The effect of elevated atmospheric CO2 concentration and nutrient supply on gas exchange, carbohydrates and foliar phenolic concentration in live oak (Quercus virginiana Mill.) seedlings

HAL Id: hal-00883281

https://hal.archives-ouvertes.fr/hal-00883281

Submitted on 1 Jan 1999

HAL is a multi-disciplinary open access

archive for the deposit and dissemination of

sci-entific research documents, whether they are

pub-lished or not. The documents may come from

teaching and research institutions in France or

abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est

destinée au dépôt et à la diffusion de documents

scientifiques de niveau recherche, publiés ou non,

émanant des établissements d’enseignement et de

recherche français ou étrangers, des laboratoires

publics ou privés.

The effect of elevated atmospheric CO2 concentration

and nutrient supply on gas exchange, carbohydrates and

foliar phenolic concentration in live oak (Quercus

virginiana Mill.) seedlings

Roberto Tognetti, Jon D. Johnson

To cite this version:

Original

article

The

effect of

elevated

_atmospheric

_CO

₂

concentration

and

nutrient

_supply

on

_gas

exchange, carbohydrates

and

foliar

_phenolic

concentration

in live oak

(Quercus virginiana

Mill.)

_seedlings

Roberto

_Tognetti

Jon

D. Johnson

a

School of Forest Resources and Conservation,

University

of _Florida, 326

_{Newins-Ziegler}

_{Hall, Gainesville,} FL 32611, USA b

Istituto per

l’Agrometeorologia

e l’Analisi Ambientale

_{applicata all’Agricoltura, Consiglio}

Nazionale delle _Ricerche, via

_Caproni

8, Florence, 50145,

Italy

c

Department

of

_{Botany, Trinity College, University}

of _Dublin, Dublin _2, Ireland

(Received 15

_July

1998;

accepted

4 November 1998)

Abstract - We determined the direct effects of

_atmospheric

CO, concentration _([CO,]) on leaf gas

_{exchange, phenolic}

and

carbohy-drate allocation in live oak

_seedlings

_{(Quercus virginiana}

_Mill.)

_grown at present

(370

)

μmol·mol

-1

or elevated ₍₅₂₀

_μmol·mol

_-1

₎

[CO,] for 6 months in open-top chambers. Two soil

_nitrogen

_(N) treatments (20 and 90

_μmol·mol

_-1

total N, low N and

_high

N

treat-ments,

respectively)

were

_{imposed by watering}

the

_plants

every 5 d with modified water soluble fertilizer. Enhanced rates of

leaf-level

_{photosynthesis}

were maintained in

_{plants subjected}

to elevated

_[CO

₂

_]

over the 6-month treatment

_period

in both N treatments.

A combination of increased rates of

_{photosynthesis}

and decreased

stomatal

conductance was

_responsible

for

_{nearly doubling}

water

use

_efficiency

under elevated [CO2]. The sustained increase in

_{photosynthetic}

rate was

_{accompanied by}

decreased dark

respiration

in elevated

_[CO

₂

_].

Elevated

_[CO

₂

_]

led to increased

_growth

rates, while total non-structural

carbohydrate

(sugars and _starch)

concentra-tions were not

_{significantly}

affected

_by

elevated

_[CO,]

treatment. The concentration of

_{phenolic compounds}

increased

_{significantly}

under elevated _{[CO,]. (© Inra/Elsevier, Paris.)}

elevated

_[CO

₂

_]

/ _gas

_exchange

/

_nitrogen

/

_phenolics

/

_{Quercus virginiana}

/ total non-structural

_{carbohydrates}

Résumé - Effet d’une concentration

_{atmosphériques}

élevée en

_CO

₂

_et

d’un apport nutritionnel sur les

_échanges

_gazeux et les concentrations en

_hydrate

de carbone et

_{composés phénoliques}

foliaires chez de

_{jeunes plants}

de

_{Quercus virginiana}

Mill. Les effets directs de deux concentrations en

_CO,

₍₃₇₀

_μmol

mol-1et 520

_μmol

_mol

_-1

₎

sur les

_échange

gazeux, les

composés phénoliques

et l’allocation

_d’hydrate

de carbone ont été étudiés sur des semis de _{Quercus virginiana} Mill. Pendant six mois dans des chambres à ciel ouvert. Deux traitement du sol N (20 et 90

_μmol·mol

_-1

des traitements totaux de N, traitements faibles en azote et traitement fort en azote

_{respectivement)}

ont été

_imposés

en arrosant les semis tous les

_{cinq jours}

avec de

_{l’engrais hydrosoluble}

modifié. Une aug-mentations de la

_{photosynthèse}

a été mise en évidence chez les semis soumis à une concentration élevée en _CO, dans les deux

traite-ments de N. Une combinaison de taux

_plus

élevé de la

_{photosynthèse}

et de la conductibilité

_stomatique

diminuée étaient

_responsable

du

_{quasi-doublement}

de l’efficacité d’utilisation de l’eau en

_CO,

élevé.

_{L’augmentation}

soutenue du taux de

_{photosynthèse}

a été

cou-plée

à une diminution de la

_respiration

en

_CO

₂

élevé. Les semis ont utilisé le carbone

_{supplémentaire principalement}

pour la croissan-ce alors que les concentrations en

_{hydrates de}

carbone non structuraux totaux (sucres et amidon) n’ont pas été _{affectées par le}

traitement élevé de CO,. (© Inra/Elsevier, Paris.)

azote /

échange

de gaz / enrichissement en

_dioxyde

de carbone /

hydrates

de carbone non-structuraux total /

_phénoliques

/

Quercus virginiana

*

Correspondence

and

_reprints

tognetti

@sunserver.iata.fi.cnr.it

** _{Present address: Intensive}

1. Introduction

In _response to elevated

_atmospheric

carbon dioxide

(CO

2

)

concentration

_([CO

₂

_]),

tree

_species

often exhibit increases in carbon

_(C)

assimilation rates

_{[36, 39],}

instantaneous water use

_efficiency

_{[25, 40]}

and

_growth

[5, 53].

Elevated

_[CO

₂

_]

_{may also} reduce dark

_respiration

[56].

