13
© New Phytologist (2001) 151 : 413 – 425 www.newphytologist.com 413 Research Blackwell Science Ltd A decline in nitrogen availability affects plant responses to ozone Douglas G. Bielenberg 1 , Jonathan P. Lynch 2 and Eva J. Pell 3 1 Intercollege Graduate Program in Plant Physiology, The Pennsylvania State University, 301 Buckhout Laboratory, University Park, PA 16802, USA; 2 Department of Horticulture, The Pennsylvania State University, 221 Tyson Building, University Park, PA, 16802, USA; 3 Department of Plant Pathology and Environmental Resources Research Institute, The Pennsylvania State University, 304 Old Main, University Park, PA 16802–1504, USA Summary The effects are presented here of a decline in nitrogen (N) availability on ozone (O 3 )-induced accelerated foliar senescence during the growing season in a hybrid poplar. Cuttings of Populus trichocarpa × maximowizii were grown in sand culture where N supply to the plant could be controlled on a daily basis and reduced in half of the plants when desired. All plants received charcoal-filtered air; half also received supplemental O 3 . Ozone and N withdrawal both reduced plant growth. Plants grown in both N treatments displayed leaf senescence and abscission in response to O 3 , but leaf abscission in the N-withdrawal treatment was approximately double that of the constant-N treatment. Ozone had differential effects on light-saturated net photo- synthesis (A sat ) and total soluble protein in the younger and older foliage depending on N treatment. A decline in N availability increased the rate of O 3 -induced accelerated senescence and was associated with plant size and possibly continued active growth. Compen- satory responses of young leaves to O 3 exposure only occured when N availability to the plant declined and O 3 -induced accelerated senescence was most severe. Key words: nitrogen, ozone, accelerated senescence, compensation, hybrid poplar, air pollution. © New Phytologist (2001) 151 : 413–425 Author for correspondence: Eva J. Pell Tel: + 1 814 863 9580 Fax: + 1 814 863 9659 Email: [email protected] Received: 18 December 2000 Accepted: 21 February 2001 Introduction Senescence is the final stage of leaf development and involves a well-described sequence of metabolic changes, the most visually obvious of which is the loss of green color of a leaf as a result of chlorophyll breakdown. Senescence can also be characterized by declines in Rubisco, associated declines in net photosynthesis, increased proteolysis, altered fine organelle structure, loss of RNA and DNA, and increased ion leakage (Noodén & Leopold, 1988). The rate at which leaf senescence progresses is sensitive to a number of external environmental and endogenous plant factors (Noodén & Leopold, 1988). The air pollutant ozone (O 3 ) causes an acceleration of the normal rate of foliar senes- cence (Reich & Lassoie, 1985) and an accelerated progression of the hallmarks associated with senescence (Ojanperä et al. , 1992; Pääkkönen et al. , 1995; Sandelius et al. , 1995) culmin- ating in abscission and leaf loss much earlier than would occur normally. Leaf exposure to O 3 has been associated with accel- erated loss of quantity and activity of Rubisco, the primary carboxylating enzyme of photosynthesis (Pell & Dann, 1991). This decrease in quantity was determined to be the result of increased proteolysis and not decreased synthesis (Brendley & Pell, 1998). As a result, individual leaf net photosynthesis and potentially, whole plant carbon (C) gain, is reduced (Greitner et al. , 1994). Nutrients remobilized from senescing leaves and internally recycled by the plant may become critical for new growth if the availability of exogenous nitrogen (N) in the root zone declines. As N becomes less available to the roots, internal N may become more important for new growth. Expression of O 3 -induced accelerated senescence of leaves is responsive to

A decline in nitrogen availability affects plant responses to ozone

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Page 1: A decline in nitrogen availability affects plant responses to ozone

©

New Phytologist

(2001)

151

: 413–425

www.newphytologist.com

413

Research

Blackwell Science Ltd

A decline in nitrogen availability affects plant

responses to ozone

Douglas G.

Bielenberg

1

, Jonathan P.

Lynch

2

and Eva J.

Pell

3

1

Intercollege Graduate Program in Plant Physiology, The Pennsylvania State University, 301 Buckhout Laboratory, University Park, PA 16802, USA;

2

Department of Horticulture, The Pennsylvania State University, 221 Tyson Building, University Park, PA, 16802, USA;

3

Department of Plant Pathology and

Environmental Resources Research Institute, The Pennsylvania State University, 304 Old Main, University Park, PA 16802–1504, USA

Summary

• The effects are presented here of a decline in nitrogen (N) availability on ozone(O

3

)-induced accelerated foliar senescence during the growing season in a hybridpoplar.• Cuttings of

Populus trichocarpa

×

maximowizii

were grown in sand culture whereN supply to the plant could be controlled on a daily basis and reduced in half ofthe plants when desired. All plants received charcoal-filtered air; half also receivedsupplemental O

3

.• Ozone and N withdrawal both reduced plant growth. Plants grown in both Ntreatments displayed leaf senescence and abscission in response to O

3

, but leafabscission in the N-withdrawal treatment was approximately double that of theconstant-N treatment. Ozone had differential effects on light-saturated net photo-synthesis (A

sat

) and total soluble protein in the younger and older foliage dependingon N treatment.• A decline in N availability increased the rate of O

3

-induced accelerated senescenceand was associated with plant size and possibly continued active growth. Compen-satory responses of young leaves to O

3

exposure only occured when N availabilityto the plant declined and O

3

-induced accelerated senescence was most severe.

Key words:

nitrogen, ozone, accelerated senescence, compensation, hybrid poplar,air pollution.

©

New Phytologist

(2001)

151

: 413–425

Author for correspondence:

Eva J. Pell Tel:

+

1 814 863 9580 Fax:

+

1 814 863 9659 Email: [email protected]

Received:

18 December 2000

Accepted:

21 February 2001

Introduction

Senescence is the final stage of leaf development and involvesa well-described sequence of metabolic changes, the mostvisually obvious of which is the loss of green color of a leaf asa result of chlorophyll breakdown. Senescence can also becharacterized by declines in Rubisco, associated declines in netphotosynthesis, increased proteolysis, altered fine organellestructure, loss of RNA and DNA, and increased ion leakage(Noodén & Leopold, 1988).

The rate at which leaf senescence progresses is sensitiveto a number of external environmental and endogenous plantfactors (Noodén & Leopold, 1988). The air pollutant ozone(O

3

) causes an acceleration of the normal rate of foliar senes-cence (Reich & Lassoie, 1985) and an accelerated progressionof the hallmarks associated with senescence (Ojanperä

et al.

,

1992; Pääkkönen

et al.

, 1995; Sandelius

et al.

, 1995) culmin-ating in abscission and leaf loss much earlier than would occurnormally. Leaf exposure to O

3

has been associated with accel-erated loss of quantity and activity of Rubisco, the primarycarboxylating enzyme of photosynthesis (Pell & Dann, 1991).This decrease in quantity was determined to be the result ofincreased proteolysis and not decreased synthesis (Brendley &Pell, 1998). As a result, individual leaf net photosynthesis andpotentially, whole plant carbon (C) gain, is reduced (Greitner

et al.

