Responses to elevated atmospheric CO2 concentration and nitrogen supply of Quercus ilex L. seedlings from a coppice stand growing at a natural CO2 spring

HAL Id: hal-00883298

https://hal.archives-ouvertes.fr/hal-00883298

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.

Responses to elevated atmospheric CO2 concentration

and nitrogen supply of Quercus ilex L. seedlings from a

coppice stand growing at a natural CO2 spring

Roberto Tognetti, Jon D. Johnson

To cite this version:

Original

article

Responses

to

elevated

_atmospheric

_CO

₂

concentration

and

_nitrogen

_supply

of

_Quercus

ilex

L.

_seedlings

from

a

coppice

stand

_growing

at

a

natural

CO

2

spring

Roberto

_Tognetti*

Jon

D. Johnson*

School of Forest Resources and Conservation,

University

of Florida, 326

_{Newins-Ziegler}

Hall, Gainesville, FL, 2611, USA

(Received 15

_September

_1998;

_accepted

1 March ₁₉₉₉₎

Abstract - Quercus ilex acorns were collected from a

_population

of trees with a lifetime exposure to elevated

_atmospheric

_CO

₂

con-centration

_(CO

₂

_),

and after

_{germination seedlings}

were

_exposed

at two

_]

₂

_[CO

(370 or 520

_{μmol mol}

-1

)

in combination with two soil N treatments (20 and 90

μmol

mol-1total N) in _open-top chambers for 6 months.

_Increasing

_[CO

₂

_]

stimulated

_{photosynthesis}

and leaf

dark

_{respiration regardless}

of N treatment. The increase in

_{photosynthesis}

and leaf dark

_respiration

was associated with a moderate reduction in stomatal conductance,

resulting

in enhanced instantaneous

_{transpiration efficiency}

in leaves of

_seedlings

in

_CO

₂

-enriched air. Elevated

_[CO

₂

_]

increased biomass

_{production only}

in the

_high-N

treatment. Fine

_root/foliage

mass ratio decreased with

high-N

treatment and increased with

_CO

₂

enrichment. There was evidence of a

_preferential

shift of biomass to

_below-ground

tissue at

a low level of nutrient addition.

_Specific

leaf area _(SLA) and leaf area ratio _(LAR) decreased

_{significantly}

in leaves of

_seedlings

grown in elevated

_[CO

_2]

irrespective

of N treatment. Leaf N concentration decreased

_{significantly}

in elevated

_[CO

₂

_]

_irrespective

of N

treatment. As a result of _patterns of N and carbon concentrations, C/N ratio

_generally

increased with elevated

_[CO

₂

_]

treatment and decreased with

_high

nutrient

_supply.

Afternoon starch concentrations in leaves did not increase

_{significantly}

with

_increasing

_[CO

₂

_],

as was the case for

_morning

starch concentrations at low-N

_supply.

Starch concentrations in _leaves, stem and roots increased with elevated

_[CO

₂

_]

_{and decreased with nutrient addition. The concentration of sugars} was not

_{significantly}

affected

_by

either

_CO

₂

or N

treatments. Total foliar

_phenolic

concentrations decreased in

_seedlings

_{grown in elevated}

_[CO

₂

_]

_irrespective

of N treatment, while nutrient

_supply

had less of an effect. We conclude that available soil N will be a

_{major controlling}

resource for the establishment and

growth

of

_Q.

ilex in

_rising

_[CO

₂

_]

conditions. © 1999

_{Éditions scientifiques}

et médicales Elsevier SAS. carbon

_physiology

/ elevated

_[CO

₂

_]

/ natural

_CO

₂

_springs

/

_nitrogen

/

_Quercus

ilex

Résumé -

Réponses

de

_{jeunes plants}

de

_Quercus

ilex L. issus d’une

_population

_poussant dans une zone naturellement enrichie

en

_CO

₂

_,

à une concentration élevée de

_CO

₂

dans l’air et à un _apport d’azote. Des

_{jeunes plants}

de _Quercus ilex _L., issus d’une

population

d’arbres _ayant

_poussé

dans une concentration élevée de

_,

₂

_CO

ont été

_exposés

à deux concentrations en

_CO

₂

(370

μmol

mol-1 ou 520

_{μmol mol}

_-1

₎

en combinaison avec deux fertilisations du sol en azote (20 et 90

_μmol

mol-1N total) dans des

chambres à ciel ouvert

_pendant

six mois.

_{L’augmentation}

de concentration en

_CO

₂

stimule la

_{photosynthèse}

et la

_respiration

nocturne

des feuilles

_{indépendamment}

du traitement en azote. Les

_{augmentations}

de

_{photosynthèse}

et de la

_respiration

nocturne des feuilles ont été associées à une réduction modérée de conductance

_stomatique,

_{ayant pour résultat}

_{d’augmenter}

l’efficience

_{transpiratoire}

instantanée des feuilles des

_{jeunes plants}

cultivés en

_CO

₂

élevé.

