Interactive effects of elevated CO2 and mineral nutrition on

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					Tree   Physiology        14,679-690
0 1994 Heron Publishing-Victoria,                      Canada

Interactive effects of elevated CO2 and mineral nutrition on
growth and CO2 exchangeof sweet chestnut seedlings(Castanea

CNRS URA 1492, Laboratoire d’Ecologie ve’gktale, Batiment 362, Universite’ Paris-Sud,
91405 Orsay Cedex, France

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Received      October      18, 1993

The effects of elevated              atmospheric         CO2 (700 pmol mol-‘)                 and fertilization        were investigated            on
2-year-old      sweet chestnut           (Castanea sativa Mill.) seedlings grown outdoors                             in pots in constantly
ventilated    open-sided         chambers.       Plants were divided          into four groups: fertilized              controls      (+F/-CO&
unfertilized     controls      (-F/-CO&          fertilized   + COz-treated            plants (+F/+CO2)            and unfertilized         + COz-
treated plants (-F/+CO$.                Dry matter accumulation             and allocation          were measured            after one growing
season and CO2 exchange                of whole shoots was measured                throughout        the growing       season.
    Shoot growth and total leaf area of unfertilized                 plants were not affected by elevated CO2, whereas both
parameters      were enhanced by elevated CO2 in fertilized                     plants. Elevated CO2 increased                  total biomass by
about 20% in both fertilized              and unfertilized      plants; however,           biomass partitioning          differed.     In unfertil-
ized plants, elevated CO2 caused an increase in root growth,                              whereas      in fertilized     plants, it stimulated
aboveground       growth.
    At the whole-shoot            and leaf levels, photosynthetic            activity       of both fertilized       and unfertilized         plants
increased    in response to elevated C02, but the seasonal pattern of this enhancement                                    varied with nutrient
treatment.     In unfertilized        plants, a downward          acclimation         of photosynthesis          was observed         early in the
season (June), and was related to reductions                  in nitrogen       and chlorophyll         content and to starch accumula-
tion. The decrease in the slope of the A/Ci curve suggested                           a decrease in Rubisco           activity.
    In both fertilized       and unfertilized         plants, shoot respiration           decreased      during the night in response                to
elevated CO2 until mid-July.               The decrease was not related to changes in sugar concentration.

Keywords: biomass partitioning, carbon budget, deciduous trees, dry weight, gas exchange, nitrogen
partitioning, photosynthesis, respiration.

Predicting forest growth response to elevated CO2 is difficult because of the interac-
tions with other environmental factors, especially nutrients and temperature. One
approach to developing a reliable method of prediction is to build mechanistic carbon
budget models in which CO2 and nutrient availability are the experimental variables
and seedlings are the experimental material (Saugier et al. 1993). The limitations of
this approach include the need to scale up from seedlings to adult trees and the need
to perform the experiments over a long period because most plants undergo a gradual
inhibition of photosynthesis during acclimation to elevated CO*. Stitt (1991) argued
that the inhibition was related to differences in the source-sink status of the plant.
Source-sink relationships are tightly controlled by the nutrient status of the plant
680                                                         EL KOHEN   AND   MOUSSEAU

(Ingestadt and Agren 1991). For example, in sugar maple, the soil nitrogen availabil-
ity influences the seasonal carbon allocation pattern (Burke et al. 1992).
    Because elevated CO;? plays a major role in plant nutrition (Conroy 1992), most
studies of the effects of elevated CO2 on plant processes have been done under
optimal nutritional conditions (Eamus and Jarvis 1989). Although several studies
have investigated the interaction between nutrients and elevated CO2 (Wong 1979,
Hocking and Meyer 1985, Wong 1990, Sinclair 1992), few studies have documented
these interactions in trees (Brown and Higginbotham        1986, Shipley et al. 1991,
Silvola and Ahlholm 1992). It has been shown that growth is enhanced by elevated
CO2 even when the nutrient supply is restricted (Norby et al. 1986a), and that CO;!

