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enrichment enhances flag leaf senescence in barley due

to greater grain nitrogen sink capacity

A. Fangmeier


*, B. Chrost


, P. Ho¨gy


, K. Krupinska


aInstitut fu¨r Pflanzeno¨kologie der Justus-Liebig-Uni

6ersita¨t,Heinrich-Buff-Ring26-32 (IFZ),D-35392Gießen,Germany bBotanisches Institut der Uni

6ersita¨t zu Ko¨ln,Gyrhofstraße15,D-50923Ko¨ln,Germany cBotanisches Institut und Botanischer Garten der Christian-Albrechts-Uni


Received 21 March 2000; received in revised form 4 July 2000; accepted 4 July 2000


Senescence is a highly regulated process which is under genetic control. In monocarpic plants, the onset of fruit development is the most important factor initiating the senescence process. During senescence, a large fraction of plant nutrients is reallocated away from vegetative tissues into generative tissues. Senescence may therefore be regarded as a highly effective salvage mechanism to save nutrients for the offspring. CO2 enrichment, besides

increasing growth and yield of C3plants, has often been shown to accelerate leaf senescence. C3plants grown under

elevated CO2experience alterations in their nutrient relations. In particular their tissue nitrogen concentrations are

always lower after exposure to elevated CO2. We used a monocarpic C3crop — spring barley (Hordeum6ulgarecv.

Alexis) — grown in open-top field chambers to test the effects of CO2enrichment on growth and yield, on nitrogen

acquisition and redistribution, and on the senescence process in flag leaves, at two applications of nitrogen fertilizer. CO2enrichment (650 vs. 366mmol mol−1) caused an increase both in biomass and in grain yield by 38% (average

of the two fertilizer applications) which was due to increased tillering. Total nitrogen uptake of the crops was not affected by CO2treatment but responded solely to the N supply. Nitrogen concentrations in grains and straw were

significantly lower (−33 and −24%) in plants grown at elevated CO2. Phenological development was not altered by

CO2until anthesis. However, progress of flag leaf senescence as assessed by chlorophyll content, protein content and

content of large and small subunit of RubisCO and of cytochrome b559 was enhanced under elevated CO2

concentrations by 4 days. We postulate that CO2enhanced flag leaf senescence in barley crops by increasing the

nitrogen sink capacity of the grains. © 2000 Elsevier Science B.V. All rights reserved.

Keywords:Chlorophyll; Grain-filling;Hordeum6ulgare; Open-top field chambers; Proteins; Redistribution; Yield

1. Introduction

Leaf senescence is a complex and highly coordi-nated developmental process under genetic con-trol (Smart, 1994; Noode´n et al., 1997) which precedes cell death (Gan and Amasino, 1997; * Corresponding author. Tel.:+49-641-9935315; fax:+


E-mail address: (A. Fangmeier).


A.Fangmeier et al./En6ironmental and Experimental Botany44 (2000) 151 – 164 152

Chrost et al., 1999). Several attempts have been made to distinguish between different stages dur-ing the leaf senescence process, and somewhat different classifications and numbers of stages have been proposed. It has been generally assumed that regulatory genes are involved in the initiation of senescence. Later on, other senescence-associated genes (SAG’s) are expressed which are encoding proteins involved in breakdown of macromolecules and, thus, in mobilization of nutrients, such as RNAses, proteinases, and lipases (Gan and Amasino, 1997; Thompson et al., 1998). Another class of SAG’s expressed at later stages appears to be related to protective or stress response functions (Bleecker, 1998). Up to now, more than 30 SAG’s have been identified, cloned and characterized (Biswal and Biswal, 1999). Recently, it has also been shown that leaf senescence — at least at late stages — is accompanied by programmed cell death (Yen and Yang, 1998).