_{L’augmentation}

de concentration du

_CO

₂

accroît la

_production

de

biomasse seulement dans le traitement élevé en azote. Le _rapport des racines fines à la masse de

_feuillage

a diminué avec le

traite-*

Correspondence

and

_reprints:

_{Istituto per}

_{l’Agrometeorologia}

e l’Analisi Ambientale

_{applicata all’Agricoltura, Consiglio}

Nazionale

delle Ricerche, via

_Caproni

8, Firenze, 50145,

Italy

** _{Present address: Intensive}

Forestry Program, Washington

State

_University,

7612 Pioneer _Way E.,

Puyallup,

WA-98371-4998,

USA

ment en azote et a

_augmenté

avec l’enrichissement en

₂

_CO

.La surface

_spécifique

de feuille _(SLA) et les taux de la surface de feuille

(LAR) ont diminé de manière

_{significative}

pour les feuilles des

jeunes plants développés

sous une concentration élevée de

_CO

₂

_,

indépendamment

du traitement en azote. La concentration en azote des feuilles a diminué de manière

_{significative}

dans le traitement élevé en

_CO

₂

_,

_{indépendamment}

du traitement en azote. En raison des

_{configurations}

des concentrations d’azote et de _carbone, le taux

C/N a

_augmenté

avec le traitement élevé en

_CO

₂

_et

diminué avec

_l’apport

d’azote. Dans

_{l’après-midi,}

les concentrations en amidon des feuilles n’ont pas

augmenté

de manière

significative

avec

_{l’augmentation}

du

_CO

₂

_,

comme _{pour les concentrations} en amidon dans le

cas du traitement limité en azote du matin. Les concentrations en amidon dans les _feuilles, la

_tige

et les racines ont

_augmenté

dans le

cas du traitement avec une concentration élevée en

_CO

₂

_et

diminué avec

_l’apport

en azote. Les concentrations en sucre _{n’ont pas été}

affectées sensiblement par les traitements de

CO

2

ou de N. Les concentrations

_phénoliques

foliaires totales ont _{diminué pour les}

jeunes plants qui

ont

_poussé

dans le traitement en

_CO

₂

_élevé,

_{indépendamment}

du traitement en _{N. Nous concluons que la}

disponibil-ité en azote dans le sol

_jouera

un rôle

_majeur

dans l’établissement et la croissance de _Q. ilex dans un _{environnement caractérisé par} un

accroissement de la concentration en

_CO

₂

dans l’air. © 1999

Éditions scientifiques

et médicales Elsevier SAS.

azote /

_CO

₂

élevé /

_physiologie

du carbone /

_Quercus

ilex / sources naturelles de de

_CO

₂

1.

Introduction

Atmospheric

carbon dioxide concentration

_[CO

₂

_]

is

currently

increasing

at a rate of about 1.5

_μmol

mol

-1

annually [52],

as a result of

increasing

fossil fuel

con-sumption

and deforestation.

Moreover,

models of future

global change

are in

_general

_agreement

_predicting

levels

reaching

600-800

_μmol

mol

-1

_by

the end of next

_century

from

_present

levels

_ranging

from 340 to 360

_μmol

mol

-1

[12].

CO

2

-enriched

atmospheres

have been shown to

increase

_{photosynthetic}

carbon

_gain,

the

_growth

of

_plants

and concentrations of total non-structural

_{carbohydrates,}

although

there is evidence of

_{species-specific}

responses

(see

reviews

_{[1, 2, 7, 16, 42]).}

The

_impact

of increased

[CO

2

]

on

_plant

_growth

is modified

_by

the nutrient level:

growth

enhancement in elevated

_[CO

₂

_]

has often been shown to decline under nutrient stress.

Indeed,

enhanced

growth

may increase

plant

nutrient

requirement,

but many Mediterranean sites are considered to have low

nitrogen (N) availability.

On the other

_hand,

it has been

proposed

that

_{plants adjust physiologically}

to low

nutri-ent

_{availability by reducing growth}

rate and

_showing

a

high

concentration of

_secondary

metabolites

[5].

The carbon-nutrient balance

_{hypothesis predicts}

that the

availability

of excess carbon at a certain nutrient level leads to the increased

_production

of carbon-based

sec-ondary

metabolites and their _precursors

_[39].

For

instance,

the often observed increase in C/N ratio under elevated

_[CO

₂

_]

has led some authors to suggest that

[CO

2

]

increases

_{might produce changes}

in the

concentra-tion of carbon-based

_{secondary compounds [29],}

thus

affecting plant-herbivore

interactions.

_Changes

in N

availability

may also alter per se the concentrations of

carbon-based

_secondary

chemicals

_[18].

A

_major

effect of

_CO

₂

_-enriched

_atmospheres

is the

reduction in the N concentration of

_plant

_tissues,

which

has been attributed to

_{physiological changes}

in

_plant

N use

_efficiency

_(e.g.

_[4,

_31]).

On the other

_hand,

there is

increasing

evidence that reductions in tissue N

concen-trations of elevated

_CO

₂

_-grown

_plants

is

_probably

a

size-dependent

phenomenon resulting

from accelerated

_plant

growth [10, 46].

It has also been documented that

reduc-tions in

_plant

tissue N concentrations under elevated

[CO

2

]

may

substantially

alter

_{plant-herbivore}

interac-tions

[30]

as well as litter

_{decomposition}

_[13].

In

fact,

insect herbivores consume _greater amounts of elevated

CO

2

-grown

foliage apparently

to

_compensate

for their

reduced N

concentration;

_again,

litter

_{decomposition}

rates _{may be slower} in elevated

_[CO

₂

_]

environments because of the altered balance between N concentrations and fiber contents.

Quercus

ilex L. is the

_{keystone species}

in the Mediterranean environment.