, 1994).Nutrients remobilized from senescing leaves and internally

recycled by the plant may become critical for new growth ifthe availability of exogenous nitrogen (N) in the root zonedeclines. As N becomes less available to the roots, internal Nmay become more important for new growth. Expressionof O

3

-induced accelerated senescence of leaves is responsive to

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the N supply at which plants are grown. However, resultsfrom different studies yield seemingly contradictory results.Pääkkönen & Holopainen (1995) demonstrated in

Betulapendula

Roth. seedlings that the rate of N supply, maintainedwith weekly doses of fertilizer, was inversely proportional tothe rate of autumnal senescence. When Pell

et al

. (1995) grew

Populus tremuloides

Michx. at six N levels and exposed theplants to O

3

, leaf loss was found to be proportional to the rateof growth of the plant, and the rate of growth was determinedby the N supply. However, this study applied a time-releasefertilizer at the beginning of the experiment, which leads to Ndepletion through the course of the experiment. While thesetwo studies seem contradictory, they serve to illustrate thecomplexity of O

3

-induced accelerated senescence in relationto nutrient magnitude and timing of nutrient supply. Whilethe greater N supply rate may mitigate O

3

-induced acceleratedsenescence, it may only be the case if N supply is maintainedat a high level throughout the growing season. If N supplydecreases as a result of depletion or seasonal factors, then theincreased demand created by optimal early nutrient supplyrates may trigger the greatest induction of O

3

-induced accel-erated senescence.

Fluctuating N supply is of particular significance becauseplants frequently receive the highest dose of N early in thegrowing season, when they are experiencing the least demand.Agricultural plants are fertilized at or near planting date, asidefrom a possible additional ‘top dressing’ of N postemergence;N is thereafter allowed to deplete. Similarly, in temperate eco-systems N levels can be highest in the spring when conditionssupport release of nutrients from litter that may have beendeposited the previous autumn (Haynes, 1986).

Taken in isolation accelerated senescence appears whollynegative; accelerated senescence of a leaf translates into ashortened period of carbon acquisition and export of photo-synthate to the rest of the plant. However, there are exampleswhere older leaves exhibit an O

3

-induced acceleration ofsenescence, but the performance of remaining tissues areseemingly unaffected, such as in mean grain yield of O

3

-exposed

Triticum aestivum

cv. Minaret (Mulholland

et al.

, 1997) orphotosynthetic performance of remaining young leaf tissue ofO

3

-exposed

Malus domestica

Borkh. (Wiltshire

et al.

, 1993).Further, several investigations have shown that there is acorrelation between a more rapid rate of senescence in olderleaves of O

3

-exposed plants and increased capacity for Cassimilation of younger tissues of the same plants. Youngfoliage of O

3

-exposed plants have been shown to have increasednet carbon assimilation (

Populus tremuloides

Michx.; Pell

et al.

,1994), synthesis and quantity of Rubisco (

Populus trichocarpa

A. Henry

×

maximowizii

Torr. and A. Gray clone ‘245’;Brendley & Pell, 1998), nitrogen content and total solubleprotein (

Pinus ponderosa

Laws.; Temple & Riechers, 1995),and amino acid concentrations (

Pinus taeda

L.; Manderscheid

et al.

, 1992). The connection between O

3

-induced acceleratedsenescence of older foliage and the increased performance of

younger foliage may lie in the fact that new leaf (or organ)growth is often supported by reallocation of nutrients such asN from internal stores or older, declining foliage. Resourcesrecaptured during O

3

-induced accelerated senescence may betranslocated elsewhere in the plant (Manderscheid

et al.

, 1992;Temple & Riechers, 1995), thus providing for compensationto the stress.

In this paper we explore the relationships between theamount of N available in the root zone of the plant and theeffects of O

3

upon the rate of senescence in older leaves asthis response relates to the ‘compensatory’ responses to O

3

observed in the younger leaves. Specifically we tested thehypotheses that: (1) O

3

-induced accelerated senescence willbe increased by a decline in N availability; and (2) potentialcompensatory responses of younger foliage will be seen onlyin treatments displaying greater O

3

-induced acceleratedsenescence. These hypotheses were tested with a hybrid poplarclone known to demonstrate O

3

-induced accelerated senes-cence. A sand culture system was used to control the timingof decreases in N available to the plant.

Materials and Methods

Plant material

Hybrid poplar (

Populus trichocarpa

A. Henry

×

maximowizii

Torr. and A. Gray clone ‘245’) cuttings were used in allexperiments. Each year, cuttings were obtained in Februaryor March from a stand at the Russell E. Larson AgriculturalResearch Farm at Rock Springs, PA, USA. Cuttings weremaintained in a dormant condition in a 4

°

C cold room untilplanting. During the experiment, all side shoots were trimmedat initiation so that the plant consisted of a single axis of growth.

Nutrient regimes

Cuttings were planted in 9 l pots containing unsifted sandderived from crushed quartz (Beavertown Block Co., PleasantGap, PA, USA). Cuttings were planted on day of year – 155,153, 154, and 153 in yr 1996, 1997, 1998 and 1999,respectively. For reference, day of year 153 is the equivalentof June 1st in 1996 and the equivalent of June 2nd in 1997,1998 and 1999. All ramets were watered daily with a solutioncontaining a complete nutrient formula, pH

=

5.7

±

0.1. Thecontainers had drainage holes and a liquid holding capacityof approximately 3–4 l of nutrient solution. All plants initiallyreceived 3.57 mM N. After 25, 30, 36, and 18 d of O

3

exposure in yr 1996, 1997, 1998 and 1999, respectively, halfof the plants in the charcoal-filtered and O

3

-added treatmentsbegan receiving 0.71 mM N daily.

The nutrient solution contained: N (3.57 mM, 3 : 1NO

3

:NH

4

); P (0.92 mM); K (2.69 mM); Mg (1.0 mM); Ca(1.1 mM); S (1.0 mM); Fe (9.474 µM); Zn (4.848 µM); Cu(2.55 µM); Mn (10.26 µM); B (8.806 µM); Mo (0.0292 µM).

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Research 415

Nitrogen was supplied in a combination of NH

4

NO

3

andKNO

3

. The N withdrawal treatments contained 0.71 mM Nobtained by reducing the NH

4

NO

3

and KNO

3

by 80%. TheK

+

lost from the reduced level of KNO

3

was replaced withK

2

SO

4

to achieve an equivalent final concentration of K

+

.In 1996, to provide a comparison with previously published

work (Pell

et al.

, 1995; Brendley & Pell, 1998), a set of cuttingswas planted in 9 l pots as above but containing Metro-Mix510 soil-less medium (W. R. Grace and Co., Cambridge, MA,USA) amended with nutrients as described in Brendley & Pell(1998). Pots were watered as needed to field capacity.