Total non-structural

_{carbohydrates}

(TNC)

have

been

_generally

shown to increase under elevated

_[CO

₂

_],

but it also _appears that this is a

_{species-specific}

_response

[29, 50].

The

_magnitude

of these _{responses may} be

affected

_by

nutrient levels

_{[15, 17].}

In most _temperate and boreal sites

_plants

are often

limited

_{by suboptimal}

soil

_nitrogen

_(N)

_availability

_[26].

Under conditions of

_optimum

_[CO

₂

_]

combined with

nutrient resource

_limitation,

which

_{restrict growth}

to a

greater extent than

_{photosynthesis,}

_plants

tend to show

an increase in C/N ratios and an excess of non-structural

carbohydrates

[6].

This excess _may then be available for

incorporation

into C-based

_secondary

_compounds

such

as

_phenolics

_[30].

The C-nutrient balance

_hypothesis

pre-dicts that the

_availability

of excess C at a certain nutrient

level leads to the increased

_production

of C-based

sec-ondary

metabolites and their _precursors

_[46].

CO

2

-enriched

atmospheres

often induce reduction in the N concentration of

_plant

_tissues,

which has been attributed to

_{physiological changes}

in

_plant

N use

effi-ciency

[5, 37, 38].

On the other

hand,

there is

_increasing

evidence that the reduction in tissue N concentrations of

high

_CO

₂

_-grown

plants

is

_probably

a

size-dependent

phenomenon

resulting

from accelerated

_{plant growth}

[11].

It has also been documented that reductions in

_plant

tissue N concentrations _may

_{substantially}

alter

plant-herbivore

interactions

[32].

In

fact,

insect

herbi-vores consume _greater amounts of

_high

_CO

₂

_-grown

foliage

apparently

to _compensate for their reduced N

concentration

_[16].

This _may

_play

an

_important

role in

seedling

survival and

_{competitive ability.}

The increase in

_{plant productivity}

_{in response} to

_rising

[CO

2

]

is

_largely

dictated

_{by photosynthesis, respiration,}

carbohydrate

production

and their differential allocation

between

_plant

_organs and the

_{subsequent incorporation}

into biomass

_[22].

For this reason _{many studies have}

investigated

the effects of elevated

_[CO,]

on

_plant

prima-ry metabolism

[ 14],

but

relatively

few studies have

investigated

the response of

plant secondary

metabolite concentrations to

_increasing

_[CO

₂

_]

and its interaction

with N

_availability

[31].

The aim of this

_study

was to

_investigate

how

_CO,

availability

alters total

_phenolics,

TNC

_(starch

_plus

sug-ars)

and to determine how elevated

_[CO

₂

_]

influences _gas

exchange

of live oak

_seedlings

_{(Quercus virginiana}

Mill.).

Live oak is an

_{important species}

in

_dwindling

southeastern United States natural _ecosystems, and is

able to withstand wind storms and hurricanes because of its

_deep

and

_strong

root

_system.

_{Extrapolation}

from stud-ies on

_seedlings

to mature trees should be

_performed

only

with extreme caution. However, the

_seedling

_stage

represents a time characterized

_by

_high

_genetic

_diversity,

great

competitive

selection and

_{high growth}

rates

_[9]

and

as such _{may represent} one of the most crucial

_periods

in the course of tree establishment and forest

_{regeneration.}

Indeed,

a small increase in relative

_growth

at the

_early

stage of

_development

_may result in a

_large

difference in

size of individuals in the successive _years, thus

deter-mining

forest

_community

structure

[3].

The null

_hypotheses

tested in this

_study

were: that

ele-vated

_[CO,]

would have no effect on _gas

_exchange,

phe-nolics

and TNC

of live oak

_seedlings;

and that interac-tions of

_CO

₂

with soil resource limitations

_(N)

would

have no effect on these variables.

2.

Materials

and methods

2.1. Plant material and

_growth

conditions

Acorns of live oak were collected in late November

from three adult

_{(open-pollinated)}

trees

_growing

in the campus

gardens

of the

_University

of Florida

_(29°43’

N and 82°12’ W;

Gainesville, FL, USA).

Seeds of each tree were broadcast in individual _trays filled with

_growing

medium

_(mixture

of _peat,

_vermiculite,

_perlite

and

_bark)

and moistened

_regularly.

The containers

_subsequently

were

_placed

in a

_growth

chamber

_(day/night

_temperature,

25

°C;

day/night

relative

_humidity

[RH],

80

%;

photo-synthetic photon

flux

_density

_[PPFD],

800

_μmol·m

_-2

_.s

_-1

_;

photoperiod,

16

_h).

Germination took

_place

at ambient

[CO,]

level in the containers.

_{Seedlings emerged}

in all

trays after 10

_days.

After 2 weeks of

_growth

in the _trays, 40

_seedlings

_per

family

were

_transplanted

into black PVC containers

(Deepots®;

25 cm

_high

x 5.5 cm in

_averaged

internal

diameter,

600

_cm

₃

₎

and maintained in the

_growth

cham-ber. The tubes were filled with a mixture

_(v/v)

of 90 %

sand and 10 % _peat; a

_layer

of stones was

_placed

in the base of each tube.

_Seedlings

in the

_growth

chamber were

watered

_daily.

While

_plants

were

_growing

in the

_growth

chamber,

the first _stage of

_growth

was

_supported

_by

adding

commercial slow-release Osmocote

(18/18/18,

N/P/K);

the nutrient additions were

_given

in two

_pulses

of 3 _g

each,

applying

the first after 1 week of

_growth

in the tubes and the second after 6 weeks. Before

_moving

the

_seedlings

to the _open-top

chambers,

the

_superficial

in order to remove accumulated salts and nutrients.

During

the 1st month of

_growth

the

_seedlings

were

fumi-gated

twice with a commercial

_fungicide.

Four months after

_germination

₍₁₇

_March),

the

seedlings

were moved to six

_open-top

chambers. Each chamber received one of two

_CO

₂

treatments: ambient

[CO

2

]

or 150

μmol·mol

-1

_exceeding

ambient

_[CO,].