One of the most obvious events occurring during early senescence at the cellular level is the transfor-mation of chloroplasts into gerontoplasts (Smart, 1994; Noode´n et al., 1997). The thylakoids are disrupted quite early whereas the plastid envelope remains intact until final stages of senescence (Thompson et al., 1998; Chrost et al., 1999). The disassembly of the photosynthetic apparatus pro-ceeds in a highly regulated manner. In field-grown

barley (Hordeum6ulgare), flag leaf senescence was

first detectable by reduced photosynthetic capacity which was accompanied by decreasing D1 protein

(8 days after anthesis). About 6 days later,

pigment content, photosystem II efficiency, cy-tochrome f and the large subunit of

ribulose-1,5-bis-phosphate carboxylase/oxygenase (RubisCO)

started to decline, whereas the small subunit of RubisCO remained high until 22 days after anthesis (Humbeck et al., 1996).

During dissassembly of the chloroplasts, 70%

of the total nitrogen contained in the cells is remobilized and allocated to sink tissues with high nutrient demand (Smart, 1994; Gan and Amasino, 1997). In monocarpic plants, developing seeds or fruits are the most important sink for nutrients allocated from senescing leaves. In experiments with wheat, we found nitrogen concentrations in flag leaves to decrease by 78% from anthesis to

maturity, and phosphorous concentrations to de-crease by 82%. Correspondingly, at maturity the wheat grains contained 85% of total shoot nitrogen and 94% of total shoot phosphorus, respectively (Fangmeier et al., 1997).

In monocarpic species the onset of fruit develop-ment is the most important factor initiating the senescence process (Noode´n et al., 1997). In addi-tion the onset and progression of senescence is influenced by many external factors such as day-length or insufficient supply of resources (light, water, nutrients) (Smart, 1994; Kleber-Janke and Krupinska, 1997). The molecular mechanism of senescence initiation in relation to fruit develop-ment is not yet clear. Nutrient demand from the developing reproductive tissues might trigger the senescence process in leaves (Kelly and Davies, 1988). Other authors, however, postulate the exis-tence of a ‘death hormone’ initiating the senescence process (Noode´n and Leopold, 1978; Wilson, 1997). Whatever the signal transduction pathway is, senescence (at least in monocarpic species) may be regarded as a highly effective nutrient salvage mechanism for plants to transfer the nutrients to their offspring. It is likely that the nutrient salvage function of senescence is the primary reason for the evolution of such a complicated, multi-factorial process, and that the end of the life-cycle of the organs concerned is just an inevitable by-product (Bleecker, 1998).

Nutrient relations in plants are affected by the

concentration of atmospheric CO2 which

repre-sents the most important plant nutrient in the

biosphere (45% of plant dry matter is carbon).

Due to human activities disturbing the global

carbon cycle, atmospheric CO2 concentrations

have been increasing from 280 mmol mol−1 in

pre-industrial times to more than 360mmol mol−1

today, and a further increase to at least 450 or,

more likely, 700mmol mol−1by the end of the next

century appears inevitable (Anonymous, 1995).

Atmospheric CO2enrichment may be regarded as

a global fertilization of the biosphere with the most abundant plant nutrient. Elevated atmospheric

CO2 concentrations do not only promote growth

and biomass of plants (most effectively in C3

species, Poorter et al., 1996) but have also conse-quences for the demand for other nutrients


ratios in tissues of C3-plants have been shown to increase considerably (Conroy, 1992; Cotrufo et al., 1998). This is not caused by a simple ‘dilution’ due to higher carbohydrate concentra-tions, but is rather due to a decreased demand for

nitrogen in green tissues. Under CO2 enrichment,

optimization of resources within the photosyn-thetic apparatus may occur (Webber et al., 1994) since ribulose-bisphosphate- (RuBP) and

phos-phate- (Pi) regeneration rather than carboxylation

by RubisCO will limit the rate of CO2

assimila-tion (Harley and Sharkey, 1991). Decreased con-tent of RubisCO (Moore et al., 1999) which comprises up to 60% of soluble leaf protein (Ja-cob et al., 1995) will reduce total nitrogen demand of green tissues. Additionally, the depression of the photorespiratory pathway (approximetaly half

at doubled CO2 concentrations, Sharkey, 1988)

will also decrease the leaf nitrogen demand be-cause of smaller contents of enzymes of the glyco-late pathway (Webber et al., 1994; Fangmeier and Ja¨ger, 1998).