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x nitrogen effects are species dependent (Wong et al. 1992). Elevated CO;? might also
affect respiration, thus changing the carbon loss component of the carbon budget
 (Amthor 1991, Bunce 1992)
    In this paper, we investigated the combined effects of elevated CO2 and nutrient
 availability on growth and CO2 exchange to obtain information on variations in the
 carbon budget of young tree seedlings in response to climate change. Sweet chestnut,
 Castanea sativa Mill., was chosen because it is a fast-growing deciduous tree with a
 relatively high photosynthetic activity (Ceulemans and Saugier 1992). In addition,
 changes in dry weight and nitrogen partitioning in sweet chestnut in response to
 elevated CO2 have already been shown to be dependent largely on nutrient availabil-
 ity (El Kohen et al. 1992).

Material   and methods
At the beginning of March, 2-year-old sweet chestnut seedlings were divided into
four treatment groups (24 seedlings per treatment): +F and-F refer to plants growing
with or without addition of fertilizers, respectively, +COz and -CO2 refer to plants
growing in elevated CO2 or ambient air, respectively. Each seedling was potted in
 12 1 of forest soil (El Kohen et al. 1992) consisting of the upper 15 cm organic layer
of a chestnut forest soil, yielding about 0.65 g N per year per pot. Each plant in the
+F treatments was fertilized monthly with 40 fertilizer granules that were spread over
the pot surface to provide 0.82 g N, 0.78 g P and 0.4 g K. These quantities were three
times as high as the final mineral content of a tree at the end of one year of growth
(El Kohen et al. 1992).
   All plants were grown outdoors in constantly ventilated open-sided chambers (2 m
long, 1 m wide, 1.25 m high) in natural light and watered daily to compensate for
evapotranspiration. Chambers providing the elevated CO2 treatments were enriched
to 700 pmol mol-’ CO2 with pure industrial CO2 (a detailed description of the
chambers and CO2 enrichment procedure is given in Mousseau and Enoch 1989).
   Plants were harvested at the end of the growing season to determine dry weight.
Leaf area (S) was computed from nondestructive measurements of length (L) and
width (I+) of all leaves, based on the relationship S = 0.65LW, previously established
on a population of similar plants.
   During the growing season, light-saturated photosynthetic activity and night res-
CO2 AND   MINERAL   NUTRITION   ON GROWTH   AND   GAS EXCHANGE                         681

piration rate were measured outdoors twice a week on four different plants of each
 treatment. Measurements of the rate of decrease or increase in CO2 concentration
 inside an acrylic chamber placed over a whole shoot for a few minutes were made
 with a portable CO2 analyzer (LCA2, Analytical Development Company,
Hoddesdon, Herts, UK) (El Kohen et al. 1993). Measurements were performed at the
 CO2 concentration in which the plants were grown. Dark respiration rate was
measured at the end of the night period with the same experimental system. A dark
cloth was placed over the acrylic chamber to insure total darkness during the
measurement. When the climatic conditions did not allow the measurements to be

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made outdoors, measurements were performed under an artificial light source in the
    For one plant per treatment, an A/Ci curve of an attached leaf in controlled
conditions was performed every month. The trees were transported in their pots to
the laboratory and the 5th leaf was enclosed in an assimilation cuvette. Measure-
ments were performed at saturating light and 25 “C in an open gas-exchange circuit.
    Leaf nitrogen concentration was determined by automatic element analysis on leaf
samples taken from the 5th leaf of five different trees. Chlorophyll content was
measured in acetone extracts (McKinney 1941) of four leaf discs (0.6 cm diameter)
sampled randomly from four different plants.
    Starch and total soluble sugars were extracted in 80% ethanol from 2 g of fresh
leaf tissue collected from five plants. The anthrone calorimetric method (Ashwell
 1957) was used to determine total soluble sugar concentration. The starch concen-
tration was determined by enzymatic hydrolysis (Thivend et al. 1965). The glucose
molecules liberated by hydrolysis were measured with an industrial sugar analyzer
(ISY 2700, Biochemistry Analyzer).
    Student t-tests were used for comparison of means.