_Q.

ilex

forests,

once

domi-nant, have shrunk as a result of fires and

_exploitation

for firewood and timber over _{thousands of years. The}

_ability

of

_Q.

ilex to

_compete

at the

_ecosystem

level as

_[CO

₂

_]

continues to increase is of concern. _{While many} studies have looked at

_seedling

_response to elevated

_[CO

₂

_],

nothing

is known _{of progeny of} trees

_growing

for

_long

term in a

_CO

₂

_-enriched

_{atmosphere. Extrapolation}

from

studies on

_{seedlings growing}

in elevated

_[CO

₂

_]

to mature trees should be made

_only

with extreme caution.

However,

the

_seedling

_{stage represents}

a time

character-ized

_{by high genetic diversity,}

_great

_competitive

selec-tion and

_high

_growth

rates

[7]

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

develop-ment _may result in a

_large

difference in size of individu-als in the successive _years, thus

_determining

forest

com-munity

structure

_[3].

As the increase in

_plant

_productivity

_{in response} to

rising

[CO

2

]

is

_largely

dictated

_{by photosynthesis,}

incorporation

of the latter into biomass

[24],

the

objec-tives of this

_study

were

_i)

to

_investigate

how

_CO

₂

avail-ability

alters

_whole-plant

tissue N

_{concentration, ii)}

to

examine the effects of increased

_[CO

₂

_]

on carbon

alloca-tion to the

_production

of

_biomass,

total

_phenolic

com-pounds

and TNC

_(total

non-structural

_{carbohydrates,}

starch

_{plus sugars),}

and

_{finally iii)}

to determine how ele-vated

_[CO

₂

_]

_{influences gas}

_exchange

rate _{in progeny of}

Q.

ilex trees

_growing

in a

_CO

₂

_-enriched

environment under two different levels of N. The

_parent

trees _{grow in}

poor soil nutrient conditions under

long-term

CO

2

enrichment and their carbon

_physiology

has been the

object

of a

previous study [48].

We

hypothesized

that the

juvenile

stage would behave like acclimated

parent

trees

when grown in

_similarly

_poor soil nutrient conditions.

2. Materials and methods

2.1. Plant material and

_growth

conditions

Acorns of

_Q.

ilex were collected in December from

adult

_{(open-pollinated)}

trees,

growing

in the

_proximity

of the natural

_CO

₂

_spring

of Bossoleto and which have

spent

their entire lifetime under elevated

_[CO

₂

_];

the

_CO

₂

vent is located in the

_vicinity

of

_Rapolano

Terme near

Siena

_{(Italy) (for}

details see

_[28]).

Seeds were

immedi-ately

sent to USA and sown in PVC

pipe

tubes

(25

cm

height

x 5.5 cm

_averaged

internal

_diameter,

600

cm

3

).

After

_germination,

_seedlings

were thinned to one _{per pot.} The tubes were filled with a mixture

_(v/v)

of 90 % sand

and 10 %

_peat,

a

_layer

of stones was

_placed

at the base of each tube. 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 one

_pulse

of 2 g,

applied

after 1 month of

_growth

in the tubes. Soil nutrients in terrestrial _systems

_suggest

that N

mineraliza-tion is sometimes limited to short

_periods

_early

_in

grow-ing

season;

furthermore,

by

giving

an initial

_pulse

of

nutrients,

we created a situation in which

_plant

require-ments for nutrients were

_increasing

_(due

to

_growth)

while

_supply

was

_{decreasing (due}

to

_{uptake) [10],}

a

phe-nomenon that _may occur

_particularly

in natural _systems

low in soil N.

_During

the first month of

_growth

(January

1995)

the

_seedlings

were

_fumigated

twice with a

com-mercial

_fungicide.

Two hundred and

forty

seedlings

were _{grown for}

6 months in six

_open-top

chambers located at the School

of Forest Resources and

_{Conservation, University}

of

Florida,

Austin

_Cary

_Forest,

_{approximately}

10 km

north-east of Gainesville. Each chamber received one of two

CO

2

treatments: ambient

_[CO

₂

_]

or 150

_μmol

mol

-1

exceeding

ambient

_[CO

₂

_].

The chambers were 4.3 m tall and 4.6 m in

diameter,

covered with clear

polyvinylchlo-ride film and fitted with rain-exclusion

_tops.

Details of the chamber characteristics _{may be} found in

_[23].

The

CO

2

,

supplied

in

_liquid

form that

_vaporized

_along

the copper

supply

tubes,

was delivered

through

metering

valves to the fanboxes of three chambers. The

_CO

₂

treat-ment was

applied

during

the 12 h

(daytime)

the fans

were

_running

with

_{delivery being}

controlled

_by

a

sole-noid valve connected to a timer. The

_CO

₂

was delivered for about 15 min after the fans were turned off in the

evenings

in order to maintain

_higher

concentrations in the chambers. The

_[CO

₂

_]

was measured

_continuously

in

both the ambient and elevated

_[CO

₂

_]

chambers

_using

a

manifold system in

_conjunction

with a bank of solenoid

valves that would _step

_through

the six chamber

_sample

lines _{every 18} min. Overall mean

_daily

_[CO

₂

_]

for the

above treatments was 370 or 520

_μmol

mol

-1

at _present

or elevated

_[CO

₂

_],

respectively

(for

details see

_[26]).

The

[CO

2

]

during

the

_night

remained

_higher

in the

_CO

₂

-enriched

chambers,

since the fans were turned

_off,

avoid-ing

air

_mixing.