Air treatments

Plants were grown in 12 (1996 and 1999, three chambersper treatment combination) or 16 (1997 and 1998, fourchambers per treatment combination) open top chambersequipped with rain exclusion caps (Brendley & Pell, 1998).Ozone was generated from pure O

2

and was dispensed andmonitored as previously described (Pell

et al.

, 1993). The airentering all chambers was charcoal-filtered 24 h d

–1

with halfof the chambers receiving a supplement of 0.08 µl l

–1

O

3

from1000 to 18.00 h daily. The O

3

treatments began 23, 17, 16,and 29 d after planting in the 1996, 1997, 1998 and 1999experiments, respectively, after buds had broken on the cuttings,and the stems had begun to elongate. Photosynthetic photonflux density, ambient air temperature, relative humidity andchamber air temperature were monitored as described by Pell

et al

. (1993).

Gas exchange

Six times during the 1996 experiment, net photosynthesis wasmeasured in the field by nondestructive gas exchange (Li-Cor6200, Lincoln, NE, USA) on leaves at positions 5 and 20 fromthe base of the plant. Measurements were made at a photosyn-thetic photon flux density (PPFD) of 1000 µmols m

–2

s

–1

orhigher. These same leaves were harvested for protein assays.

Total soluble protein and Rubisco determination

Six times during the 1996 experiment, leaves 5, 10 and 20 fromthe base of the plant were harvested for total soluble protein(TSP) determination. During the 1997 and 1999 experimentsleaves 6 and 21 from the base of the plant were harvested everyfourth day from initiation until senescence or the end of theexperiment. These samples were analyzed for total solubleprotein content and Rubisco content as described by Brendley& Pell (1998). The gels were digitally imaged with an Eagle

Eye

IIphotoimaging system utilizing EagleSight software, ver. 3.2(Stratagene, Inc., La Jolla, CA, USA). The relative abundanceof the large and small subunits of Rubisco within the TSP ofthe sample was determined via digital image analysis usingOneD-Scan software (Scanalytics, Inc., Billerica, MA, USA).

Biomass and leaf area

Six times during the 1996 experiment one plant was randomlyselected from each chamber to be destructively harvested. Leaveswere removed from the plant and individually measured for leafarea on a Li-Cor 3000 leaf area meter with a Li-Cor 3050 Atransparent conveyor belt accessory (Lincoln, NE). The plantwas divided into five fractions for biomass and N determination:upper, expanding leaves, middle leaves, lower leaves, stems androots. The leaf positions (marked by the leaf scar in case ofabscission) not included in the upper, expanding categoryof leaves were evenly divided between middle and lower leaffractions. Petioles were included in the leaf fractions. Plantfractions were dried in a 70

°

C drying oven for 7 d, after whichthe d. wt of each fraction was determined.

Whole plant destructive harvests were conducted in thesame manner in 1997 and 1998 with the exceptions that leafarea was not measured on destructively harvested plants andnine harvests were conducted in 1998. Also, petioles wereincluded with the stem fraction rather than with the respect-ive leaf fractions. Leaf area was monitored by nondestructiverepeated measures in the 1997 and 1998 experiments. Eightplants were selected from each treatment at the initiation ofthe experiment and measured twice weekly for the course ofthe experiment. Midrib length (L, point of petiole attachment toblade to blade tip) and blade width (W) at the widest pointof each leaf were measured from the time that the leaf separ-ated from the apical meristem until no changes in size weremeasured on two or three successive measurements. Theproduct of L

×

W was converted to leaf area using a linearregression equation obtained from destructively harvestedleaf samples whose area was determined using a Li-Cor 3000leaf area meter. Leaves sampled for the regression analyseswere obtained across the range of leaf sizes and ages and atseveral times during the experiment. In 1997 the equationarea

=

0.7158

×

(L

×

W) – 7.091 (

r

2

=

0.997) was used forconversions. In 1998 the relationship between the productof L

×

W and area was determined to be more accuratelydescribed by two regression equations representing populationsof relatively large or small leaves. The equation area

=

0.585

×

(L

×

W)

+

0.222 (

r

2

=

0.995) was used for leaves with a L

×

W < 50 cm

2

. The equation area

=

0.678

×

(L

×

W) – 3.904(

r

2

=

0.983) was used for leaves with a L

×

W > 50 cm

2

. Leafabscission was also recorded in these plants.

In 1999 one whole plant destructive harvest was performedat the end of the experiment. Plants were divided as beforeinto five plant fractions and dried for biomass determination.Whole plant leaf area was measured as in 1996.

Total nitrogen determination

In the 1996 and 1997 experiment total N was determined inthe dried plant fractions by an EA 1108 elemental analyzeroperating in CHN mode using acetinilide as a standard

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(Fisons Instruments, Milan, Italy). Before analysis the sampleswere dried and ground in a Wiley mill through a 40 meshscreen. Plant fractions from the 1998 experiment were analyzedfor total N content by micro-Dumas combustion analysis atthe Institute of Ecology’s Ssotope/Soil Biology Laboratory atthe University of Georgia, Athens, GA, USA.

Statistics

The experimental design was a 2

×

2 factorial design (two Ntreatments and two O

3

levels) replicated three (1996 and 1999)or four (1997 and 1998) times. Treatment combinationswere assigned to chambers and the chambers randomizedin the field. When necessary, data were normalized by trans-formations. All plant d. wt values were Log

e

transformed. Allleaf abscission data were arcsin transformed. Treatment effectson variables were evaluated using ANOVA with N, O

3

, andharvest date (day of year) as class variables. Data were analyzedby repeated measures with date as the repeated factor andchamber as the subject. A first-order autoregressive covariancestructure was used to adjust for correlations between adjacentharvest dates. The mean square associated with the O

3

× Ninteraction nested within replicate was used to test significanceof the main effects and their interaction. The residual error termwas used to test significance of date alone, and interaction

with the treatment effects. Analyses were performed for eachyear separately, using the Mixed Models procedure of SAS(SAS Institute, Inc., Cary, NC, USA). Only harvest dates thatoccured after the N withdrawal were analyzed.

Tests of significance for slopes of the number of leavesabscised g–1 d. wt regression lines and slopes of [TSP] regres-sion lines were performed using the regression procedure ofSAS (SAS Institute, Inc., Cary, NC, USA).

Results

The experiment was repeated in four consecutive years. Therelative responses between the treatment combinations weresimilar in all years of experiments. Therefore, only repres-entative data are shown in the figures; statistics from all yearshave been presented in the tables. When data for more than oneyear are shown, it is to illustrate where differences do exist (i.e.Fig. 1 and Fig. 2) or to highlight trends that arise from a syntheticconsideration of the pooled experiments (i.e. Fig. 4 and Fig. 6).