Biomass accumulation and partitioning
The increase in total dry matter in response to elevated CO2 did not differ signifi-
cantly between fertilized and unfertilized plants (20 and 25% for -F/+COz and
+F/+COz plants, respectively). Fertilization altered the allocation pattern of the
increase in dry matter induced by elevated CO*. In unfertilized plants, the total
increase in dry weight was allocated to roots (Table l), suggesting that unfertilized
plants were nutrient limited, whereas in unfertilized plants, it was allocated to
aboveground parts (Table 1).
   In unfertilized plants, elevated CO2 did not have any significant effect on total leaf
area, whereas in fertilized plants elevated CO2 caused a significant enhancement of
foliage production by increasing both the number of leaves and the mean leaf area
(El Kohen 1993).

Net photosynthesis
Seasonal changes in shoot net photosynthetic activity are presented on a shoot basis
682                                                                                                                   EL KOHEN               AND     MOUSSEAU

Table 1. Biomass        (gow & SE) of different       plant parts and total leaf area (dm2) of young sweet chesmut
seedlings    after one year of growth       in ambient (-CO2)        or elevated    CO2 (+COz)   on fertilized   (+F) or
unfertilized    soil (-F). R/S = root/shoot     ratio, n = 24, * = differing    from the control (ambient CO2) at P <
0 .05 , ** = no SE could be attributed       to these numbers because the litter was collected       as a whole.

                        Initial    DW         Unfertilized         (-F)                                     Fertilized         (+F)

                                              Ambient        (-CO2)         Elevated     (+CO2)             Ambient           (-CO2)          Elevated     (+COz)

Litter                                        11.5**                        13.8                             30.4**                               40.9**
Shoot                    4.1 f0.8             16.1 +4.1                     17.8 f     6.7                   46.2        k   10.3                 61.5 * 21.5*
Root                     8.6 k 1.8            32.9 k 11.5                   40.6 f     10.2*                 67.8        5   21                   80.5 3125.1

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Total                   12.7 k 1.8            60.5 xi 14.7                  72.4 f     15.2*                144.6        +   26.5                182.9 * 40.7*
Leaf area                                     18.9+4                        18.0+      8                     30.2        A   8*                   37.3 f 8
R/s                                             1.19                          1.28*                            0.88                                 0.78*

in Figure la and on a leaf area basis in Figure lb (whole-shoot photosynthetic
activity divided by total leaf area) for the sweet chestnut plants in the four treatments.
During April and May, young leaves in the elevated CO2 treatment had a higher net
photosynthetic activity than young leaves in the ambient CO2 control treatment. The
low photosynthetic rates of control plants in early spring suggest that, functionally,
leaves were still not fully developed.
   From July, the elevated-COZ-induced increase in photosynthetic rate of unfertil-
ized plants was not statistically significant (as shown by SE bars in Figure la),
although photosynthesis of COZ-enriched plants remained slightly higher than that
of control plants. A second increase in carbon fixation occurred at the end of the
summer, and was correlated with an unusual regrowth of the terminal bud, probably
caused by a warm period (which enhanced the CO2 effect) prevailing at that time.

                                         Unfertilized                                   Fertilized

7          20
     E     10

    0          0   -...-.--....-....-.......~....~.................--......,~................................----.--
     f                 0                                                       P
         -10       -       9
         -20       I                                                         I                                                               1
                       APT        May   Jun        JUI       Aug      Sep        APT    MOY          JUll    Jul             Aug       SSP

 Figure     1. Effects    of elevated     CO2 in two contrasting          nutrient   treatments  on the light saturated       net
 assimilation      rate of young chestnut         seedlings.    The measurements        were made in situ on whole shoots
 twice a week during the growing              season. Each point represents        the mean of at least four measurements
 (*SE).     Solid symbols      = COz-enriched        trees; open symbols      = ambient CO:! (control)   trees. (a) Assimila-
 tion on a whole shoot basis; (b) assimilation               on a leaf area basis.
CO2 AND     MINERAL        NUTRITION          ON GROWTH      AND   GAS EXCHANGE                                  683