Details of the chamber characteristics and the

_CO

₂

treat-ment

_application

_may be found elsewhere

_{[20, 27].}

Overall mean

_[CO

₂

_]

was 370 or 520

_μmol·mol

_-1

at pre-sent or elevated

_CO

₂

concentrations

_(daytime),

respec-tively.

Ten

_days

after

_transferring

the

_plants

to the

_open-top

chambers,

two different nutrient solution treatments were initiated and

_seedlings

of each

_family

were

ran-domly

assigned

to a

_CO

₂

× nutrient solution treatment

combination.

_Thus,

the two

_CO

₂

treatments were

repli-cated three

times,

with the two nutrient solution treat-ments

_replicated

twice within each

_CO

₂

treatment. The

seedling

containers were assembled in racks and

wrapped

in aluminum foil to avoid root _system

_heating,

and set in

_trays

_{constantly containing}

a

_layer

of nutrient

solution to avoid desiccation and minimize nutrient

loss,

thus

_limiting

nutrient

_{disequilibrium}

_[24].

Plants were fertilized _{every 5}

_days

to saturation with

one of the two nutrient solutions obtained

_{by modifying}

a water-soluble Peters fertilizer

_{(Hydro-Sol®,}

Grace-Sierra

_Co.,

_Milpitas,

CA,

USA):

complete

nutrient

solu-tion

_{containing high}

N

₍₉₀

_μmol·mol

_-1

_NH

₄

_NO

₃

_),

or a

nutrient solution with low N

₍₂₀

_μmol·mol

_-1

_NH

₄

_NO

₃

_).

Both nutrient solutions contained

_[in

_μmol·mol

_-1

_]:

_PO

₄

(20.6),

K

_(42.2),

Ca

(37.8),

Mg

(6),

_SO

₄

(23.5),

Fe

_(0.6),

Mn

_(0.1),

Zn

_(0.03),

Cu

_(0.03),

B

_(0.1)

and Mo

_(0.02),

and were

_adjusted

to

_pH

5.5;

every 5 weeks

supplemen-tary Peters

_(STEM)

micronutrient elements

(0.05

g·L

-1

)

were added. Deionized water was added to saturation every other

day

in order to _prevent salt accumulation. Plant containers were moved

_frequently

in the chambers

to avoid

_positional

effects.

2.2. Gas

_exchange

Measurements of stomatal conductance

_(g

_s

₎

and C

exchange

rate

_(CER)

were made at the

_growth

_[CO

₂

_]

with a

_{portable gas-analysis}

_system

_(LI-6200,

Li-cor

Inc.,

Lincoln, NE, USA)

on

_{mid-canopy fully expanded}

leaves of the same

_stage

of

_development

of

_randomly

selected

_plants;

each time labeled leaves

(two

per

plant)

were measured twice. Measurements were

_performed

on

different occasions

_during

the

_{experiment, starting}

from

d 5 of _exposure

_{(after plant}

acclimation to the new

envi-ronment)

to d

178,

to

_investigate

the time-course of _gas

exchange.

Measurements of

_{daytime g}

_s

and

photosyn-thetic rate

_(A

_n

₎

were

_performed

under

_{saturating light}

conditions

_(PPFD

1 200-1 500

_μmol·m

_-2

_·s

_-1

_),

between 10:00 to 15:00 hours

_(temperature

25-35

_°C).

Measurements of dark

_respiration

_(R

_d

₎

were

_performed

on d 178

(CER

was measured before

_sunrise,

04:00-06:00

_hours).

Intrinsic water use

_efficiency

(WUE)

was calculated as

_A

_n

_/g

_s

_.

On several

occasions,

in

order to

_investigate

_daily

course

_during

_sunny

_days,

CER

were monitored from

_predawn

to dusk. Air

_temperature,

RH and PPFD in the leaf cuvette were

_kept

at

_growth

conditions.

Groups

of six different

_plants

were selected for

har-vest

_{(d 7)}

from each treatment for

_growth

measurements,

at the start of

_CO

₂

and nutrient treatments and continued

every 5-7 weeks until

_September.

Harvested

_plants

were

analyzed

for total

_phenolic

concentration

_{(fresh leaves)}

and total non-structural

_{carbohydrates}

after oven

_drying

plant

material at 65 °C to constant

_weight.

2.3. Phenolics

_analysis

Equal-aged

leaves

_(three

_per

_plant)

were taken for

total

_{phenolic compounds analysis.}

Leaves were treated in

_liquid

N at the field

_site,

then

_transported

to the

labo-ratory

and stored in the freezer at -20 °C until

_analysis.

The leaf blades were

_punched

on either side of the main

vein. Five

_punches

_{(0.2 cm}

2

_each)

_per leaf were

_analyzed

for

_{phenolics by modifying}

the insoluble

polymer-bond-ing procedure

of Walter and Purcell

_[55].

Other

_punches

from the

_remaining

leaf blades were used for

_{dry weight}

(DW) determination,

as described earlier. Leaf tissue

was

_homogenized

in 5.0 mL of hot 95 %

ethanol,

blend-ing

and

_boiling

for 1-2 min.

_Homogenates

were cooled

to room _temperature and

_centrifuged

at 12

_{000 g}

for 30 min at 28 °C.

_Supernatants

were decanted and

evaporat-ed to

_dryness

in N at 28 °C.

_Eight

milliliter

_aliquots

of the

_sample

in 0.1 M

_phosphate

buffer

_(KH

₂

_PO

₄

_,

_{pH 6.5)}

were mixed with 0.2 _g of Dowex resin

_(Sigma

Chemical

Co.,

St.

_{Louis, MO, USA)}

_{by agitating}

for 30 min

(200

g, 28

°C). Dowex,

a

_strong

basic

_{anion-exchange}

resin

_(200-400

_dry

_mesh,

medium

_porosity,

chloride ionic

_form),

was

_purified

before use

_{by washing}

with 0.1

N NaOH

solution,

distilled water and 0.1 N HCL

_and,

finally,

with distilled water. Absorbance at 323 nm

(A

323

)

was measured

_{spectrophotometrically}

both before

and after Dowex treatment,

representing

the absorbance

by phenolic compounds.