At the

_beginning

of March

_(1995),

two different

nutri-ent solution treatments were initiated. Within a

_chamber,

equal

numbers of

_{pots (21)}

were

_randomly

assigned

to a

high-

or low-N treatment. Before

_starting

the nutrient treatment, the

_superficial

layer

of Osmocote was

removed from the tubes and the latter flushed

_repeatedly

for 1 week with deionized water in order to remove

accumulated salts and nutrients. 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

desicca-tion and minimize nutrient loss

_limiting

nutrient

disequi-librium

_[25].

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.,

Yosemite Drive

Milpitas, CA, USA):

com-plete

nutrient solution

_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

_PO

₄

(20.6

μmol

mol

-1

),

K

_{(42.2 μmol}

_mol

_-1

_),

Ca

_(37.8

_μmol

mol

-1

),

Mg (6 μmol

mol

-1

),

SO

4

(23.5

μmol

mol

-1

),

Fe

(0.6 μmol

mol

-1

),

Mn

_(0.1

_μmol

_mol

_-1

_),

Zn

_(0.03

_μmol

mol

-1

),

Cu

_{(0.03 μmol}

_mol

_-1

_),

B

_(0.1

_μmol

_mol

_-1

₎

and Mo

_(0.02

_μmol

mol

-1

),

and were

_adjusted

to

_pH

5.5.

Every

5 weeks

_{supplementary}

Peters

_(S.T.E.M.)

micronutrient elements

_(0.05

_g

_dm

₃

₎

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 in order to avoid

2.2. Gas

_exchange

measurement

Measurements of stomatal conductance

_(g

_s

₎

and leaf carbon

_exchange

rate were made with a

_portable

_gas

analysis

system (LI-6200,

Li-cor

Inc.,

Lincoln, NE,

USA)

on _upper-canopy

fully

expanded

leaves of the same

_stage

of

_development

of

_randomly

selected 18

plants

for each

_CO

₂

x N treatment combination

_(all

mea-surements were made in

_duplicate

and each leaf was

measured

_twice).

Measurements of

_{daytime g}

_s

and

pho-tosynthetic

rate

_(A)

were

_performed

under

_saturating

light

conditions

(PAR

1 000-1 500

_μmol

m

-2

s

-1

),

between 10:00 to 15:00 hours on

_August

27-29

_(air

tem-perature

27-30

°C,

relative

_humidity

70-75

_%).

Leaf dark

_respiration

_(R

_d

₎

was measured before sunrise

(04:00-06:00 hours)

on

_August

26-28

_(air

_temperature, 23-25

_°C).

Instantaneous

_{transpiration efficiency (ITE)}

was calculated as

_A/g

_s

_.

Air

_temperature,

relative

humidi-ty and PPFD in the leaf cuvette were

_kept

at

_growth

con-ditions.

2.3. Biomass allocation

Heights

and root-collar diameters were measured on all the

_{plants (240)}

on

_September

4. On

_September

6 all

plants

were harvested and were

_separated

into

_leaves,

stem, and coarse

_(>

2

_mm)

and fine

_(<

2

_mm)

roots.

Surface area of each leaf and total

foliage

area of each

seedling

were measured with an area meter

_(Delta-T

Devices

_{Ltd, Cambridge, UK).}

Plant material was dried

at 65 °C to constant

_weight

and

_dry

mass

_(DW)

measure-ments were made. Leaf area

_ratio,

LAR

_(m

₂

_g

_-1

_),

was

calculated as the ratio of total leaf area to total

_{plant dry}

mass;

_specific

leaf area, SLA

(m

2

g

-1

),

as the ratio of

total leaf area to leaf

_dry

mass;

partitioning

of total

plant

dry

mass,

LWR,

SWR and RWR

(g

g

-1

),

as the fraction of

_plant

_dry

mass

_belonging

to

leaves,

stem and roots,

respectively.

In

addition,

root/shoot

_dry

mass

ratio,

RSR

(g

g

-1

),

was determined.

2.4.

_{Carbohydrate,}

carbon and N

analysis

The amount of total non-structural

_{carbohydrates}

(TNC),

including

starch and sugars, was measured

using

the anthrone method on 12

_seedlings

for each

_CO

₂

x N

treatment combination. These

seedlings

were harvested either at dawn or in late

afternoon,

and

_immediately

(after

leaf area

_{measurements) placed}

into a drier

(see

above).

Previously

dried

_plant

materials

_(leaves,

stem

and

_roots)

were

_ground

in a

Wiley

mill fitted with 20 mesh screen.

_{Approximately}

100 _mg of

_ground

tissue was extracted three times in

boiling

80 %

ethanol,

cen-trifuged

and the

_{supernatant pooled.}

The

_pellet

was

digested

at 40 °C for 2 h with

_{amyloglucosidase}

from

Rhizopus (Sigma

Chemical

_{Co., USA)}

and filtered. Soluble sugars and the

glucose

released from starch were

quantified spectrophotometrically

following

the reaction with anthrone. All

_samples

were

_prepared

in

_duplicate.

Total carbon and N concentrations

_(mg

_g

_-1

_DW)

were

determined for all 240

_{seedlings (leaves,}

stem and

_roots)

by

catharometric measurements

_using

an elemental

analyser (CHNS 2500,

Carlo

_{Erba, Milano, Italy)}

on

5-9 mg of

powder

of dried

_samples.

2.5. Phenolic

_analysis

Equal-aged

leaves

_(three

leaves _per

_plant)

were taken

from all 240

_seedlings,

the

_day

before the

harvest,

for

total

_phenolic

_compounds

_analysis.