   Because elevated CO2 had no effect on total leaf area of unfertilized plants, the unit
leaf assimilation rate of COz-enriched plants was strongly enhanced during April and
May (Figure lb). After May, the stimulation decreased and became nonsignificant.
A downward acclimation of photosynthesis was observed after May, suggesting
nutrient deficiency.
   The effect of elevated CO2 on fertilized plants differed from the effect on unfertil-
ized plants. As a result of the combined effects of CO2 on leaf area and leaf
photosynthetic activity, the total carbon fixation of fertilized plants in elevated CO2
remained higher than that of +F/-CO2 plants until the end of the season (Figure la).

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Total carbon fixation of fertilized plants decreased when leaf abscision began. On a
leaf area basis, no limitation in photosynthetic rate was observed before mid-August
(Figure lb).
   Evidence for a down regulation of photosynthesis in unfertilized plants compared
to fertilized plants was indicated by the A/Ci curves (Table 2). In the ambient CO2
treatments, fertilization caused an increase in the COzsaturated photosynthetic rates.
In May, the rates doubled in response to CO2 enrichment in both nutrition treatments.
In unfertilized plants, there was little effect of CO2 enrichment on the COz-saturated
photosynthetic rate in July, but a slight decrease was observed in August (Table 2).
   In unfertilized plants, the slope of the A/Ci curve increased in response to elevated
CO2 only in May and then decreased from July to the end of the season. Because the
slopes of the A/Ci curves are thought to represent plants’ carboxylation efficiency, it
may be inferred that, in unfertilized plants, there was a decrease in carboxylation
efficiency in response to elevated CO*. In contrast, in the fertilized plants, the slope
values increased in response to elevated CO* until the end of the season.

Starch and sugar concentrations
The down regulation of photosynthesis could be due to a negative feedback resulting
from the accumulation of starch in the chloroplasts (Yelle et al. 1989). Figure 2 shows
the seasonal variation in starch and soluble sugar concentrations of leaves in the

Table 2. Characteristics     of the A/Ci curves of the 5th leaf of 2-year-old      sweet chestnut  trees grown in
ambient (-CO2) or elevated CO2 (+CO2) on fertilized       (+F) or unfertilized    soil (-F). Each curve was made
from measurements        on the same leaf each month.

                                                      -F/+CO2                                          +F/+CO2
Month            -F/-CO2               -F/+CO2        p-              tF/-CO2         +F/tCO2
                                                      -F/-CO2                                          +FJ-CO2

A at saturating C, (~01 mm2SF’)
May             4.1         11.3                      2.8              5.5            11.0            2.2
July             13.2                  13.9           1.1             10.0            15.0             1.5
August           15.0                   9.9           0.7             19.3            14.8            0.8

Slope ofAICi curve (mmol mm2s-l)
May           12           19                          1.6            10              17               1.7
July            86                 74                 0.9             19              82              4.3
August          69                 19                 0.3             61              86               1.4
684                                                                                    EL KOHEN     AND   MOUSSEAU

                          Unfertilized                                    Fertilized

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            May   Jun     JUI     Aug    S.=P   act    MOY    Jun   Jul        Aug      SeP   act

Figure 2. Seasonal changes     in starch and total soluble sugar concentrations      of young chestnut       leaves
grown in elevated CO2 (closed symbols)        or ambient CO2 (open symbols)     in two nutritional    treatments.
No error bars are indicated   because each analyzed      sample (2 g of fresh material)    represents    the mean
value of several leaf disks punched     on 5-8 different leaves and pooled together.