Phenolic concentration

_(mg·g

_-1

DW)

was determined from a standard curve

_prepared

with a series of

_chlorogenic

acid standards treated

simi-larly

to the tissue extracts and

_comparing

_changes

in

absorbance measured for the standards and those caused

2.4.

_{Carbohydrates}

_analysis

The amount of

TNC,

_including

starch and sugars, was

carried out

_using

the anthrone method.

_Previously

dried

plant

materials were

_separated

and

_ground

in a

_Wiley

mill fitted with 20 mesh screen.

_{Approximately}

100 _mg of

_finely

_ground

tissue were extracted three times in

boiling

80 %

_ethanol,

_centrifuged

and the _supernatant

pooled.

The

_pellet

was

_digested

at 40 °C for 2 h with

amyloglucosidase

from

_{Rhizopus (Sigma}

Chemical

_Co.)

and filtered. Soluble sugars and the

glucose

released from starch were

_quantified

_{spectrophotometrically}

fol-lowing

the reaction with anthrone.

2.5. Statistical

_analysis

Three-way analysis

of variance

_(ANOVA)

with

har-vest

time,

_[CO

₂

_]

and N

_availability

as the main effects was conducted for all

_{parameters except}

for those rela-tive to the last harvest date which were tested

_by

two-way ANOVA. Two- and/or

three-way

interactions were

included in the model.

3. Results

3.1. Gas

_exchange

All _gas

_exchange

_parameters showed variations

(P

<

_0.0001)

with the course of the

_growing

season and

the relative _stage of

_development

of the leaves

_(figure

1).

Periodic measurements

_throughout

the

_growing

sea-son indicated a consistent

_(P

<

_0.0001)

_pattern of

_higher

photosynthetic

rate in leaves _grown at

_higher

_[CO

₂

_]

(when

measured at the

_growth

environment;

figure

1 and

table

_I),

with the _greatest differences

_occurring

_by

the

end of the

_experiment.

Plants _{grown in low N} had lower

(P

<

_{0.0001 )}

_{photosynthetic}

rates when

_compared

with

high

N

_{plants (figure}

1 and table

I).

There was no

signif-icant interaction between N and

_CO,

treatment

_{(table I).}

The effects of N and

_CO,

treatment increased over time

(figure

1)

and the interaction between measurement date and N

_(P

<

_0.001)

or

_CO

₂

_(P

<

0.05)

treatment was

sig-nificant

_{(table I).}

Stomatal

conductance, overall,

was

_{significantly}

reduced

_(P

<

_0.0001)

at

_higher

_[CO,]

_(figure

1 and table

I),

although, by

the end of

_experiment,

the differences between

_CO

₂

treatments tended to be lower when

com-pared

with

the

other measurement dates. Nutrient

avail-ability

did not

_{significantly}

affect stomatal conductance

(figure

I and table

_I),

even if

_high

N

_plants

showed

high-er values

_by

the end of the

_experiment.

The increases in

_{photosynthetic}

rate and decreases in

stomatal conductance combined to increase

(about

dou-bled,

P <

0.0001)

leaf-level water use

_efficiency

with

[CO

2

]

at _every date measured

_(figure

I and table

_I).

Nutrient

_availability

had a

_significant

_(P

<

_0.0001)

and

positive

effect on intrinsic water use

_efficiency

_(figure

1

and table

_I)

and resulted in a

significant

_(P

<

0.05)

inter-action between N and

_CO

₂

treatment

_{(table I).}

The increase in leaf-level water use

_efficiency

with

increasing

_[CO

₂

_]

was confirmed

_{by examining}

the

_slopes

of the lines shown in the

_graph

of

_{photosynthetic}

rate

against

leaf conductance

_(figure

_2).

The

_regressions

between

_CO

₂

treatments were

_{significantly}

different

(P

<

_0.001)

and

showed a lack of acclimation of

photo-synthetic

rate under elevated

_CO,

concentration. The effect of N treatment on the

_{regression slope}

was less

evident.

The

_C

_i

_/C

_a

ratio intercellular

_[CO

₂

_]

to ambient

_[CO

₂

_]

ratio increased

(P

<

_0.0001)

in

_plants

_grown at

_higher

[CO

2

]

(figure

1 and table

_I).

N

_availability

had less effect

on

the

_C

_i

_/C

_a

ratio

_(figure

1 and table

_I).

the

_day

_(data

not

shown).

Plants grown at elevated

_[CO

₂

_]

had lower

_predawn

dark

_respiration

_regardless

of N

availability.

When leaves were stratified as either old

_(spring

leaves) or new

_{(summer leaves)}

and

_analyzed

as two

groups, new leaves had

_higher

_(P

<

0.0001)

photosyn-thetic rates

_{(25-30 %),}

_predawn

dark

_respiration

_(30-70

%)

and stomatal conductance

_(20-30

%),

regardless

of N

or

_CO

₂

treatment

_{(table II).}

Intrinsic water use

_efficiency

was not influenced

_{significantly}

_by

_{age. N}

_availability

significantly

(P

<

_0.001)

affected all _parameters but

predawn

dark

_respiration.

The

_latter,

in

_particular,

decreased 45 and 62 % in old and new

_leaves,

respec-tively,

in response to

_increasing

_[CO

₂

_].

3.2. Phenolics

Overall,

total

_{phenolic compound}

concentration was

increased

_{significantly}

_(P

<

_0.0001)

_by

elevated

_[CO

₂

_]

(figure

3 and table

I),

although

the increment was

much

more evident

_by

the end of the

_experiment

(35 %)

than

during

the

_previous

harvests. Harvest

date,

in

fact,

had clear influences on the

_phenolic

concentration

(P

<

_0.0001).

N

_availability

did not influence

_phenolic

concentration

_{significantly,}

and there were no

_significant

3.3.

_{Carbohydrates}

Generally,

soluble sugars, starch and TNC

concentra-tions were

significantly

affected

_by

time of harvest

(tables

III and

_IV).

However,

carbohydrate

concentration

was not

_{significantly}

affected

_by

both N and

_CO

₂

treat-ment

_(tables

III and

_IV).