In previous studies with cereal crops, we could demonstrate that nitrogen uptake by the crops

was not affected by CO2 enrichment, but was

dependent on nitrogen supply (Fangmeier et al., 1997). At the same time, grain yield was

signifi-cantly increased under CO2 enrichment. We also

observed a faster progress of senescence, and an earlier remobilisation of proteins, in flag leaves of

wheat crops under CO2 enrichment (Vermehren

et al., 1998). Similar observations have been made by Sicher and Bunce (1998).

We assume that enhanced flag leaf senescence

during grain filling in cereals under CO2

enrich-ment may be triggered by the different effects of

elevated [CO2] on grain production (which is

thought to be increased) on the one hand, and on the acquisition and storage of nitrogen in vegeta-tive pools used during grain filling (which are thought to be not affected), on the other hand. By

this means, CO2 enrichment might accelerate

senescence via increased grain nutrient sink capac-ity. We also speculate that the effect of elevated

CO2 on flag leaf senescence will not be mitigated

by additional nitrogen fertilization as additional N will lead to increased biomass and yield rather than to higher nitrogen pools that would be

avail-able per unit grain yield.

To test these hypotheses, we used open-top field

chambers to expose spring barley (H. 6ulgare cv.

Alexis) crops to CO2 enrichment at two nitrogen

supplies and assessed the effects on growth and yield, on nitrogen acquisition and redistribution by the crops, and on the progress of senescence in barley flag leaves.

2. Materials and methods

2.1. Plant culture

On May 3, 1997, (Julian Date (JD) 123) spring

barley (H. 6ulgare L. cv. Alexis) was sown into

circular containers with a volume of 90 l (diame-ter 61 cm, depth 40 cm) which were placed in open-top field chambers (OTC). Sowing density

was 500 plants per m2. One week after seedling

emergence (JD 127) the stands were thinned to

250 plants per m2. Soil was taken from an

agricul-tural field site with vega (fluvisol) as prevailing soil type and mixed with sand 1:1 (vol:vol) to get

a substrate low in organic matter (B1%). The

crops were supplied with two levels of NPK-fertil-izer, also containing micronutrients,

correspond-ing to 140 kg N ha−1

ha−1on JD 140, 148, and 163, respectively, which

corresponded to growth stages (according to Tottman and Broad, 1987) 13, 22 and 34. OTC were equipped with a rain exclusion top. The plants were regularly irrigated using deionized water to avoid any drought stress. Green side shading panels, which reduced diffuse radiation by 50%, were raised as the crop grew. The pots were covered with white isolating panels to avoid high soil temperatures.

2.2. CO2 exposure

Plants were exposed to CO2for 24 h per day in

circular 3.15 m diameter OTC as described by Fangmeier et al. (1992), as soon as seedlings emerged (on JD 127) until canopy maturity (on

JD 231) at final harvest. CO2mean target


A.Fangmeier et al./En6ironmental and Experimental Botany44 (2000) 151 – 164 154

(elevated) mmol mol−1 (see Fig. 1). Air samples

for CO2 monitoring were taken 5 cm above the

top of the canopies. The air sampling lines were moved up as the canopies grew. Microclimatic conditions in OTC were monitored continuously throughout the season and logged as hourly means.

2.3. Assessments

2.3.1. Har6ests for biomass and yield estimations An inner circle of the canopies 50 cm in diame-ter was used for final harvest at maturity to gain biomass and yield data. Samples were separated into main stem ear, tiller ears, and straw (leaves and stems). Because appropriate sampling of roots from cereal crops is extremely time-consum-ing and only restricted man-power was available, roots were not harvested. Straw samples were dried at 70°C, and ears at 35°C, until weight constancy. Ears were threshed to estimate yield, grain number, thousand grain weight, and glume weight, separately for main stem ears and for tiller ears. Nitrogen concentrations were estimated in these samples by Kjeldahl.