elevated and ambient CO2 treatments.
   The fertilization treatment lowered leaf starch concentrations of plants in the
ambient CO2 treatment (Figure 2). Elevated CO:! enhanced starch accumulation by a
factor of four to live in both fertilized and unfertilized plants during the middle of
the vegetative season. However, starch accumulation occurred earlier in the unfertil-
ized plants than in the fertilized plants.
   The accumulation of soluble sugars was slightly greater in plants in the elevated
CO2 treatment than in plants in the ambient CO2 treatment. Soluble sugars accumu-
lated early in spring in unfertilized plants, whereas they accumulated only at the end
of the season in fertilized plants. In response to elevated CO2, the fertilized plants
seemed to transform all their starch to soluble sugars, so that their soluble sugar
concentration increased greatly before leaf fall. Norby et al. (19866) also noted that
abscised leaves from seedlings of Quercus alba in elevated CO2 contained higher
concentrations of soluble sugars than abscised leaves from seedlings in ambient

Nitrogen and chlorophyll concentrations
Figure 3 shows that, although the fertilization treatment increased the overall nitro-
gen content of the leaves, N concentrations were reduced by elevated CO2 in both
nutrient treatments during the entire vegetative season. In fertilized plants, elevated
CO2 caused a 20% reduction in nitrogen compared to plants in ambient CO2, whereas
in unfertilized plants, it caused a progressive reduction from 20% in the spring to
40% in the fall (Figure 3).
   In unfertilized plants, elevated CO2 caused the amount of chlorophyll per unit leaf
area to decline (Table 3). The reduction was even greater when chlorophyll concen-
tration was expressed on a dry weight basis (Table 3) because the leaf mass per unit
CO2 AND      MINERAL          NUTRITION           ON GROWTH           AND        GAS EXCHANGE                                              685

                            Unfertilizad                                             Fertilized


                                                                 I         I         I
      01          ’

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            May       Jun   Jul    Aug     Sap     Ott     Nov       May       Jun       Jul      Aug   Ssp    Ott

Figure 3. Seasonal change in leaf nitrogen concentration       of young chestnut leaves grown in elevated CO2
(closed symbols)    or ambient CO2 (open symbols)        in two nutritional   treatments. For each date, values
represent the nitrogen concentration    of pooled leaf disc samples of at least five plants.

Table 3. Leaf chlorophyll      concentrations     (* SE, n = 4) of sweet chestnut               trees grown in four CO:! x
nutrient  treatments:    +COz = COS-enriched         plants, -CO2 = ambient air, -F = plants growing               on sandy
forest soil, and +F = plants growing          on fertilized     forest soil. Ratio refers to the ratio of -F/-CO2          to
+F/+COz.     Date of measurements      was July 7 for -F/+CO2          and -F/-CO2        plants and July 17 for +F/-CO2
and +F/+COa      plants. An asterisk (*) indicates        a value statistically   different      (P < 0.05) from that of the
control (ambient CO2 ).

Treatment     Chl a               Chl b          Chl a/h     Chl total           Chl a                  Chl b        Chl a/b   Chl total
              (mg mm2)            (mg me2)                   (mg mm2)            (mg gow-‘)             (mg gow-‘)             (mg DW-‘)

-F/-CO2       190 f 22             60f3          3.16        259 + 25            2.67                   0.85         3.14      3.52
-F/+CO2       127+ ll*             56f8          2.87        183 k 19*           1.29                   0.57         2.26      1.86
Ratio         1.49                1.07                       1.42                2.07                   1.49                   1.89

+F/-CO2       344 ?r 34            123f4         2.79        467 f 38            4.41                   1.58         2.29      5.99
+FJ+CO2       375 k 6              135f3         2.77        510f9               3.83                   1.38         2.77      5.20
Ratio         0.92                0.91                       0.92                1.15                   1.14                   1.15

area increased in response to elevated CO2 (Mousseau and Enoch 1989). The
elevated CO2 treatment had no effect on leaf chlorophyll concentrations of fertilized
trees (Table 3). This result confirms findings already documented for other tree
species (Wullschleger et al. 1992). The elevated CO2 treatment caused a substantial
shift in the ratio of chlorophyll a to chlorophyll b (Chl a/b) in the unfertilized plants
but had no effect on the ratio in fertilized plants (Table 3).