_Although

the interactions

between harvest

_day

and

_CO

₂

_{(and N)}

treatment were

sometimes

_significant,

it is

_{not possible}

to

_identify

a

spe-cific trend. The effect of

_CO

₂

and N treatments on

carbo-hydrate

concentration in

the

_tap

and fine roots

_sampled

at the end of the

_experiment

was also not

_significant

(table

V).

4.

Discussion

Both

_atmospheric

_CO

₂

and nutrient

_{supply greatly}

affected the

_{photosynthetic}

rate of

_Q.

_virginiana

seedlings.

The increase in ambient

_[CO

₂

_]

elicited a simi-lar increase in

_{photosynthesis}

in both nutrient treatments

[45].

The

_higher

values of net assimilation rate at

_higher

N

_supply

are consistent with those

_reported

in other

stud-ies

[34, 43].

The effect of elevated

_[CO

₂

_]

on the

photo-synthetic

rate

_{persisted during}

the whole

_study

_period,

despite

reductions in N concentration

_[52].

The

_relatively

low starch content of leaves in all treatments

_might

sug-gest

that there was no limitation to

_{photosynthesis}

at

ele-vated

_[CO

₂

_]

_imposed

_by

excessive

_{carbohydrate loading.}

The absence of downward

_{photosynthetic}

acclimation is similar to the

_findings

of other studies on

_{woody species}

[2].

No downward trend of

_{photosynthesis}

was shown

through length

of exposure,

portion

of

_growing

season

and age of

foliage

[10,

19].

Declines in response to ele-vated

_[CO

₂

_]

have been

_reported

to occur in older

_foliage

[18, 21],

late

in the

_growing

season

_[44]

and after weeks of exposure to elevated

_[CO

₂

_]

_{[12, 48, 54].}

Our

_findings

contrast _{with responses in many}

_experiments

with

_potted

plants

[14, 47]

in which the observed

_declining

_response

changing

developmental

sink

_strength.

Samuelson and Seiler

_[49]

found that

_seedlings

of

_{Abies fraseri growing}

in 1 000

cm

3

_pots

showed no

_depression

in net

photosyn-thesis _{after 12 months of exposure} to elevated

_[CO

₂

_]

while in 172

cm

3

pots

photosynthetic

acclimation was

evident after 5 months.

_{Q. virginiana}

_seedlings

_{grew in} 600

cm

3

_pots for about 6 months. However, the

_large

tap-root, characteristic of

_seedlings

of this

_species,

showed a

_positive

_response to elevated

_[CO

₂

_]

and this

might

constitute an

_adequate

sink for additional C.

_CO

₂

stimulated

_growth

of all

_plant

_compartments

of

_Q.

vir-giniana

seedlings

(the

accumulation of total biomass increased 30-40 %

_by

the end of the

_experiment)

[52].

Greater C assimilation in _response to

_CO

₂

often stimu-lates new sinks for C

[23].

The diurnal measurements of

increase in C

_gain

was maintained in elevated

_[CO

₂

_]

[51].

There

was also no indication from the

_pattern

of

the

photosynthetic

rate over the course of the

_day

that there

was an accumulation of

_{carbohydrates}

in the afternoon in elevated

_[CO

₂

_]

_causing

_temporary feedback inhibition.

Stomatal conductance of

_{Q. virginiana}

_generally

decreased with

_CO,

enrichment in both N treatments

(less

evidently

at

the

end of the

_experiment).

Nutrient

availability

did not affect stomatal conductance

_except

for the last harvest

_day.

_{Stomatal response} to

_CO,

is a common

_phenomenon

and stomatal conductance in

many

plants

decreases in _response to

_increasing

atmos-pheric

[CO

2

]

[2, 9, 14],

despite

several documented

exceptions

[8,

19, 33].

At elevated

_[CO,],

intercellular

[CO

2

]

should rise if stomata close

_{consistently,}

conse-quently leading

to an increase in assimilation rate.

Indeed,

in

_{Q. virginiana}

_seedlings

the ratio of

intercellu-lar to

_atmospheric

_[CO

₂

_]

_{increased up} to 14 % at elevat-ed

_atmospheric

_[CO

₂

_].

As a result of increased assimilation rate and decreased stomatal

conductance,

water use

_efficiency

of

leaves increased

_strongly

at elevated

_[CO

₂

_]

in

_Q.

virgini-ana

_seedlings.

This increase is a common _response to

elevated

_{[CO,] [13, 14, 35].}

A

_significant

interaction

between

nutrient

_supply

and

_[CO,]

led to a

_higher

pro-portional

increase in water use

efficiency

in

_seedlings

The effect of nutrient

_supply

and

CO,

treatment on

assimilation rate and stomatal conductance did not

change

when

_spring

and summer leaves of

_Q.

_virginiana

were

_{compared, despite}

a

_large

effect of leaf _age

_(the

lat-ter not evident for water use

_efficiency).

This

_finding

may

support

the

_hypothesis

of a lack of acclimation of

gas

exchange

at elevated

_[CO

₂

_].

Dark

_respiration

as

mea-sured on

_spring

_(maintenance

_{respiration only)}

and

sum-mer leaves at the end of the

_experiment

was

_{significantly}

reduced

_{by [CO}

₂

_]

but not affected

_by

nutrient

_supply.

Dark

_respiration

was

affected

_by

_age, and the interaction

between

CO,

treatment and nutrient

_supply

was

signifi-cant,

resulting

in a

_larger

reduction due to

_CO

₂

treatment

in summer leaves

_{(recently expanded)}

in which the

growth respiration

component should be still

_important.

Direct

_(short-term)

and indirect

_(long-term)

inhibition of

respiration by

_CO

₂

is a _common,

_although

not universal

phenomenon

[1, 7].

Lower leaf N, and

_presumably

pro-tein,

was observed in

_{Q. virginiana seedlings}

_{[52] and,}

therefore,

it is

_possible

that the amount of _{energy needed} for leaf construction _may be reduced in elevated

_[CO,]

relative to ambient

_{[CO,] [56]. However,}

reduced

leaf N

concentration in

_plants

_grown

at elevated

_[CO

₂

_]

does not

necessarily

indicate

_parallel

differences in construction

costs

_[57].