Plant growth and leaf abscission

Date of harvest exerted a significant effect upon biomassaccumulation (Table 1 and Fig. 1) each year. Both O3 and Ntreatments affected plant biomass accumulation although

0

60

120

180

240

300

360

Who

le p

lant

d. w

t (g) 0

60

120

180

240

300

360

0

60

120

180

240

300

360

0

60

120

180

240

300

360(c)

(a) (b)

(d)

190 200 210 220 230 2400

60

120

180

240

300

360

Day of year

190 200 210 220 230 2400

60

120

180

240

300

360(e) (f)

Fig. 1 Whole plant biomass of hybrid poplars grown in open-top chambers receiving charcoal-filtered air (open circles, solid lines) or charcoal-filtered plus supplemental O3 to a concentration of 0.08 µl−1 (closed circles, dotted lines). Plants were grown in sand with nutrient solution provided daily. (a 1996), (c 1997) and (e 1998) received a 3.57-mM N solution daily for the entire experiment. (b 1996), (d 1997) and (f 1998) received a 3.57-mM N solution daily for 3–4 wk of O3 exposure after which N concentration was reduced to 0.71 mM N in the daily nutrient solution. Arrows mark time of nutrient withdrawal. (mean ± SE, n = 3 (1996) or 4 (1997 and 1998)). For reference, day of year 190 is the equivalent of July 8th in 1996 and July 9th in 1997, 1998 and 1999.

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Research 417

statistical significance could not always be detected at P > F =0.05 (Fig. 1 and Table 1). Ozone exposure and N withdrawalboth reduced whole plant d. wt accumulation. The relativedifferences among whole plant d. wt of the O3 exposure andN withdrawal treatments were the same in the separateyears of experiments, despite differences in maximum biomassachieved among years (Fig. 1). In 1996 there was a significantN × O3 interaction and no interactions with date (Table 1).In 1997, the O3 effect and the N × date interaction were signi-ficant (Table 1). In 1998, the effect of O3 was only significantat the 0.0693 level; the N × date interaction was also significant.

In 1996, an additional set of plants was grown in Metro-Mix soilless media with time-release fertilizer to provide acomparison with previously published work (Pell et al., 1995;Brendley & Pell, 1998). The O3 response of the Metro-Mixgrown plants in this experiment was similar to those previouslypublished by Brendley & Pell (1998) and Pell et al., (1995).These plants did not achieve the same level of biomass accu-mulation as the plants grown in sand culture, but biomassdecline in response to O3 was similar. At the final harvest theMetro-Mix grown plants had accumulated only 56 and 77%of the biomass of the constant N and N withdrawal treatmentplants, respectively.

Whole plant leaf area was determined by frequent repeatednondestructive measures in the 1997 and 1998 experiments(Fig. 2). Treatment differences in the 2 yr form a similarpattern but vary greatly in magnitude. Nitrogen withdrawalgreatly reduced whole plant leaf area as a result of its effectupon individual leaf size. In both N treatments O3 reducedwhole plant leaf area. In 1997, the N × O3 × date interactionwas significant (Table 2). In 1998, the N × date and O3 × dateinteractions were significant (Table 2). The pattern of leaf area

Pla

nt to

tal l

eaf a

rea

(m2)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Day of year

170 180 190 200 210 220 230 2400.00

0.08

0.16

0.24

0.32

(a)

(b)

Fig. 2 Whole plant leaf area in charcoal-filtered air (open symbols, solid lines) and charcoal-filtered air supplemented with O3 (closed symbols, dotted lines), and constant N (circles) and N withdrawal (squares) treatments for the 1997 (a) and 1998 (b) experiments. Points are mean ± SE (n = 5–8). Note difference in y-axis scale between years.

0.0

0.1

0.2

0.3

0.4

0.5

Fra

ctio

n of

leav

es a

bsci

sed

(abs

cise

d le

aves

[abs

cise

d +

atta

ched

leav

es]–1

)

0.0

0.1

0.2

0.3

0.4

0.5

190 200 210 220 230 2400.0

0.1

0.2

0.3

0.4

0.5

Day of year

190 200 210 220 230 2400.0

0.1

0.2

0.3

0.4

0.5(c)

(a) (b)

(d)

Fig. 3 Leaf abscission (fraction of leaves abscised = abscised leaves [abscised plus attached leaves]–1) of hybrid poplars grown in open-top chambers receiving charcoal-filtered air (open symbols, solid lines) or charcoal-filtered plus supplemental O3 to a concentration of 0.08 µl l–1 (closed symbols, dotted lines), constant N (circles) or N withdrawal (squares) treatments for the 1996 (a, b) and 1997 (c, d) experiments. Arrows mark time of nutrient withdrawal. Points are mean ± SE, n = 3 (1996) or 4 (1997).

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Research418

by treatment was similar in both experiments: charcoal-filteredconstant N > O3-exposed constant N > charcoal-filteredN withdrawal > O3-exposed N withdrawal. The reduction inwhole plant leaf area with harvest date in response to O3 was aresult of leaf abscission (Fig. 3) and slightly reduced individualleaf size in the upper canopy (data not shown). Total leafnumber produced was not affected by any of the treatmentsin any of the experiments (data not shown).

Leaf abscission was greatly increased by O3 exposure inboth N treatments (Fig. 3). Charcoal-filtered treatments dis-played little or no leaf abscission in either N treatment, except

in 1996 (Fig. 3). The N withdrawal treatment produced greaterleaf abscission in response to O3 than the constant N treatment.There were significant N × date and O3 × date interactions inall years, except in 1999 when N × date was not significant(Table 1). In 1997 and 1999, the N × O3 interaction was alsosignificant (Table 1). The differences among treatments forleaf abscission response of the constant N and N withdrawaltreatments to O3 exposure were the same in all years, but themagnitude of leaf abscission varied between experiments.

Variations in magnitude of leaf abscission between yearsare reduced when the number of abscised leaves is expressedper unit of whole plant biomass (Fig. 4), which also variedbetween years (Fig. 1). The relationship between harvest dateand number of leaves abscised g–1 total d. wt for the constantN treatment was not significant (F = 2.24, P > F = 0.1395,Fig. 4a). The slope of the charcoal-filtered N withdrawal treat-ment was significantly different from zero (slope = 0.0009,r2 = 0.0827, F = 6.13, P > F = 0.0158; Fig. 4a) The leafabscission in response to O3 was greater in all years in the Nwithdrawal treatment when between year variation in wholeplant size was taken into account (Fig. 4b). The N withdrawaltreatment increased O3-induced leaf abscission with harvestdate (slope = 5.690, r2 = 0.4848, F = 64.0, P > F = 0.0001) ata faster rate than the constant N treatment (slope = 1.297,r 2 = 0.0875 F = 6.52, P > F = 0.0129; Fig. 4b). The slopeof the two O3-exposed N treatments differed significantly(F = 25.26, P > F = 0.0001).