Leaves were _put into

liquid

N at the field

site,

then

_transported

to the

laborato-ry 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

_bonding

procedure

of Walter and Purcell

_[51].

Other

_punches

from the

_remaining

leaf blades were used for

_dry

mass

determination, as described above. Leaf tissue was

homogenized

in 5.0

cm

3

of hot 95 %

_ethanol,

_blending

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

evaporated

to

dryness

in N at 28

_(C.

_Aliquots

₍₈

_cm

₃

₎

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

solu-tion,

distilled water and 0.1 N HCL

_{and, finally,}

with distilled water. Absorbance at 323 nm

_(A

₃₂₃

₎

was

mea-sured

_{spectrophotometrically}

both before and after the

Dowex treatment,

_representing

the absorbance

_by

pheno-lic

_compounds.

Phenolic concentration was determined

from a standard curve

prepared

with a series of

chloro-genic

acid standards treated

_similarly

to the tissue

extracts and

_comparing

_changes

in absorbance measured for the standards and those caused

_by

the treatment.

2.6. Statistical

_analysis

Individual measurements were

_averaged

_per

_plant,

and

_plants

measured with _respect to each

_CO

₂

x N treat-ment combination were

_averaged

across the

_open-top

analysis

of variance

_(ANOVA)

for randomized

_design

and Duncan’s mean

_separation

test for the measured

parameters

(5

%

_significant

_level);

_CO

₂

and N were

treated as fixed variables. A

_{preliminary analysis}

showed that differences between chambers within the same

_CO

₂

treatment were never

_significant.

_Proportions

and

per-centages were transformed

_using

the arcsine of the square root

_prior

to

_analysis.

3. Results

3.1. Gas

_exchange

rate

Increasing

[CO

2

]

had a

significant

effect on leaf

car-bon

_exchange

rate

_{(table I).}

_Comparison

of assimilation

rates at the

_growth

_[CO

₂

_]

showed that

_increasing

_[CO

₂

_]

from 370 to 520

_&mu;mol

mol

-1

resulted in 33 % increase in A for

_plants

_{grown with low-N}

_supply

and in 36 % increase for

_plants

grown with

high-N supply.

Nutrient

supply

also

_{significantly}

_{affected the response} of A. Plants _grown with

_{high-N supply}

had 25 and 29 %

high-er A than

_plants

_grown with low-N

_supply

at ambient and elevated

_[CO

₂

_],

_{respectively.}

There was no _strong inter-action between

_CO

₂

and N treatment

(P

=

0.084),

i.e.

increase in

_[CO

₂

_]

_elicited

a similar increase in A in both N treatments.

Comparison

_{of g}

_s

at the

_growth

_[CO

₂

_]

showed that

increasing

[CO

2

]

from 370 to 520

_&mu;mol

mol

-1

led to a 14 % decrease in _gs for

_plants

_{grown with low-N}

_supply

and to 10 % decrease with

_plants

_{grown with}

_high-N

supply (table I).

Nutrient

_supply

treatment and the

inter-action between

_CO

₂

and N treatment did not affect

signi-ficatly

gs.

As a result of increases in A and decreases in _gs, ITE of leaves increased with

_[CO

₂

_]

in both N

_supply

treat-ments

_{(table I).}

ITE was

_{significantly}

different _among

the four

_CO

₂

_x

N treatment combinations

_(ITE

was

high-er with

_{high-N supply),}

and there was a

_significant

inter-action between

_CO

₂

_and

N treatment

_(P

<

_0.05)

reflected

by

a marked increase in ITE in

_plants

_grown in elevated

[CO

2

]

with a

_{high-N supply.}

The ratio of internal

_[CO

₂

_]

_(C

_i

₎

to ambient

_(i.e.

exter-nal)

_[CO

₂

_]

_(C

_a

₎

decreased

_(P

<

0.0001)

with both

_CO

₂

-enrichment and

_{high-N supply,}

while the interaction between

_CO

₂

_and

N treatment was not

_significant

(table I).

The increase in A was associated with a

_significant

increase in

_R

_d

_(table

I).

Comparison

of

_R

_d

_at

the

_growth

[CO

2

]

showed that

_increasing

_[CO

₂

_]

from 370 to 520

&mu;mol

mol

-1

led to 48 % increase for

_plants

_grown with

low-N

_supply

and to 36 % for

_plants

_grown with

_high-N

supply.

The increase in N

_supply

and the interaction between

_CO

₂

_and

N treatment had less of an effect on the increase in

_R

_d

_.

3.2. Growth and biomass

_partitioning

Basal stem

diameter,

number of leaves _per

_plant

and

foliage

area were increased

_by

elevated

_[CO

₂

_]

treatment

only

when

_Q.

ilex

_seedlings

were _grown in the

_high-N

treatment

_{(table II, figure I).}

Shoot

_length

and individual leaf area were not influenced

_by

_[CO

₂

_]

treatment but increased with N

_supply.

After 6 months of

_CO

₂

_x

N

treatment combination there were

_significant

increases in

ambient

_[CO

₂

_],

_irrespective

of N treatment. The interac-tion between

_CO

₂

and N treatment was

_significant

for

total,

stem, fine root and

_foliage

biomass. As a result of

this,

effects of

_CO

₂

_-enriched

air on whole

_seedling

growth,

stem, fine root and

_foliage

biomass were

signifi-cant

_only

in the

_high-N

treatment

_(figures

1 and

_2).