Q. virginiana seedlings

were

_{using photosynthates}

mainly

for

_growth

_[52]

and thus non-structural

carbohy-drates

_(sugars

and

_starch)

did not accumulate in _any

plant

compartment. Soluble sugars and starch

concentra-tions in stem and roots have

_already

been found not to

increase in other

_experiments

_{[4, 28].}

In contrast with

our

_findings,

starch and total non-structural

_carbohydrate

accumulation in

_foliage

_(and

other

_{compartments)}

of

plants

grown at elevated

_[CO

₂

_]

is a much more common

phenomenon

[2, 42, 43],

although

it has been

_reported

to

be a _strong

_{species-specific}

_response

_{[29, 50].}

We

sam-pled

the

_plant

material in the afternoon and

_Wullschleger

et al.

_[56]

found no

_large

differences between ambient

[CO

2

]

and ambient + 150

_&mu;mol·mol

_-1

_[CO

₂

_]

_(a

similar

CO,

treatment to that used in our

_experiment)

in starch

and

sucrose of leaves of

_{yellow poplar}

and white oak

seedlings

collected in the

_evening.

The response of foliar

phenolic

concentration to

_CO

₂

enrichment has been found to be variable

_{[28, 31, 41]. In}

our

_experiment

the

_CO

₂

effect on

_{increasing phenolic}

concentration took

_place

without a

_parallel

increase in

total non-structural

_{carbohydrates}

at elevated

_[CO,]

that otherwise would have

_presumably

diluted

_phenolics.

An

increase in the C/N

_ratios,

which also occurred in our

plant

material

_[52],

due to a decrease in N content in

seedlings

grown under elevated

_[CO

₂

_],

is in accordance with increases in C-based

_compounds

_[32].

The increased foliar

_phenolic

concentration in

_conjunction

with increased C/N ratios may alter the

performance

of

herbivores of

_Q.

_virginiana

in the

_{regeneration phase,}

in view of

_projected

increases in

_atmospheric

_[CO

₂

_].

Foliar

phenolics

decreased

_following

leaf maturation

_[28].

Nutrient treatment did not affect

_phenolic

concentration. This is in contrast with the C-nutrient balance

_hypothesis

[6],

which

_predicts

that

_{plants adjust}

_{physiologically}

to

low nutrient

_{availability by reducing growth}

rate and

showing

a

_high

concentration of

_secondary

metabolites.

Nevertheless,

several _{different responses} to

_CO

₂

enrich-ment

_reported

in the literature and nutrient

_availability

effects on C-based

_{secondary compounds}

are in _apparent

contradiction with the C-nutrient balance

_hypothesis

[28].

It is

_possible

that when

_growth

is

_suppressed

under

insufficient N

_supply

conditions for new tissue

forma-tion,

recycling

of the

_enzymatic

N

_required

for

sec-ondary

metabolism _may occur,

making

increased

pheno-lic accumulation

_possible

_[28].

The _{lack of response} found in the _present

_study

can be attributed to the low N

treatment not

_being

_sufficiently

_{growth limiting}

_[52].

Results from this

_study

_suggest that the establishment

and

_growth

of

_{Q. virginiana}

on sites with poor nutrition

will benefit

_{substantially}

from elevated

_[CO

₂

_]

as a result

of more C

_gain.

The sustained increase in

_{photosynthetic}

rate,

coupled

with decreased dark

_respiration

in elevated

[CO

2

],

provides

the

_potential

for increased C

_acquisition

by the

whole crown. Raised

_[CO

₂

_]

may have a real

impact

on the defensive

_chemistry

of

_{Q. virginiana}

seedlings.

Acknowledgement:

The technical assistance of D.

Noletti is

_{greatly appreciated.}

References

[1] Amthor J.S.,

Respiration

in a _future,

_higher

_CO,

_world, Plant Cell Environ. 14 (1991) 13-20.

[2] Amthor J.S., Terrestrial

_higher-plant

_response to

increasing atmospheric

[CO,] in relation to

_global

carbon

cycle,

Global

_Change

Biol. 1

(1995)

243-274.

[3] Bazzaz F.A., Miao S.L., Successional status, seed size,

and responses of tree

_seedlings

to

_CO,,

_light,

and _nutrients,

Ecology

74 ₍₁₉₉₃₎ 104-112.

[4]

Bhattacharya

N.C.,

Bhattacharya

S., Strain _B.R., Biswas P.K., Biochemical

_changes

in

_{carbohydrates}

_and pro-teins of sweet _potato

_{plants (Ipomea}

batatas _{[L.] Lam.)} in response to enriched _CO, environment at different _stages of

growth

and

_development,

J. Plant

_Physiol.

135 ₍₁₉₈₉₎ 261-266.

[5] Brown K.R., Carbon dioxide enrichment accelerates the

[6]

Bryant

J.P., Feltleaf willow-snowshoe hare

interac-tions:

_plant

carbon/nutrient balance and

_foodplain

_succession,

Ecology

68 (1987) 1319-1327.

[7] Bunce _J.A., Short- and

_long-term

inhibition of

respira-tory carbon dioxide efflux

_by

elevated carbon dioxide, Ann. Bot. 65 (1990) 637-642.

[8] Bunce J.A., Stomatal conductance,

photosynthesis

and

respiration

of _temperate deciduous tree

_seedlings

grown

out-doors at elevated concentration of carbon dioxide, Plant Cell Environ. 15 ₍₁₉₉₂₎ 541-549.

[9] Ceulemans R., Mousseau M., Effects of elevated

atmos-pheric

CO

2

on

_{woody plants,}

New

_Phytol.

127 ₍₁₉₉₄₎ 425-446.

[10] Ceulemans R.,

Taylor

G., Bosac C., Wilkins D.,

Besford R.T.,

Photosynthetic

acclimation to elevated

_CO

₂

_in

poplar

grown in

glasshouse

cabinets or _{in open top} chambers

depends

on duration of exposure, J.

Exp.

Bot. 48 (1997) 1681-1689.