Table 2 Results of statistical analyses for effects of air (O3), N treatment (N) and measurement date (D) on whole plant leaf area (m2) determined by nondestructive repeated measures on sand culture grown hybrid poplars in the 1997 and 1998 experiments. Bold P-values are ≤ 0.05

Source of variance

1997 1998

df F P > F df F P > F

N 1 27.76 0.0002 1 2.59 0.1332O3 1 23.39 0.0004 1 4.61 0.0529N × O3 1 4.70 0.0512 1 0.00 0.9712D 10 62.25 0.0001 7 124.96 0.0001N × D 10 9.86 0.0001 7 11.44 0.0001O3 × D 10 3.41 0.0003 7 7.68 0.0001N × O3 × D 10 3.42 0.0003 7 0.92 0.4897

Table 1 Results of statistical analyses for effects of air (O3) and N treatment (N), and harvest date (D) on whole plant biomass (loge g total biomass) and percent leaf abscission [no. of abscised leaves no.−1 of leaves produced] of hybrid poplars for the 1996, 1997, 1998 and 1999 experiments. Bold P-values are ≤ 0.05

Source of variance N O3 N × O3 D N × D O3 × D N × O3 × D

Biomass1996 df 1 1 1 4 4 4 4

F 20.83 28.31 11.00 67.15 1.28 0.95 1.01P > F 0.0018 0.0007 0.0106 0.0001 0.2974 0.4459 0.4175

1997 df 1 1 1 4 4 4 4F 10.45 44.57 0.02 150.76 5.89 1.81 0.43P > F 0.0072 0.0001 0.8957 0.0001 0.0006 0.1428 0.7889

1998 df 1 1 1 7 7 7 7F 6.07 4.17 0.11 6.82 2.81 0.14 0.61P > F 0.0298 0.0639 0.7427 0.0001 0.0111 0.9950 0.7446

Abscission1996 df 1 1 1 4 4 4 4

F 0.94 17.73 0.23 50.81 11.35 5.31 2.36P > F 0.3608 0.0030 0.6448 0.0001 0.0001 0.0021 0.0743

1997 df 1 1 1 4 4 4 4F 87.49 380.63 9.53 68.59 5.49 26.63 0.70P > F 0.0001 0.0001 0.0094 0.0001 0.0010 0.0001 0.5943

1998 df 1 1 1 7 7 7 7F 34.63 77.83 0.55 65.23 5.44 10.80 0.96P > F 0.0001 0.0001 0.4713 0.0001 0.0001 0.0001 0.4674

1999 df 1 1 1 7 7 7 7F 12.33 130.77 8.72 40.26 0.54 16.81 0.46P > F 0.0080 0.0001 0.0183 0.0001 0.7998 0.0001 0.8620

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Leaf photosynthesis and protein content

Light-saturated net photosynthesis (Asat, µmol CO2 m–2 s–1)

was measured on a lower and upper canopy leaf several timesthrough the 1996 experiment. Patterns of Asat through timewere similar in both N treatments in response to O3 exposurein the lower canopy leaf (Fig. 5c,d). Ozone exposure resultedin a decline in Asat with time, ultimately ending with thecomplete senescence and abscission of the lower leaf. Ozoneand date both significantly affected Asat but no effect of Nwas observed for the lower leaf (Table 3). The lower leavesfrom charcoal-filtered plants in both N treatments showed acharacteristic developmental pattern of Asat with a peak followedby gradual decline. Amongst the upper canopy leaves, Nwithdrawal significantly reduced Asat in the charcoal-filteredtreatment (Table 3). The upper leaves of the two N treatmentsdiffered in response to O3 exposure (Fig. 5a,b). The upper leafof the constant N treatment responded variably to O3 exposure,

Table 3 Results of statistical analyses for effects of air (O3), N treatment (N) and date (D) on light saturated net photosynthesis (µmol CO2 m

−2 s−1) of an older, lower canopy leaf (5th from base) and a younger, upper canopy leaf (20th from base) of sand culture grown hybrid poplars in the 1996 experiment. Bold P-values are ≤ 0.05

Source of variance

Leaf 5 Leaf 20

df F P > F df F P > F

N 1 2.21 0.1757 1 22.46 0.0015O3 1 86.12 0.0001 1 3.75 0.0890N × O3 1 0.61 0.4562 1 0.19 0.6709D 4 39.57 0.0001 3 5.49 0.0061N × D 4 0.42 0.7920 3 0.39 0.7591O3 × D 4 0.64 0.6382 3 2.31 0.1053N × O3 × D 4 1.91 0.1332 3 1.08 0.3795

0

10

20

30

Day of year190 200 210 220 230 240

0

10

20

30

A (

µmol

s C

O2

m–2

s–1

)

0

10

20

30

190 200 210 220 230 2400

10

20

30

(b)

(d)

(a)

(c)

Fig. 5 Light-saturated net photosynthesis (µmol CO2 m

−2 s−1) in upper (a, b) and lower (c, d) canopy leaves of hybrid poplar plants exposed to charcoal-filtered air (open symbols, solid lines) or charcoal-filtered air plus supplemental O3 to a concentration of 0.08 µl−1 (closed symbols, dotted lines), and constant N (a, c) or N withdrawal (b, d) in the 1996 experiment. The lower leaf is the fifth leaf from the base of the plant, the upper leaf is 15 positions above leaf 5. Arrow marks time of nutrient withdrawal. (Points are mean ± SE, n = 3, except where missing as a result of leaf abscission).

Day of year

200 210 220 230 240

Num

ber

of a

bsci

sed

leav

es g

–1 d

. wt

0.00

0.05

0.10

0.15

0.20

0.25

0.00

0.05

0.10

0.15

0.20

0.25 (a)

(b)

Fig. 4 Linear least square fit lines through number of abscised leaves g–1 whole plant d. wt vs time for hybrid poplars exposed to charcoal-filtered air (a) or charcoal-filtered air with O3 added (b). Data from 4 yr of experiments are included: 1996 (triangles) 1997 (circles) 1998 (squares), and 1999 (inverted triangles). Points shown are mean values of 3–4 replicate values, regression significance tests were performed using all points. Plants were grown in sand culture receiving constant N (open symbols) or N withdrawal (closed symbols).

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generally not differing from the charcoal-filtered leaf. In the Nwithdrawal treatment, O3 exposure increased Asat above thatof the charcoal-filtered treatment in a sustained fashion. Theeffect of O3 was statistically significant at the P > F = 0.0890level (Table 3). Date did have a significant effect upon Asat inthe upper canopy leaves (Table 3), which resulted from thetrend of all treatments to decline with leaf age.