Night respiration
Figure 4 shows a typical set of curves of hourly changes in dark respiration (Rd) of
leaves in the spring. The Rd rate of attached leaves at constant temperature (16 “C)
decreased during the night period. The decrease was always significantly less in
leaves in the elevated CO2 treatment than leaves in the ambient CO2 treatment. At
the beginning of the night, the difference due to CO2 enrichment was greater in
fertilized plants than in unfertilized plants. Because Rd rates were steady during the
final hours of the night in all treatments, we chose this time to compare Rd of plants
686                                                                                                                    EL KOHEN   AND   MOUSSEAU

              1.2         ,    ,     ,    ,      ,      ,    ,        ,      ,         ,    ,     ,       ,   ,    ,
    ;cl                                         d-v.,
              1.0   -                                       -.p
         E                                                                ’ v.
        o     0.6   -                                                             ‘cr .y
                         r      I                                                               -.
         ..   0.6   -

                                                                                                                                                   Downloaded from by on July 9, 2010
i                   -                                                                        Unfertilized
         II   0.9        Obo-
:                                   Q-O-0-Q..

         E    0.8   -                                       0-a             o-‘o-Q~o~

        1..   0.7
              0.6   :*
              0.5         I     ’    ’    ’      ’      1    1        1      1     ’        ’     ’     ’     1    I
                         20         22          00          02              04             06         06          10
                                                     Time        of   day        (h)

Figure 4. Typical       curve of the hourly   change in dark respiration      rate (& = CO2 output)  of attached
chestnut    leaves during the night. Measurements         were made at growth CO2 concentration     and constant
temperature      (16 “C) in an open gas analysis    system in the laboratory.     Closed symbols = COa-enriched
trees; open symbols        = ambient CO2 (control)   trees.

from different treatments.
   For each CO:! and nutrition treatment, whole-shoot Rd was measured in situ at the
end of the night during the entire leafy period. Figure 5 shows the results plotted on
a whole shoot basis (Figure 5a) and on a leaf area basis (Figure 5b). Respiration rates
were high in spring, which was especially warm in the study year. At the beginning
of the growing season, COz-enriched plants had a significantly lower Rd rate than
plants in the ambient CO2 treatment in both nutrition treatments (Figure 5). This
difference decreased with time and was negligible by June in unfertilized plants, but
persisted until mid-July in fertilized plants.

Biomass accumulation increased in response to elevated CO2 regardless of nutrient
treatment. The response (20 to 25% total biomass increase per tree) was of similar
magnitude to that reported for other woody species (Eamus and Jarvis 1989).
   In both fertilized and unfertilized plants, the rate of carbon fixation approximately
doubled in response to elevated CO2. The relative increase in photosynthesis due to
elevated CO2 was greater than the corresponding increase in biomass, whereas
fertilization had a greater effect on biomass (Table 1) than on photosynthetic rate.
This response to elevated CO2 and fertilization is identical to that of willow (Silvola
and Ahlholm 1992), which is probably because the photosynthetic measurements
were performed at light saturation and, therefore, do not represent the real carbon
COz AND MINERAL NUTRITION ON GROWTH AND GAS EXCHANGE                                                                       687

                                                      Unfertilized                           Fertilized

                                                                             (4         6                       (4
     n    0.6   -         &..
i                                    ..                      6
                                          ..            :,       ‘6
    z0    06.
    x                                          “.lY                   ‘6
    'ij   0.4

     5    0.2
          0.0                                                                                                          +
r          20

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                                      Jun                         JUI             May       JlJn          JUI   Aug
                    May                                                    Aug

Figure 5. Seasonal variation in the end-of-night dark respiration rate (& = CO2 output) expressed per
shoot (a) or per unit leaf area (b) of sweet chestnut seedlings under two CO2 x nutrition treatments.
Closed symbols = C02-enriched trees; open symbols = ambient CO2 (control) trees. Each point
represents the mean of at least four measurements (& SE).