Fine

root/foliage

mass ratio decreased with N treatment and increased with

_CO

₂

enrichment

_{(table II, figure 2).}

As a result of increased allocation to

_below-ground

tissue,

RSR and RWR were increased

_{significantly by}

CO

2

treatment, while SWR and LWR were

_decreased,

only

at a low level of N

_supply

_{(table II, figure 3).}

More

biomass was

_partitioned

to

_above-ground

tissue in the

high-N

treatment

_irrespective

of

_CO

₂

treatment; as a result RSR and RWR

decreased,

while

_conversely,

SWR and LWR increased

_{significantly}

at a

_high

level of N

supply.

SLA and LAR decreased

_{significantly}

in leaves of

_seedlings

_{grown in} elevated

_[CO

₂

_]

_irrespective

of N

treatment, while N

_supply

affected LAR

_(only

in elevat-ed

_[CO

₂

_]

but not SLA

_(table

_{II, figure 3).}

3.3. Carbon and N concentrations

Overall,

carbon concentrations in

leaves,

stem and

roots were not

_{significantly}

affected

_by

either

_CO

₂

or

nutrient treatment

_{(table III).}

Leaf N concentration

decreased

_{significantly}

in elevated

_[CO

₂

_]

_irrespective

of

nutrient treatment, while N concentrations in stem and

roots were decreased

_by

elevated

_[CO

₂

_]

in the

high-nutri-ent treatmhigh-nutri-ent. Nutrient

_supply

treatment affected N

con-centration

_{significantly}

in leaves

_irrespective

of

_CO

₂

treatment, and in stem and roots

_only

in the ambient

[CO

2

]

treatment. As a result of

_patterns

of N and carbon

concentrations,

C/N ratio

_generally

increased with ele-vated

_[CO

₂

_]

and decreased with

_high

nutrient

_supply

(table III).

3.4. Total non-structural

carbohydrate

and total

_phenolic

concentrations

Morning

starch concentrations were

_{higher (P}

<

_0.01)

in leaves of

_seedlings

_{grown in}

_CO

₂

_-enriched

air

_(table

IV),

but

_particularly

at low level of N

_supply.

Afternoon starch concentrations did not increase

_{significantly}

with

increasing

[CO

2

].

Both

_morning

and _{afternoon sugars}

concentration did not increase

_{significantly}

with

_rising

[CO

2

].

Both

_morning

and afternoon starch

concentra-tions decreased

_(P

<

_0.001)

with

_increasing

N addition while _sugars concentration was not affected

_by

N treat-ment

_{(table IV).}

Overall starch concentrations in

leaves,

stem and roots

affected

_{significantly by}

either

_CO

₂

_or

N treatment. As a

result,

TNC concentrations were influenced

_by

both

_CO

₂

enrichment and N treatment because of

_changes

in starch concentrations.

Total

_phenolic

concentrations decreased in leaves of

seedlings

grown in elevated

_[CO

₂

_]

irrespective

of N treatment, while N

_supply

treatment and the interaction between

_CO

₂

_and

N treatment had less of an effect

(fig-ure

_4).

4.

Discussion

Photosynthesis

of

_Q.

ilex

_seedlings

was stimulated

_by

elevated

_[CO

₂

_]

even in the low level of

_supplemental

fer-tilization and

_despite

_declining

foliar N

concentration,

as

for other broad-leaved trees

_(e.g.

_[34]).

The increase in

leaf dark

_{respiration expressed}

on a leaf area basis in

CO

2

-enriched

air may be correlated with the enhanced

carbohydrate

content

_[44].

_Although

many studies show

significant

reductions in

_plant

_respiration

in elevated

[CO

2

]

(e.g.

[47];

see

_[1]

for a

review),

accordingly

with these

_{seedlings, parent Q.}

ilex trees at the natural

_CO

₂

spring

in

_Italy

_{grow in} a N _{poor soil} and have also been

found to show

_{higher photosynthesis}

and dark

respira-tion than trees at ambient

_[CO

₂

_]

_{[9, 48].}

Stomatal

response to

_CO

₂

_is

a common

_phenomenon

and stomatal conductance in _many

_plants

decreases in response to

increasing

[CO

2

]

(see

reviews

[1, 7, 42],

and references cited

_therein).

In our

_study,

_however,

elevated

_[CO

₂

_]

Similar results have been

reported

for other

_species

when

plants

were _grown at

_high

irradiances

_(e.g.

_{[6, 21, 31,}

49].

Stomatal

_sensitivity

to

_CO

₂

in our

_seedlings,

_grown

at full irradiances with an

_{adequate supply}

of soil water,

may have been reduced

[16]. Indeed,

the ratio of internal

[CO

2

]

(demand)

to external

_[CO

₂

_]

_(supply)

decreased with

_CO

₂

enrichment while intercellular

_[CO

₂

_]

remained

relatively

constant,

despite

at elevated

_[CO

₂

_]

intercellu-lar

_[CO

₂

_]

should rise if stomata close

_{consistently.}

This

rate

and,

secondarily

decreased stomatal

_conductance,

instantaneous

_{transpiration efficiency}

of leaves

_markedly

increased at elevated

_[CO

₂

_]

_[15].

The elevated

_[CO

₂

_]

treatment increased

_seedling

growth only

when nutrient

_availability

was

_high.

Similar

findings

have been

_reported

for Pinus taeda L.