2.3.2. Chlorophyll content

Chlorophyll contents of flag leaves were esti-mated non-destructively at the mid position of flag leaf blades using a SPAD 502 chlorophyll meter (Minolta, Japan) on dates when, and just before, flag leaf harvests took place (see below).

2.3.3. Har6ests for estimations of protein

degradation during flag leaf senescence

Flag leaf harvesting and SPAD measurements took place on 7 occasions over a period of 20 days from full maturity of flag leaves (JD 189) until nearly complete senescence (JD 209). Flag leaf ligules were visible on JD 166 (growth stage 39, Tottman and Broad, 1987), and anthesis (growth stage 65) was on JD 178 in all treatments. Twenty two border plants per pot, growing out-side the 50 cm diameter circle, were used for the intermediate harvests. Only plants from high nu-trient supply treatments were analysed. Harvests were done between 1:00 and 2:00 p.m. Then, fresh weight and length of flag leaves were measured immediately and samples were frozen in liquid nitrogen within one minute after harvest. Samples

were stored at −80 °C until analysis.

Data on leaf blade length were used to calculate leaf area by regression analysis of 30 flag leaves harvested from sparse plants in ambient plots.

Best fit (adjusted R2

=0.982) was achieved using

the formula:


wherexis the length of flag leaf blade (cm) andy

is the leaf area (cm2).

Proteins were extracted from flag leaf samples as described earlier (Humbeck et al., 1996). Protein concentrations were estimated using the

method of Lowry et al. (1951). Aliquots of 30mg

protein were subjected to SDS – PAGE, and

incu-bated with antibodies directed towards

cy-tochrome b559 (Cyt b559), and large subunit (LSU) and small subunit (SSU) of RubisCO. Gel photographs were analysed for protein contents using a Kodak Electrophoresis Documentation and Analysis System 120 in order to yield relative contents of Cyt b559, LSU, and SSU, based on flag leaf area.

Fig. 1. (A) Daily mean air temperature and cumulative daily global radiation, and (B) CO2concentrations (daily means) in


2.4. Statistical design and data e6aluation

CO2 treatments were carried out in three

repli-cate OTC, respectively. For each nutrient supply, one barley pot was exposed within each OTC. OTC means served as input data for analysis of

variance, usingSPSS 8.0 for Windows (SPSS Inc.,

Chicago). Biomass and nitrogen data represent

CO2 treatment means9standard deviation based

on three chamber replicates.

SPAD measurements were taken both on plants in the inner circle left for final harvest and on border plants. There were no significant differ-ences between these data-sets. SPAD data given for any particular date represent means of eight to 15 single measurements per OTC and each nutri-ent supply. Standard deviation bars shown in the graph were calculated using means of three repli-cate OTC, respectively.

Because of restricted number of border plants available for intermediate flag leaf harvests, no harvests in replicate OTC could be carried out. Rather, at any harvest date four flag leaves taken from all three replicate OTC were combined to yield one sample.

3. Results

3.1. CO2 concentrations and climatic conditions

Seasonal CO2 daily averages measured on top

of the canopy from barley crop emergence (on JD

127) to final harvest (on JD 231) were 36690.9

mmol mol−1 in ambient OTC and 650920 mmol

mol−1 in elevated OTC (Fig. 1). Since no

feed-back control was used in the CO2-dispensing

sys-tem, concentrations in OTC with elevated CO2

were somewhat influenced by ambient wind speed. With high ambient windspeeds, air in OTC

en-riched with CO2 was mixed with intruding

ambi-ent air which caused CO2 concentrations to fall.