[11] Coleman _J.S.,

_McConnaughay

K.D.M., Bazzaz _F.A., Elevated

_CO

₂

and

_{plant nitrogen-use:}

is the tissue

_nitrogen

con-centration

_{size-dependent?, Oecologia}

93 ₍₁₉₉₃₎ 195-200.

[12] De Lucia E.H,. Sasek T.W., Strain B.R.,

Photosynthetic

inhibition after

_long-term

_exposure to elevated levels of

_CO

₂

_,

_Photosynth.

Res. 7 (1985) 175-184.

[13] Eamus D., The interaction of

_rising

_CO, and

tempera-tures with water use

_efficiency,

Plant Cell

Environ.

14 ₍₁₉₉₁₎ 843-852.

[14] Eamus D., Jarvis P.G., The direct effects of increase in the

_{global atmospheric CO,}

concentration on natural and

com-mercial temperate trees and forests, Adv. Ecol. Res. 19 (1989)

1-55.

[15] El Kohen _A., Mousseau M., Interactive effects of ele-vated

_CO

₂

and mineral nutrition on

_growth

and _CO,

_exchange

of sweet chestnut

_seedlings

_{(Castanea sativa),} Tree

_Physiol.

14

(1994) 679-690.

[16]

Fajer

E.D., Bowers M.D., Bazzaz F.A., The effects of enriched CO,

atmospheres

on the

_{buckeye butterfly,}

Junonia

coenia,

Ecology

72 (1989) 751-754.

[17] Griffin _K.L., Thomas _R.B., Strain B.R., Effetcts of

nitrogen supply

and elevated carbon dioxide on construction

cost in leaves of Pinus taeda _(L.)

_{seedlings, Oecologia}

95

(1993) 575-580.

[18] Gunderson C.A.,

Wullschleger

S.D.,

Photosynthetic

acclimation in trees to

_{rising atmospheric}

_CO,:

a _broader

per-spective, Photosynth.

Res. 39 (1994)

369-388.

[19] Gunderson C.A.,

Norby

R.J.,

Wullschleger

S.D.,

Foliar gas

exchange

responses of two deciduous hardwoods

during

3 years of

_growth

in elevated _CO,: no loss of

photosyn-thetic enhancement, Plant Cell Environ.

16

(1993) 797-807.

[20]

Heagle

A.S., Philbeck R.B., Ferrell R.E., Heck W.W.,

Design

and

_performance

of a

_large,

_{field exposure chamber} to

measure effects of air

_quality

on

_plants,

J. Environ. Qual. 18

(1989) 361-368.

[21] Hicklenton _P.R., Jolliffe _P.A., Alterations in the

physi-ology

of

_CO,

_exchange

in tomato

_plants

grown in CO2

-enriched

_atmospheres,

Can. J. Bot. 58 ₍₁₉₈₀₎ 2181-2189.

[22]

Hollinger

D.Y., Gas

_exchange

and

_dry

matter alloca-tion response to elevation to

_atmospheric

CO, concentration in

seedlings

of three tree

_species,

Tree

_Physiol.

_{3 (1987)}

193-202.

[23] Idso S.B., Kimball B.A., Allen S.G.,

_CO,

enrichment of sour _orange trees: _{2.5 years into} a

_long

term

_experiment,

Plant Cell Environ. _{14 (1991) 351-353.}

[24]

Ingestad

T., Relative addition rate and external con-centration:

_driving

variables used in

_plant

nutrition research,

Plant Cell Environ. 5 (1982) 443-453.

[25] Johnsen K.H., Growth and

_{ecophysiological}

_responses of black spruce

seedlings

to elevated CO2 under varied water

and nutrient additions, Can. J. For. Res.

_{23 (1993)}

1033-1042.

[26] Johnson D.W.,

Nitrogen

retention in forest soils, J. Environ. Qual. 21 (1992) 1-12.

[27] Johnson J.D., Allen E.R.,

Hydrocarbon

emission from southern

_pines

and the

_potential

effect of

_global

climate

change,

in: Final Technical

_Report,

SE

_Regional

Center

-NIGEC, Environmental Institute Publication No. 47, The

University

of Alabama, Tuscaloosa, AL, USA, 1996; 26 p.

[28] Julkunen-Tiitto R., Tahvanainen J., Silvola J.,

Increased CO, and nutrient status

_changes

affect

_phytomass

and the

_production

of

_plant

defensive

_secondary

chemicals in Salix

_myrsinifolia

(Salisb.),

Oecologia

95 (1993) 495-498.

[29] Körner C.,

Miglietta

F.,

_Long

term effects of

_naturally

elevated

_CO,

on Mediterranean

_grassland

and forest trees,

Oecologia

99 (1994)

343-51.

[30] Lambers H.,

Rising

CO

2

,

secondary plant

metabolism,

plant-herbivore

interactions and litter

_{decomposition.}

Theoretical considerations,

Vegetatio

104/105 ₍₁₉₉₃₎ 263-271.

[31] Lavola A., Julkunen-Tiitto _R., The effect of elevated carbon dioxide and fertilization on

_primary

and

_secondary

metabolites in birch, Betula

_pendula

(Roth.),

Oecologia

99

(1994) 315-321.

[32] Lawler I.R.,

_Foley

W.J., Woodrow I.E., Cork S.J., The effects of elevated CO,

atmospheres

on the nutritional

_quality

of

_{Eucalyptus foliage}

and its interaction with soil nutrient and

light availability, Oecologia

109 (1997) 59-68.

[33] Liu S.,

Teskey

R.O.,

_Responses

of foliar gas

exchange

to

_long-term

elevated

_CO

₂

_{concentrations}

in mature

_loblolly

pine

trees, Tree

Physiol.

15 ₍₁₉₉₅₎ 351-359.

[34] McDonald A.J.S., Lohammar T.,

Ingestad

T., Net assimilation rate and shoot area

_development

in birch _(Betula

pendula

Roth.) at different

_steady-state

values of nutrition and

photon

flux

_density,

Trees 6 (1992) 1-6.

[35]

Norby

R.J., O’Neill E.G., Growth

_dynamics

and water

use of

_seedlings

of _Quercus alba L. in

_CO

₂

_-enriched

atmos-pheres,

New

_Phytol.