_[20],

Betula

_{populifolia Marsh.,}

Fraxinus americana

L.,

Acer

rubrum L.

_[3]

and Pinus

_palustris

Mill.

_{[38]. However,}

positive growth

responses to

_CO

₂

_-enriched

air even

under conditions of low soil nutrient

_availability

have been

_reported

for Castanea sativa Mill.

[17],

Pinus

pon-derosa

_Dougl.

ex Laws.

_{[27], Eucalyptus grandis}

W. Hill ex Maiden

_[11]

_{and Quercus virginiana}

Mill.

_[46];

in this latter case the

_experimental

conditions were the

same as in the

_{present study.}

These

_contrasting

results

(even

between studies with identical

_experimental

proto-cols)

indicate that the interactive effects of

_CO

₂

and

nutrient

_availability

are

_{species dependent.}

The lack of a

growth

response to elevated

_[CO

₂

_]

in

_seedlings

in the low-N treatment is of interest because

_suboptimal

con-centrations of N are common in the Mediterranean

envi-ronment.

Indeed,

responses in the

parent Q.

ilex trees at

the natural

_CO

₂

_springs

in

_Italy

do not _appear to be

clear-ly

more evident than in trees at ambient

_[CO

₂

_]

_[22].

Coarse root

_(and

total

_root)

biomass

_responded

posi-tively

to elevated

_[CO

₂

_]

_irrespective

of nutrient

availabil-ity,

while fine root biomass increased

_{significantly}

under low nutrient

_{availability. Partitioning}

of resources was

reflected

_{by adjustments}

in shoot and root

_growth

and in RSR. Low nutrient

_supply

enhanced overall biomass

par-titioning

to roots

_(higher

RWR and

RSR,

lower

_SWR),

while

_{high-N availability}

resulted in a

_greater

_proportion

of biomass

_being

distributed

to stem and leaves

_{[19, 32,}

38].

Preferentially

induced distribution

_{of photosynthates}

below-ground

as carbon

_supply

increases in _response to

CO

2

-enriched

air is a common

_{phenomenon [7, 38, 43].}

Such a

_pattern

was detected in our

_experiment

at a low level of nutrient

_{supply only,}

which was reflected

_by

increased RSR and _{RWR. This may allow}

_seedlings

in

CO

2

-enriched

air to

_explore

the soil in order to attain

more resources such as water and nutrients to meet

growth

demands.

_{Conversely, seedlings}

_{grown in}

ambi-ent

_[CO

₂

_]

had a _greater

_proportion

of biomass distributed

to

_above-ground

tissues at low level of nutrient

_supply

only,

which was reflected

_by

decreased RSR and

increased SWR and LWR. Increased stem biomass in

seedlings

grown under elevated

_[CO

₂

_]

and

high

nutrient

and

_height,

while in the low soil nutrient

_availability

treatment,

_seedlings

in elevated

_[CO

₂

_]

were even shorter than those in ambient

_[CO

₂

_].

These

_{findings support}

the idea that

_plants

allocate

photosynthate

to tissues needed to

_acquire

the most

lim-iting

resources

_[8].

Such shifts to below- and/or

above-ground

tissues,

may have

implications

during

the

regen-eration

_phase

in terms of

_competition

for

_light

and water

with other

_{woody species}

of the Mediterranean vegeta-tion.

_CO

₂

treatment also increased the fine

_root/foliage

mass ratio while N treatment had the

_opposite

effect

[46].

This

_change

in allocation

_{might represent}

a substi-tution between

_potential

carbon assimilation and nutrient

acquisition

[34]. However,

conflicting

results are

report-ed in the literature

[37, 45].

LAR and LWR decreased in response to elevated

_[CO

₂

_]

_suggesting

that

_canopy-level

adjustment

in carbon assimilation did occur in these

seedlings.

It must be

_pointed

out that our

_seedlings

_grew in _pots and

_growth

_responses to elevated

_[CO

₂

_]

_may sometimes be influenced

_by

_{pot size,}

_though

the issue of

pot

size is far from

_being

resolved. Soil nutrient

disequi-librium

_(which

we tried to

_minimize)

may be more

important

than

_pot

size in

_{affecting growth}

_response to

elevated

_[CO

₂

_].

Plants in natural environments do not

have unlimited

_below-ground

resources with which to

maximize

_growth

in elevated

_[CO

₂

_]

_[1],

and _{the presence} of shallow bedrock at the site of

_origin

of

_Q.

ilex

_parent

trees

is,

in this sense, a

good example.

The observed increased leaf biomass and area in response to

_CO

₂

_enrichment

_(at

a

_high

level of soil

observed in

_plants

grown in

_CO

₂

_-enriched

air

(e.g.

[14])

and have been attributed to an additional cell

layer [40]

or starch accumulation

[36].

In our

_study,

lower SLA at

elevated

_[CO

₂

_]

was

_partly

attributable to

_higher

carbohy-drate

concentrations,

a

_{large part}

of which was starch.

The reduction in N concentration in leaves of

seedlings

grown at elevated

_[CO

₂

_],

_irrespective

of the N

availability

in the

soil,

agrees with other studies on tree

species

[7, 42].

Similarly,

mature

_Q.

ilex trees grown

long

term under elevated

_[CO

₂

_],

from which the acorns were

collected,

showed a decreased N concentration in

leaves when

_compared

to trees grown at ambient

_[CO

₂

_]

[48].