Fig. 1 also presents daily averages for air tem-perature and global radiation monitored in OTC. Average temperature during crop growth (JD 127 to 231) was 19.8°C, cumulative temperature (at baseline of 0°C) amounted 2079 day-degrees, and

cumulative radiation sum was 1103 MJ m−2.

Average vapour pressure deficit (VPD) in OTC

during daylight hours (global radiation \50 W

m−2) from crop emergence to final harvest was

15.04 hPa (no VPD data shown in Fig. 1).

3.2. Treatment effects on biomass and yield

Barley crops grown under CO2 enrichment

ac-quired 38% more shoot biomass than those grown

under ambient CO2 concentrations (Table 1).

Crops fertilized with 14 g N m−2

increased shoot

biomass by 13% compared with 8 g N m−2

. CO2

enrichment and nitrogen supply interacted

posi-tively, i.e. biomass increase due to CO2

enrich-ment was larger at high N supply than at low N supply (45 vs. 30%) and, vice versa, additional nitrogen had greater impact on shoot biomass at

650 mmul mol−1 CO2 than at ambient CO2

(in-crease 18 vs. 7%).

Treatment effects on total grain yield were sim-ilar to shoot biomass responses. Correspondingly, there were no significant treatment effects on

har-vest index. Increases in grain yield due to CO2

enrichment and additional nutrient supply were solely attributable to increased tillering. Neither

CO2 enrichment nor additional fertilization had

any significant effect on grain yield from main stems. However, yield from tiller ears was

en-larged 2.3-fold under 650mmol mol−1CO2.

Addi-tional fertilization increased tiller grain yield by

29%. As for shoot biomass and total yield, CO2

enrichment and nitrogen supply interacted posi-tively on tiller grain yield.

Thousand grain weight (TGW) of main stem

grains was slightly reduced by CO2 enrichment

(−7.5%). This caused also a decline in the

aver-age TGW for all grains. There was no such effect on grains from tiller ears. Fertilization did not affect TGW. Thus, the sink capacity for carbon of one single grain was only slightly affected by the treatments.

3.3. Treatment effects on nitrogen acquisition

In contrast to the effects on shoot biomass and

on yield, CO2enrichment did not affect the




















Table 1

Yield and biomass of barley crops exposed to CO2 enrichment at two levels of nitrogen supply

Elevated CO2 (650919

Ambient CO2 (36690.9

Nitrogen supply Results of analysis of variancea(based on open-top field


chamber means)

mmol mol−1) mmol mol−1)

(g m−2)

N CO2N Adjusted R2 CO



PB0.001 P 1400927

967973 P=0.021 14

Shoot biomass acquisition (g 0.947 (PB0.001) m−2)

=0.001 8 909923 1190948


14 0.934 (PB0.001) PB0.001 P=0.013 Grain yield total (g m−2) 515948 762910

=0.002 49297.5 631932



Grain yield tillers (g m−2) 14 170949 420917 0.938 (PB0.001) PB0.001 P=0.032

=0.004 8 14894.3 310927

51.490.4 0.604 P=0.003 n.s. n.s. Thousand grain weight all 14 54.291.2

grains (g) (P=0.015) 8 53.790.5 49.692.6

n.s. n.s. n.s. n.s.

Thousand grain weight tiller 14 46.590.8 48.291.3 grains (g)

8 44.692.7 46.592.2


Fig. 2. Chlorophyll breakdown (assessed as SPAD values) during flag leaf senescence in spring barley crops exposed to ambient or elevated CO2 at two levels of nitrogen supply.

Data represent means9standard deviation from three repli-cate OTC. Regression lines were calculated using logistic func-tions.

with 8 g N m−2contained 11.2 g N m−2in their

shoots. Thus, not only nitrogen from fertilization but also from pools in the soil and from mineral-ization served as sources for the crops. Crops

provided with 14 g N m−2contained less nitrogen

in the shoots (13.3 g m−2) than was supplied as

mineral fertilizer. Most of the nitrogen found in the shoots at final harvest was allocated to the grains which contained 77% of total shoot N on average. This nitrogen allocation pattern was not influenced by the treatments (Table 2).