111 (1989) 491-500.

[36]

Norby

R.J., O’Neill E.G., Leaf area

_compensation

and nutrient interactions in CO2-enriched

seedlings

of

_yellow

poplar

(Liriodendron

tulipifera

L.), New

_Phytol.

117 ₍₁₉₉₁₎ 515-528.

[37]

Norby

R.J., Pastor J., Melillo _J.M.,

_{Carbon-nitrogen}

interactions in

_CO

₂

_-enriched

white oak:

_{physiological}

and

long-term perspectives,

Tree

_Physiol.

2 ₍₁₉₈₆₎ 233-241.

[38]

Norby

R.J., O’Neill _E.G., Luxmoore R.J., Effects of

nutri-tion of _Quercus alba

_seedlings

in nutrient poor soil, Plant

Physiol.

82 (1986) 83-89.

[39]

Norby

R.J., Gunderson C.A.,

Wullschleger

S.D.,

O’Neill E.G., McCracken _M.K.,

_Productivity

and compensato-ry responses of

yellow-poplar

trees in elevated

_CO

₂

_,

_Nature

357 (1992) 322-324.

[40] Picon C., Guehl J.-M., Aussenac G., Growth

dynam-ics,

transpiration

and water-use

_efficiency

in _Quercus robur

plants

submitted to elevated

_CO

₂

_and

_drought,

Ann. Sci. For.

53 (1996) 431-446.

[41] Pe&ntilde;uelas J., Estiarte _M., Kimball B.A., Idso S.B.,

Pinter Jr P.J., Wall _G.W., Garcia _R.L., Hansaker D.J., LaMorte

R.L., Hendrix D.L.,

Variety

of responses of

plant phenolic

con-centration to

_CO

₂

_enrichment, J.

_Exp.

Bot. 47 (1996)

1463-1467.

[42] Pettersson R., McDonald _J.S., Effects of elevated car-bon dioxide concentration on

_{photosynthesis}

and

_growth

of small birch

_plants

(Betula

pendula

Roth.) at

_optimal

_nutrition,

Plant Cell Environ. 15 (1992) 911-919.

[43] Pettersson R., McDonald A.J.S.,

Stadenberg

I.,

Response

of small birch

_plants

(Betula

pendula

Roth.) to ele-vated

_CO

₂

_and

_{nitrogen supply,}

Plant Cell Environ. 16 (1993)

1115-1121.

[44] Radin J.W., Kimball _B.A., Hendrix _D.L.,

_Mauney

J.R.,

Photosynthesis

of cotton

_{plants exposed}

to elevated levels of carbon dioxide in the _field,

_Photosynth.

Res. 12 (1987)

191-203.

[45]

Radoglou

K.M.,

Aphalo

P., Jarvis _P.J.,

_Response

of

photosynthesis,

stomatal conductance and water use

_efficiency

to elevated

_CO

₂

and nutrient

_supply

in acclimated

_seedlings

of Phaseolus

_vulgaris

L., Ann. Bot. _70(1992) 257-264.

[46] Reichardt _P.B.,

_Chapin

F.S. III,

Bryant

J.P., Mattes

B.R., Clausen T.P., Carbon/nutrient balance as a

_predictor

of

plant

defence in Alaskan balsam

_{poplar: potential importance}

of metabolite _turnover,

_Oecologia

88 (1991) 401-406.

[47]

Sage

R.F., Acclimation of

_{photosynthesis}

to

_increasing

atmospheric

CO

2

:

the gas

exchange perspective, Photosynth.

Res. 39 ₍₁₉₉₄₎ 351-368.

[48]

Sage

R.F.,

Sharkey

T.D., Seemann _J.R., Acclimation of

_{photosynthesis}

to elevated

_CO

₂

_in

five

_C

₃

_species,

Plant

Physiol.

89 ₍₁₉₈₉₎ 590-596.

[49] Samuelson _L.J., Seiler J.R., Fraser fir

_seedling

_gas

exchange

and

_growth

_{in response} to elevated

_CO

₂

_,

Environ.

Exp.

Bot. 32 ₍₁₉₉₂₎ 351-356.

[50]

Schäppi

B., Körner Ch., In situ effects of elevated

_CO

₂

on the carbon and

_nitrogen

status of

_{alpine plants,}

Funct. Ecol. 11 _{(1997) 290-299.}

[51]

Teskey

R.O., A field

_study

of the effects of elevated

CO

2

on carbon assimilation, stomatal conductance and leaf and branch

_growth

of Pinus taeda trees, Plant Cell Environ. 18

(1995) 565-573.

[52]

Tognetti

R., Johnson J.D.,

Responses

of

_growth,

nitro-gen and carbon allocation to elevated

_atmospheric

_CO

₂

concen-tration in live oak _{(Quercus virginiana Mill.)}

_seedlings

in rela-tion to nutrient

_supply,

Ann. Sci. For. 56 (1999) 91-105.

[53] Vivin P., Gross P., Aussenac _G., Guehl _J.-M.,

Whole-plant

_CO

₂

exchange,

carbon

_partitioning

and

_growth

in

Quercus robur

_{seedlings exposed}

to elevated

_CO

₂

_,

Plant

Physiol.

Biochem. 33 (1995) 201-211.

[54] Vivin P., Martin F., Guehl _J.-M.,

_Acquisition

and

within-plant

allocation of 13C and 15N in

_CO

₂

_-enriched

Quercus robur

_{plants, Physiol.}

Plant. 98 (1996) 89-96.

[55] Walter Jr. W.M., Purcell A.E., Evaluation of several methods for

_analysis

of sweet potato

phenolics,

J.

_Agric.

Food Chem. 27 (1979) 942-946.

[56]

Wullschleger

S.D.,

Norby

R.J., Hendrix D.L., Carbon

exchange

rates,

chlorophyll

content, and

carbohydrate

status of

two forest tree

_{species exposed}

to carbon dioxide enrichment,

Tree

_Physiol.

_{10 (1992) 21-31.}

[57]

Wullschleger

S.D., Ziska L.H., Bunce J.A.,

Respiratory

responses of

higher plants

to

_atmospheric

_CO

₂