We

found, however,

that N concentration in stem

and roots did not decrease in the

_CO

₂

_-enriched

air and low-N treatment combination. The decrease in tissue N

concentration may be an indirect and

size-dependent

effect of elevated

_[CO

₂

_]

[10, 46], alternatively

it has been

_proposed

that starch accumulation dilutes N and

lowers its concentration in the tissues

_{(e.g. [53]).}

Carbon concentration was not

_strongly

affected

_by

either

treat-ment

_(despite

an increase in carbon concentration in the

CO

2

-enriched

air and low-N treatment

_{combination).}

The

C/N ratio in leaves was enhanced

by

increasing

[CO

2

]

and

_diminishing

nutrient

_supply;

this trend was

confirmed in stem and roots but to a lesser extent. The increase in the C/N ratio could increase carbon storage

and the concentration of carbon-based

_secondary

com-pounds [30],

and nutrient

_cycling

in this as in other

Quercus

species

[50].

In the current

_experiment

foliar

phenolic

concentrations decreased in

_CO

₂

_-enriched

air.

A

_variety

of

_phenolic

concentration _responses to

_CO

₂

enrichment have been observed

_(e.g.

_[35]).

The

_parallel

increase in TNC at elevated

_[CO

₂

_]

_{may have caused} a

dilution of

_phenolics.

If increased

_[CO

₂

_]

reduces in the

long-term

both the N and

_phenolic

concentrations of leaves the consequences for interactions between

Q.

ilex and insect _{herbivores may be great.}

Nevertheless,

tannin concentrations in leaves of the

_parent

trees were

posi-tively

affected

_by

elevated

_CO

₂

_[48].

Enhancement of mineral nutrient

_supply

alone

_(which

caused

_{significant growth}

_stimulation)

reduced starch and TNC concentrations in

_leaves,

stem and roots.

_CO

₂

enrichment stimulated the accumulation of

TNC,

by

strongly

increasing

starch

formation,

starch

_storage

seems to be

_particularly

_important

in

_Q.

ilex. The lack of carbon flow to soluble _sugars

_suggests

a limitation in the

partitioning

of carbon to this intermediate

_{[54]. Rising}

concentrations of TNC in the leaves may occur, among

other reasons

_[43],

because of reductions in sink

_activity

(e.g.

as a _{consequence of}

_limiting

resources other than

CO

2

).

The accumulation of starch in

_{leaves, particularly}

in the

_CO

₂

_-enriched

air and low-N treatment

combina-tion, however,

was not

_{accompanied by}

the reduction in

photosynthetic

rate,

suggesting

that the demand for

car-bohydrates

in these

_seedlings

is

_high.

This

_study,

which

_represents

the first one on

proge-nies of trees _grown

_long

term in

_CO

₂

_-enriched

air,

reports

data similar to those conducted on mature trees at

the natural

_CO

₂

_spring

in

_Italy

_{in soil with poor N}

avail-ability

[48],

in that lack of a

_growth

_responses to

elevat-ed

_[CO

₂

_]

in

_seedlings

in the low-N treatment occurred

despite

increased rate of net

_{photosynthesis.}

All the

para-meters studied in the

_present

_experiment

_(except

for

car-bon-based defense

_compounds)

follow the

_findings

on

parent

trees

_exposed

to

_CO

₂

_-enriched

air when

_seedlings

grown in elevated

_[CO

₂

_]

and low-N

availability

are taken

into account. It is

_possible

to

_hypothesize

that the avail-able soil N will be a

_{major controlling}

resource for the

establishment and

_growth

of this

_species

in

_rising

_[CO

₂

_].

Q.

ilex _{stands established in soils poor in N will}

_probably

not exhibit a

_larger

increase in

above-ground

productivi-ty in the

_predicted

_CO

₂

_-enriched

_atmosphere,

but

below-ground

processes and interactions between trees and

tree-feeding

insects

_might

be altered.

Acknowledgement:

The technical assistance of Dave

Noletti is

_{greatly appreciated.}

References

[1] Amthor J.S., Terrestrial

_higher-plant

_response to

increas-ing atmospheric

[CO

2

]

in relation to

_global

carbon

_cycle,

Global

Change

Biol. 1 ₍₁₉₉₅₎ 243-274.

[2] Bazzaz F.A., The response of natural ecosystems to the

rising global

CO

2

levels, Annu. Rev. Ecol.

_Syst.

21 (1990)

167-196.

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

and responses of tree

_seedlings

to

_CO

₂

_,

_light,

and nutrients,

Ecology

74 ₍₁₉₉₃₎ 104-112.

[4] Brown _K.R., Carbon dioxide enrichment accelerates the decline in nutrient status and relative

growth

rate of

Populus

tremuloides Michx.

_seedlings,

Tree

_Physiol.

8 (1991) 61-173.

[5]

Bryant

J.P., Feltleaf willow-snowshoe hare interactions:

plant

carbon/nutrient balance and

_foodplain

succession,

Ecology

68 ₍₁₉₈₇₎ 1319-1327.

[6] 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.

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

atmos-pheric

_CO

₂

on

_woody

_plants,

New

_Phytol.

127 (1994) 425-446.

[8]

Chapin

F.S. III, Bloom A.J., Field _C.B.,

_Waring

R.H.,

Plant responses to

_multiple

environmental _farctors, BioScience

37 (1987) 49-57.

[9] Chaves M.M., Pereira J.S., Cerasoli _S., Clifton-Brown