Nitrogen concentrations (expressed as % of dry weight) both in straw and in grain samples were not significantly affected by nutrient supply. This suggests that no nitrogen deficiency occurred in

crops provided with only 8 g N m−2. CO


enrich-ment, on the other hand, reduced the nitrogen concentration of the straw by 34% and that of grains by 24%. Since nitrogen concentrations in grains from main stems and from tillers, and treatment effects on it, were nearly identical, only average grain nitrogen concentrations are pre-sented in Table 2. As a consequence of decreased nitrogen concentrations in the tissues, the nitro-gen use efficiency of barley crops was improved

by 35% due to CO2 enrichment.

3.4. Treatment effects on flag leaf senescence

3.4.1. Chlorophyll breakdown

Chlorophyll content was assessed non-destruc-tively during flag-leaf senescence using a Minolta SPAD meter. SPAD values obtained from JD 191 to 209 are shown in Fig. 2. Assessments started right before chlorophyll breakdown began. Before breakdown, chlorophyll content in crops grown

under elevated CO2 were significantly lower than

under ambient CO2(ANOVA results not shown).

However, no significant effects of nutrient supply on chlorophyll degradation were observed. Pro-gress of chlorophyll breakdown was significantly

enhanced by CO2 enrichment. In crops grown at

650 mmol mol−1CO2, chlorophyll degradation to

50% of the maximum values was already achieved on JD 199, whereas it took until JD 203 to get the

same degree of degradation at ambient CO2.

found in barley shoots was 13.2 g m−2at ambient

CO2 and 13.4 g m

−2 at elevated CO

2 when the

plants had high nutrient supply (Table 2). At low

fertilization, the shoots contained 11.2 g m−2

nitrogen (ambient CO2) or 11.3 g m



(elevated CO2). The amount of nitrogen in the

grains of the crops at maturity was 9.83 (ambient

CO2) and 10.7 g m


(elevated CO2) at high N

supply, and 8.60 and 8.75 g m−2at low N supply,

respectively, without any significant impact of

CO2enrichment. However, nitrogen acquisition in

grains on main stems decreased by 26% under

CO2 enrichment corresponding to the decrease in

nitrogen concentration in grains (see below). Ni-trogen acquisition by tiller grains increased by

75% when the plants were grown at 650 mmol


CO2 which results both from the 2.3-fold

increase in tiller grain yield, and the 26% decrease

in grain nitrogen concentration, at elevated CO2.




















Table 2

Nitrogen acquisition of barley crops exposed to CO2enrichment at two levels of nitrogen supply

Parameter Nitrogen supply Ambient CO2 Elevated CO2 Results of analysis of variancea(based on open-top field



(g m−2) chamber means)




Adjusted R2


n.s. PB0.001 n.s. 13.490.2

14 13.290.4 0.826 (P=0.001) Shoot nitrogen acquisition (g


8 11.290.8 11.390.3

0.44090.017 0.787 (P=0.001) PB0.001 n.s. n.s. Straw nitrogen content (% dry 14 0.73790.100



8 0.46790.029

PB0.001 n.s. n.s. 0.865 (PB0.001)


Main stem grain nitrogen 6.6290.49 4.8990.32 acquisition (g m−2)

8 6.1290.30 4.5390.22

1.4090.04 0.904 (PB0.001) PB0.001 n.s. n.s. Grain nitrogen content (% dry 14 1.9290.10


8 1.7590.11 1.3990.02

PB0.001 n.s. n.s. 0.932 (PB0.001)

Nitrogen use efficiency (g DM (g 14 73.493.6 104.093.6 N)−1)

8 81.395.6 104.591.4


3.4.2. Time course of soluble protein content

Time-course of total soluble proteins in barley flag leaves (Fig. 3) was very similar to that of chlorophyll. Concentrations were in general lower

when the plants were grown under CO2

enrich-ment. Under ambient CO2, a reduction of protein

contents to 50% of maximum was achieved on JD

197.5, whereas at 650 mmol mol−1 CO2 protein

content was reduced to 50% already on JD 196. Thus, protein breakdown appeared to be

acceler-ated by CO2 enrichment, but was somewhat less

affected than chlorophyll disassembling.