Total soluble protein (TSP) concentration was determinedin upper and lower canopy leaves throughout the life of theleaf in three separate years of experiments. The relative effectof O3 upon [TSP] in the leaves was determined by dividingthe mean [TSP] of the O3-exposed leaves in a N treatment bythe mean [TSP] of the CF leaves in the same N treatment ateach measurement date. When all three years were combined,both N treatments showed nearly identical relative response

of [TSP] to O3 exposure in the lower leaf (Fig. 6a). Early in theleaf ’s development there was little effect of O3 (O3 : CF ≅ 1.0);thereafter, the impact of O3 became steadily greater with leafage. The timing of N withdrawal coincided roughly with theearly stages of O3-induced protein decline in both treatments(Fig. 6b). Slopes of linear least squares trendlines through theconstant N (slope = −0.021, r2 = 0.7074, F = 48.36, P > F =0.0001) and N withdrawal treatments (slope = −0.028,r 2 = 0.7005, F = 46.79, P > F = 0.0001) were significant.The slopes of the two treatments did not significantly differ(F = 2.11, P > F = 0.1545).

In the upper leaf, the relative response of the two N treatmentsto O3 differed. The [TSP] of leaves receiving constant Ntreatment appeared not to be affected by O3 exposure (e.g.O3 : CF ≅ 1.0), however, the O3-exposed leaves subjected toN withdrawal exhibited increased [TSP] (e.g. O3 : CF > 1.0)relative to the charcoal-filtered control (Fig. 6a). This increaseoccured primarily early in leaf development in individual

Day of year

180 190 200 210 220 230 240

Rel

ativ

e (O

3 : C

F)

tota

l sol

uble

pro

tein

con

cent

ratio

n

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

(b)

(a)

N withdrawal: 1.22 (0.03)Constant N: 1.00 (0.05)

Fig. 6 Relative concentration (mg protein g–1 d. wt, O3 : CF) of total soluble protein in an upper (a) and lower (b) hybrid poplar canopy leaves of plants from the constant N treatment (open symbols) and the N withdrawal treatment (closed symbols) from initiation to senescence or the end of the experiment. Data are mean values for plants grown in charcoal-filtered (CF) and O3-added air in 1996 (circles), 1997 (squares) and 1999 (triangles). Lower leaf trendlines are linear least squares fit lines through data from all 3 yr combined (solid line, constant N; broken line, N withdrawal); trendlines include only dates following N withdrawal, as indicated by the appearance of the closed symbols. No trends with time were significant for upper leaves (a). Text in (a) shows the mean (SE) of pooled values of relative total soluble protein content in upper leaves. Means differ significantly (F = 14.14, P > F = 0.0007).

0.01

0.02

0.03

0.04

0.01

0.02

0.03

0.04

Nitr

ogen

con

cent

ratio

n (g

tota

l N g

–1 d

. wt)

0.01

0.02

0.03

0.04

0.01

0.02

0.03

0.04

Day of year

190 200 210 220 230 2400.01

0.02

0.03

0.04

0.01

0.02

0.03

0.04

(a)

(b)

(c)

Fig. 7 Leaf nitrogen concentration (g N g–1 d. wt) in upper (a), middle (b) and lower (c) canopy sections of hybrid poplar grown with constant N (circles) or with N withdrawn (squares) and exposed to charcoal-filtered air (open symbols, solid lines) or to charcoal-filtered air with O3 added (closed symbols, dotted lines) in the 1997 experiment. Arrow marks time of N withdrawal. (mean ± SE, n = 4, except where missing as a result of abscission).

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years (data not shown). There was no trend to increase ordecrease relative [TSP] with time in either treatment asslopes of linear least squares trendlines though the constantN (F = 0.44, P > F = 0.5194) and N withdrawal (F = 1.04,P > F = 0.3227) treatments did not differ significantly fromzero (Fig. 6a). Since there was no significant trend with time,upper leaf total soluble protein ratios were pooled across alldates and the means of the two N treatments compared. Themean of the N withdrawal treatments (1.22 ± 0.03, mean ± SE)was significantly (F = 14.14, P > F = 0.0007) greater than theconstant N treatment (1.00 ± 0.05, mean ± SE).

Rubisco concentration was measured in the same leaves aswere measured for [TSP]. In both the upper and lower leavesin all 3 yr, the response of Rubisco to N and O3 treatmentreflected that of the TSP (data not shown).

Canopy N concentration

Leaf [N] in the upper, middle, and lower canopy sections ofhybrid poplars was determined in 1996, 1997 and 1998 andillustrated for 1997 (Fig. 7). In general, [N] in all leaf fractionsdeclined with time in both N treatments and the N withdrawaltreatment generally enhanced this decline in [N] relative tothe constant N control (Fig. 7). This was reflected in the signi-ficant effect of date upon the [N] of all the leaf canopy sectionsin each year except for the upper leaves in 1998 (Table 4). Ozoneexposure reduced [N] in lower leaves of both N treatments,with a significant N × O3 × date interaction in 1997 (Table 4).

In 1996, the effects of O3, date and N × date were significant(Table 4). In 1998, O3, date and N withdrawal all independ-ently and significantly reduced [N] (Table 4). The converseoccured in the upper, expanding leaves of both N treatments,where O3 increased the tissue [N] in 1996 and 1997 (Table 4and Fig. 7). Ozone did not significantly increase upper leaf[N] in 1998, when the only significant treatment effect wasthat of N treatment (Table 4). Middle leaves showed no effectof O3 in 1997 and 1998, with date and N withdrawal independ-ently and significantly reducing [N] in 1997 and a significantN × date interaction in 1998 (Table 4). In 1996, the effects ofN × O3 and N × date were significant.

Discussion

This study addressed two hypotheses: O3-induced acceleratedsenescence will be increased by a decline in N availability;and potential compensatory responses of younger foliagewill be seen only in treatments displaying greater O3-inducedaccelerated senescence. Both hypotheses were supported. Thedegree of O3-induced accelerated senescence was also associatedwith plant size and/or active growth.

Will a change in N availability influence O3-induced accelerated senescence?

A sand culture system was developed for the growth of hybridpoplar, which enabled careful control of the daily N supply to

Table 4 Results of statistical analyses for effects of air (O3) and N treatment (N) and harvest date (D) on leaf total N concentration (g N g dry weight leaves–1) for canopy sections of hybrid poplars in the 1996, 1997 and 1998 experiments. Canopy sections were divided at harvest as detailed in the Materials and Methods. Bold P-values are ≤ 0.05

Source of variance N O3 N × O3 D N × D O3 × D N × O3 × D

1996 df 1 1 1 4 4 4 4Upper F 74.03 37.82 9.89 35.70 0.17 1.70 1.05

P > F 0.0001 0.0003 0.0137 0.0001 0.9529 0.1744 0.3975Middle F 104.96 0.33 11.54 41.21 7.09 1.06 1.53

P > F 0.0001 0.5826 0.0094 0.0001 0.0004 0.3950 0.2164Lower F 51.58 46.55 0.18 6.37 4.33 1.20 2.34

P > F 0.0001 0.0001 0.7208 0.0007 0.0066 0.3318 0.0765

1997 df 1 1 1 4 4 4 4Upper F 77.95 22.95 0.12 90.96 3.49 2.86 0.34

P > F 0.0001 0.0004 0.7368 0.0001 0.0139 0.0333 0.8527Middle F 51.16 2.12 2.05 36.75 2.76 1.54 0.72