fixation in fluctuating environmental conditions.
   Down regulation of photosynthesis was observed in unfertilized plants in the
elevated CO2 treatment. This reduced photosynthetic activity cannot be attributed to
a restriction of root growth, i.e., a sink limitation, because the pot volume was about
12 1; however, increases in the length of fine roots, which can be enhanced several-
fold by elevated CO;! (Berntson et al. 1993, Pettersson et al. 1993), could have been
restricted by the pots. The decline in photosynthetic rate was correlated with a
decrease in leaf nitrogen concentration (Figure 3), and corresponded to declines in
both the slope and plateau of the A/Ci curves (Table 2), indicating possible limitation
by Rubisco (Von Caemmerer and Farquhar 1981) and limitation as a result of a
reduction in end product synthesis, respectively.
   The accumulation of starch in COz-enriched leaves has been found in many species
(Koch et al. 1986, Downton et al. 1987, Wong 1990). Our finding that the extent of
starch accumulation in the fertilized and unfertilized plants was similar could
indicate that the seedlings were unable to utilize all of the carbohydrates that they
had assimilated. The stimulation of net carbon assimilation was not paralleled by a
stimulation of other metabolic pathways. For example, respiratory activity decreased
in response to elevated CO*. In the unfertilized plants, starch accumulation may
indicate a lack of new sinks to incorporate the carbohydrate surplus, i.e., the -F/+CO;!
plants did not increase their leaf area in response to elevated CO2, whereas the
+F/+COz plants increased their leaf area.
   The lowering of the respiration rate in COZ-enriched plants is an intriguing
phenomenon. It has been found in many woody species (Bunce 1992, Idso and
Kimball 1992, Wullschleger et al. 1992) and some herbaceous species (Amthor et al.
688                                                                                           EL KOHEN        AND    MOUSSEAU

 1992, Bunce and Caulfield 1991), and has been shown to be an instantaneous and
reversible response (El Kohen et al. 1991, Amthor et al. 1992). Our results show that
the percentage reduction in respiration rate followed a diurnal pattern, with maxi-
mum values at the beginning of the night and minimum values at the end. The plateau
in the nighttime respiration rate in the elevated-CO2 treatment is probably associated
with the elevated-CO*-induced decreases in sucrose export and metabolism at night
(Wullschleger et al. 1992).
   The elevated-COZ-induced decrease in Rd also showed a seasonal pattern; the
reduction was important when the plant was growing vigorously in May and June,

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and much less so at the end of the season. The decrease occurred earlier in the season
for unfertilized plants than for fertilized plants and did not seem to be related to the
seasonal change in leaf sugar concentration (Figure 2). The mechanism underlying
this decrease in Rd is not known. Wullschleger and Norby (1992) concluded that~both
growth and maintenance components of respiration should be affected. In many
cases, maintenance respiration is related to the nitrogen content of the tissue (Ryan
1991). However, in Castanea, the decrease in Rd in response to elevated CO2 was
evident even when the results were expressed on a nitrogen basis (El Kohen et al.
   To build a whole-plant carbon balance response to elevated COZ, it will be
necessary to consider mycorrhizal growth and maintenance. Rouhier et al. (1994)
found increased rhizospheric activity in elevated CO2. This could be due to increased
fine root activity and turnover (Khmer and Amone 1992), or stimulation of microbial
activity due to an increase in carbohydrate root exudates (Norby et al. 1987), or both.
A plant-soil system model will be needed to reconcile all of these observations.

This study was supported      by an EC research program       (EPOCH      COTU    13). It represents   part of the Ph.D.
research   of A. El Kohen.       The authors      acknowledge     Bernard    Saugier for stimulating        discussions,
Jean-Yves    Pontailler for technical assistance,     and Bernard Legay and Jacqueline         Liebert for their help in
the field and in the laboratory.

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   roots in small growth containers.          Oecologia     94:558-564.
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