However, we observed enhanced rates of photosynthesis in elevated CO2-treated plants throughout the 19 month
exposure period, and only the reductions in total Rubisco
activity, which occurred in the first year of CO2 exposure but
disappeared in the second year, indicated the occurrence of
photosynthetic acclimation in response to elevated CO2. In
studies of loblolly pine grown in nonlimiting nutrient conditions, photosynthesis was enhanced and Rubisco activity reduced after 4 months (Thomas et al. 1994) and 2 years (Tissue
et al. 1993) of exposure to elevated CO2. Although the reductions in Rubisco activity are involved in the acclimation response of loblolly pine to elevated CO2, the strength of
Rubisco-mediated control of photosynthesis appears to be
small. Similarly, Quick et al. (1991) found that Rubisco activity of tobacco grown at elevated CO2 could be reduced 49%
with only a 14% reduction in photosynthesis. These results
suggest that, in the presence of elevated CO2, Rubisco capacity
is excessive, perhaps because Rubisco also functions as a form

GROWTH AND PHOTOSYNTHESIS OF PINE IN ELEVATED CO2

of N storage (Millard 1988), and hence reductions in Rubisco
may occur with little effect on photosynthesis. In general,
photosynthesis is controlled by many factors, including Rubisco, stomatal regulation, RuBP regeneration, and end-product synthesis, and the degree to which each factor regulates
photosynthesis depends on prevailing environmental conditions (Stitt 1991).
Enhanced photosynthetic rates have been observed in fieldgrown trees after 3 years of CO2 enrichment (Idso et al. 1991b,
Gunderson et al. 1993), although in some studies, the initial
increase in photosynthesis was not maintained during longterm exposure to elevated CO2 (Samuelson and Seiler 1992,
Mousseau 1993). Differences in photosynthesis (source activity) due to elevated CO2 are often dependent on carbon use
(sink activity) in growth. If there are inadequate sinks for the
additional carbon assimilated at elevated CO2, then photosynthesis may be reduced as a result of starch accumulation or
biochemical down-regulation of Rubisco, or both (Stitt 1991).
In this study, seasonal shifts in sink strength affected photosynthetic rates, especially in plants grown at elevated CO2. Root
growth in loblolly pine occurs primarily in two peaks, one in
late spring or early summer and one in late summer or early
fall, whereas shoot growth occurs primarily in April through
September (Wahlenberg 1960). Loblolly pine exhibited 60-125% increases in photosynthetic rate at elevated CO2 during
periods of high sink activity, induced by favorable environmental conditions and rapid plant growth, and 14--33% increases in photosynthetic rate in the winter. Starch
accumulation occurred during the period of peak photosynthesis and growth indicating some limitation in sink strength even
during periods of maximum sink activity, but not sufficient to
greatly affect photosynthesis. These seasonal differences in the
magnitude of the photosynthetic response to elevated CO2,
which were maintained over two growing seasons, indicate the
importance of frequent and periodic measurements in accurately assessing long-term plant response to elevated CO2.
At the canopy level, total plant leaf area may increase as a
result of accelerated ontogeny of the plant rather than as a
specific response to CO2 (Tolley and Strain 1984a, Conroy et
al. 1986, Berryman et al. 1993). If there were a specific leaf
area response to elevated CO2, then a reduction in LAR may
indicate a canopy-level adjustment in carbon assimilation that
may not be accompanied by leaf-level adjustments in photosynthesis at the biochemical level (Norby et al. 1992, Gunderson and Wullschleger 1994). In loblolly pine, total plant leaf
area increased in response to elevated CO2 but there was no
change in LAR suggesting that canopy-level adjustment in
carbon assimilation did not occur and that total plant leaf area
increased as a result of accelerated ontogeny. Enhanced leaflevel photosynthesis coupled with increased total leaf area
indicate that net carbon assimilation for the whole plant was
greater in elevated CO2 than in ambient CO2. However, in some
trees such as L. tulipifera and Maranthes corymbosa Blume,
reductions in LAR at elevated CO2 without reductions in
leaf-level photosynthesis demonstrate canopy-level reductions
in carbon assimilation (Norby et al. 1992, Berryman et al.
1993).

57

In summary, an early and positive response to elevated CO2
rapidly and substantially increased total plant biomass. Enhanced rates of leaf-level photosynthesis were maintained in
plants subjected to elevated CO2 over the 19-month treatment
period despite reductions in Rubisco activity and leaf N concentration. Reductions in Rubisco activity indicated photosynthetic adjustment to elevated CO2, but Rubisco-mediated
control of photosynthesis was small. Seasonal shifts in sink
strength affected photosynthetic rates, greatly magnifying the
positive effects of elevated CO2 on photosynthesis during periods of rapid plant growth. Greater carbon assimilation by the
whole plant accelerated plant development and stimulated new
sinks for carbon through increased plant biomass, secondary
branching and new leaf production. We conclude that elevated
CO2 will enhance photosynthesis and biomass accumulation in
loblolly pine seedlings under high nutrient conditions, but
reductions in the relative biomass response of elevated CO2treated plants over time complicate predictions of the eventual
magnitude of carbon storage.
Acknowledgments
We thank Will Cook, Alex Hanafi, Heather Hemric, John King, Jim
Lewis and Jeff Pippen for assistance in the field and in the lab. Renate
Gebauer, John King, Jim Lewis and Joy Ward provided valuable
comments on an earlier draft of this manuscript. This research was
supported by the Department of Energy, CO2 Research Division,
contract DE-FGO5-87ER60575, the Electric Power Research Institute
Forest Response to CO2 Program and by an NSF grant DEB-9112571
for support of the Duke University Phytotron.
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