P > F 0.0001 0.1707 0.1773 0.0001 0.0380 0.2063 0.5818Lower F 70.98 45.42 2.85 23.17 4.67 3.14 2.70

P > F 0.0001 0.0001 0.1173 0.0001 0.0029 0.0227 0.0416

1998 df 1 1 1 7 7 7 7Upper F 14.15 0.35 0.00 1.81 0.69 1.45 1.97

P > F 0.0027 0.5651 1.00 0.0968 0.6818 0.1948 0.0695Middle F 32.43 2.76 0.11 4.36 0.31 0.39 0.88

P > F 0.0001 0.1226 0.7503 0.0004 0.9433 0.9031 0.5232Lower F 20.44 13.65 0.34 5.01 1.97 0.04 0.26

P > F 0.0007 0.0031 0.5722 0.0001 0.0694 0.9999 0.9688

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the plant. The O3-induced accelerated senescence response ofplants grown in a Metro-Mix soilless media with time-releasefertilizer as a N source was reproduced (Pell et al., 1995), con-firming the earlier observation that declines in N availabilitywere consistent with O3-induced accelerated senescence.

Many studies have investigated the influence of N supplyupon the response of plants to O3 exposure. Nitrogen treatmentsranged from fertilized field plots (Menser & Street, 1962),soilless media mixes with time-release fertilizer (Pell et al., 1995)and sand culture systems (Maurer et al., 1997; Volin et al.,1998). In general, when plants are grown in constant highfertility or N, there is a higher level of O3-induced leaf abscis-sion than in constant low fertility grown plants (Einig et al.,1997; Maurer et al., 1997). Previous studies (Pell et al., 1995;Brendley & Pell, 1998) suggested that when available N inthe root environment is in decline, the intensity of O3-inducedaccelerated senescence may be increased. Data reported hereinsupport these observations. When the N supply was reducedto 20% of its previous supply, leaf abscission was increasedrelative to plants that had been maintained at a constant Nconcentration. Similarly, Pääkkönen & Holopainen (1995)demonstrated that when Betula pendula Roth. seedlings wereexposed to O3, autumnal senescence was seen earlier in treesfor which N supply (maintained by weekly doses) was decreasedby half than in plants that had their supply doubled.

Ozone and N both significantly affected hybrid poplar leafabscission (Table 1). These main effects interacted with harvestdate, indicating that the nature of the responses was dynamic,probably as a result of growth processes and growth-inducedchanges in the internal nutrient status of the plants as indicatedby the foliar [N] (Fig. 7). While a significant impact of thefour treatments on biomass and leaf abscission could not bedetected in each of the four experiments (Table 1), the sametrends were always observed. Maximum leaf abscission andplant weight varied over the 4 yr in which the experimentswere conducted. The cause of variation between years is notknown. There were variations in environmental conditionsover the years (data not shown), but a more likely explanationfor the reduced biomass accumulation in 1998 is reducedvigor of the parent trees from which cuttings were made. In allyears the cuttings were made from the same parent trees, butin 1997 experiment the parent trees suffered from an outbreakof Septoria leaf spot disease. Control measures were under-taken to remove all of the infected plant material and noinfected material was used in the experimental plants. Noevidence of any disease (leaf spots or stem cankers) was foundon plants grown in our experimental chambers. However, it ispossible that the infection of the parent plants reduced thevigor of the cuttings used in the experiment, resulting inincreased time to budbreak and slower initial growth rates.The reproducibility of the O3-induced accelerated senescenceand abscission in the 1998 experiment, despite reducedgrowth of the plants, emphasizes the robustness of the leafabscission response to O3. Previous work has highlighted the

potential effects of plant size upon plant response to environ-mental stresses, particularly gaseous air pollutants (Coleman& McConnaughay, 1995; Pell et al., 1995). When leafabscission was adjusted for plant size, plant response to O3was greater in the N withdrawal treatment than in the Nconstant treatment in all years (Fig. 4). This indicated thatthe leaf abscission response to O3 was sensitive to plant size:smaller plants lost a smaller proportion of leaves than didlarger plants. These results are similar to those reported byPell et al. (1995) in which hybrid poplars fertilized at differentrates exhibited a graded response to O3 exposure in proportionto the growth rates of the plants as controlled by N supply.

While year to year variation in leaf abscission can beaccounted for by plant size, the mechanistic explanation maylie in a link with growth (or rate of growth), as has been pos-tulated in the past (Pell et al., 1995) to account for differencesin O3 responsiveness of plants. Evaluation of plant growthdata in this experiment supports the idea that O3-inducedaccelerated senescence may require continued plant growth.Although O3 reduced whole plant leaf area in these experimentscompared with the charcoal-filtered controls, especially in theN withdrawal treatment, at no point did the extant wholeplant leaf area decline within a treatment (Fig. 2). Despitesevere leaf abscission (up to 50% of the total leaves producedin 1997), increase in whole plant leaf area only slowed, or wasmaintained at a constant level (Figs 2, 3). The finer timescaleof sampling leaf abscission by nondestructive-repeated meas-ures, revealed that when the poplars set bud at the end of theexperiment (thereby ending new leaf addition), leaf abscissionwas also arrested. This supports the hypothesis that demandof resources for new growth may be a driving factor for theseverity of O3-induced accelerated senescence of older foliage.

Why is O3-induced accelerated senescence seen in both of the N treatments?

When a single lower canopy leaf was followed throughout itsdevelopment we observed that there were no differences inthe physiological (Asat) or biochemical ( [TSP]) impact of O3relative to the charcoal-filtered control between N treatments(Figs 5, 6). Leaf [Rubisco] followed a pattern of response similarto that of [TSP] (data not shown). This finding agrees withthat reported by Einig et al. (1997) and Maurer et al. (1997)in their studies on the O3 response of birch trees utilizingconstant low and constant high fertility treatments in sandculture. The fact that the decline in Asat and [TSP] in responseto O3 in older leaves was similar in the two N regimes, despitelarge differences in eventual magnitude of leaf senescence andabscission of the whole plant, suggests that there is differentialcontrol of senescence and abscission by environmentalconditions. Since the rate of decline in Asat and [TSP] did notdramatically differ in the two N treatments in the face ofapparently different conditions of plant demand for newgrowth, it could be possible that the rate of decline at a

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particular O3 exposure is rather fixed and involves only theexpression of a senescence program. The N available to theplant (or the internal N status) may only serve to modulatethe sensitivity of the cue for initiating the expression of theprogram. Alternatively, O3 could be exerting the same impactupon leaf senescence and nutrient remobilization in bothN treatments, but the N withdrawal may provide a signalto ultimately complete the process by triggering abscission.Nitrogen availability in the root zone can influence the pro-duction and export of abscission promoting hormones such asABA to the shoot (Aiken & Smucker, 1996). Root zone NO3

availability also strongly influences production and export ofthe senescence-preventing hormone cytokinin to the shoot(Aiken & Smucker, 1996).

The O3-induced reductions in Asat (Fig. 5) and [TSP](Fig. 6) observed in older leaves are similar to those reportedpreviously (Reich & Lassoie, 1985; Maurer et al., 1997; Brendley& Pell, 1998). However, the observation that O3 exposureelicited similar responses in both N treatments seems to con-tradict our hypothesis that O3 impacts would be greater in theN withdrawal than the constant N treatment. We observedthat plants grown in constant N had a lower degree of leafsenescence and abscission in response to O3. The similar shiftsin Asat and [TSP] in both N treatments may result fromanalysing a single leaf in the lower canopy. The leaf measuredwas within the group that senesced and abscised, for even theconstant N treatment exhibited some leaf abscission, but itwas much less than in the N withdrawal treatment. The pooledlower leaf [N] data (Fig. 7) demonstrates that when the lowercanopy leaves are considered as a pool, the [N] of the two O3treatments is similar at the beginning of the experiment. There-after leaf [N] declines in the N withdrawal treatment until allof the leaves ultimately abscise. The number of leaf positionsin the lower leaf fraction of the two N treatments was approx-imately the same (data not shown). The [N] data provide someevidence that the loss of N from lower canopy leaves as a resultof O3-induced accelerated senescence is more severe in the Nwithdrawal than the constant N treatment.

Are potentially compensatory physiological phenomenalinked to accelerated senescence of older foliage? Nitrogentreatment had a primary effect upon the performance of theyounger leaf in the upper canopy. In most cases, N withdrawalsignificantly lowered absolute levels of Asat and [TSP] relativeto the constant N treatment. The effect of O3 was subtle, andvariable with harvest date, depending on the response para-meter measured. Ozone appeared to elevate Asat above the CFcontrol in the N withdrawal treatment, especially at two of themeasurement dates, but the effect was statistically significantonly at the P = 0.0890 level (Fig. 5). The Asat result was weakerthan expected, but the trend supported previous observations(Brendley & Pell, 1998; Landry & Pell, 1993). Using the samehybrid poplar clone reported herein, these authors observedelevated Asat in response to O3 in remaining foliage undergoingO3-induced accelerated senescence.

Although Asat was only measured in 1996, another parameterof leaf performance, [TSP], was evaluated in 3 yr of experiments.With some variation between years, [TSP] declined in responseto N withdrawal, a commonly observed response to low Navailability (Marschner, 1995). Individually, the [TSP] responseobserved in different years varied somewhat in relation to Nwithdrawal and O3 exposure. In 1996 there was a clear increasein response to O3 exposure while in 1997 and 1999 elevated[TSP] in response to O3 were only detected transiently in theearliest part of the lifespan of the leaf. Despite the transienceof the effect in 1997 and 1999, an c. 20% increase in [TSP]in the O3-exposed N withdrawal leaves over charcoal-filteredcontrols was seen when all dates were pooled (Fig. 6). Thiswas significantly different from the constant N plants whereno difference was seen between the O3-exposed and charcoal-filtered leaves (Fig. 6). This difference is evidence of a responseby the N withdrawal plants that could result in compensatoryC assimilation by the younger leaves. The transient and earlyresponse of young leaf protein concentration to O3 was alsoobserved by Brendley & Pell (1998).

It may be that a developmentally transient physiologicalresponse will be missed if leaf sampling is only performed onone leaf position through time. Therefore, the [total N] of theupper, expanding leaves was also determined throughoutthe experimental period (Fig. 7). This leaf pool represents amoving window over time that stays centred upon the leavesthat have not yet reached full expansion. [Total N] is elevatedin response to O3 exposure in both of the N treatments (Fig. 7and Table 4). This is further evidence of compensatoryperformance by younger leaves in response to O3 as has beenreported by previous studies (Pell et al., 1994). The fact thatan increase in [N] in the younger leaves was observed whensampled in a manner that evaluates only the currentlyexpanding leaves at any timepoint supports the hypothesisthat elevated performance of younger foliage will only be seenin the treatment where there is a large degree of O3-inducedaccelerated senescence.

Conceptual diagram

A simplified diagram of N pools within the plant, and thephysiological transformations among these pools is presentedbased upon the data obtained from this study and from the liter-ature concerning the effects of O3 and reduced N availabilityon plant growth (Fig. 8). In the diagram, O3 induces leafsenescence, which promotes the remobilization of N from olderleaves to new leaf growth via the stems and roots. Leaf senescencemay eventually culminate in leaf abscission resulting in a lossof N from the plant system. The N loss is a consequence of thefact that remobilization of nutrients from senescing leavesis never complete; the efficiency with which nutrients areremobilized from senescing leaves is dependent upon geneticand environmental factors (Killingbeck, 1996). Decreased Nin the root environment will decrease the N taken up by the

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plant roots and consequently reduce the supply of N for usein new leaf growth. Decreased external N may also be sensedby the roots, altering the synthesis and export of hormonessuch as abscisic acid and cytokinins to the leaves of the plant.These hormones could potentially modify various processesinvolved in O3-induced accelerated senescence. Effects couldinclude an increase in sensitivity to O3 as an initiator of leafsenescence, directly regulating the rate of senescence itself,and potentially providing a signal to promote leaf abscission.Although the data (e.g. xylem hormone concentrations) toascertain the mechanism of an interaction were not collected,it is evident that N availability to the plant is modulating theresponse of the plant to O3 since the N withdrawal treatmentdid not induce appreciable leaf loss in the absence of O3.

Conclusions

When N availability decreases, the rate of O3-induced acceleratedsenescence increases. This response appears to be associatedwith plant size and, possibly, continued active growth. Com-pensatory responses of young leaves to O3 exposure onlyoccured when N availability was restricted and O3-inducedaccelerated senescence was most severe. In an upcoming pub-lication we report on further experiments in which 15N wasused to determine the source (newly acquired or remobilized)of N for new leaf growth in plants experiencing O3-induced

accelerated senescence, and net N flux in different organs inthe plant.

Acknowledgements

DGB thanks Judith P. Sinn and Christian Vinten-Johansen fortechnical expertise and assistance and Dr David M. Eissenstatfor use of the elemental analyzer. This research was supportedby NSF Grant IBN-961030 and NSF Training Grant BIR-9413204. This research was also supported in part by thePennsylvania Agricultural Experiment Station and the Environ-mental Resources Research Institute, Contribution no. 2074,Department of Plant Pathology, The Pennsylvania StateUniversity, USA.

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Fig. 8 Simplified diagram of N flow between plant N pools with reference to the effects of O3-induced accelerated senescence and a decline in N availability. Subscripts identify N pool: NExt, external N concentration; NS,R, stem and root N; NYL, young leaves; NOL, old leaves; NLitter, N lost to the plant via leaf abscission. Solid arrows represent flux of N between pools within/without the plant. Where paired arrows are present, thickness indicates relative flux. Dotted arrows indicate regulatory influence; circle with plus sign indicates promotion.

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