3.4.3. Degradation of RubisCO LSU, SSU, and cytochrom b559

LSU breakdown was clearly accelerated under

CO2enrichment (Figs. 4 and 5). No LSU could be

detected any longer on JD 195, and afterwards, in

plants grown under 650 mmol mol−1 CO2,

whereas flag leaves from plants grown under

am-bient CO2 still had nearly maximum LSU

con-tents at that time. Decline of the SSU level differed signficantly from that of LSU. At

ele-vated CO2 concentrations, degradation of SSU

started only after JD 192. On JD 198, there was

still some SSU (6% of maximum contents)

de-tectable. At ambient CO2, no clear degradation of

SSU towards the end of the sampling period could be observed. Cyt b559 was longer detectable than LSU, but its level started to decline some-what earlier than the level of SSU. Opposite to LSU and SSU, levels of Cyt b559 appeared to

decline somewhat more rapidly at ambient CO2

than at elevated CO2.

4. Discussion

Biomass and yield of barley crops increased by

38% when grown under 650 instead of 366 mmol

mol−1 CO

2 which resulted solely from an

en-hancement of tiller production and tiller survival.

This CO2-fertilization is lower than reported in

previous studies. Poorter et al. (1996) compiled

data from various CO2 enrichment experiments

and cite one study for H. 6ulgare where an

in-crease in biomass of 104% occurred. However, only plants that were not in the reproductive phase were included into that data compilation. Thus, limitations occurring at later stages of de-velopment (nutrients, competition for light) were not included in the evaluation. In a season-long

study under CO2 enrichment, Weigel et al. (1994)

found increases in spring barley yield of up to 89%. However, these authors used plants in small pots with a low plant density. This might cause an

over-estimation of CO2 effects due to a lack of

LAI limitations (Ko¨rner, 1995). The results pre-sented in this study were obtained under condi-tions closely resembling field condicondi-tions. Thus, the 38% increase in yield appears to be realistic.

Plants from the high fertilizer treatment were not subjected to nitrogen limitation, as (1) there was no increase in tissue nitrogen concentrations when nitrogen supply was increased from 8 to 14

g N m−2

(compare Table 2), (2) the crops

fertil-ized with 14 g N m−2did not take up all of the N

applied (total nitrogen amount in the crop at final

harvest: 13.3 g m−2), whereas crops receiving

only 8 g N m−2had a higher nitrogen content on

an area basis (11.2 g m−2) than was applied. In

spite of sufficient nitrogen being available (at least

in the high N fertilizer treatment), CO2

Fig. 3. Concentrations of total soluble protein during flag leaf senescence in spring barley crops exposed to ambient or elevated CO2 and fertilized with 140 kg N ha−1. Data are


A.Fangmeier et al./En6ironmental and Experimental Botany44 (2000) 151 – 164 160

Fig. 4. Contents of different proteins (relative units per cm2leaf area) during flag leaf senescence in spring barley crops exposed to

ambient or elevated CO2and fertilized with 140 kg N ha−

1. Data are from four flag leaves randomly selected from three replicate

OTC at each harvest date, respectively. LSU, large subunit of RubisCO; SSU, small subunit of RubisCO; Cyt b559, cytochrome b559.

enrichment significantly depressed nitrogen con-centrations found in the tissues. In a recent data

compilation, CO2 enrichment has been found to

decrease the nitrogen concentration in green

leaves of non-woody C3 plants by 17%, and in

litter by 9% (Cotrufo et al., 1998). We found even greater reductions (40% in straw at high nitrogen supply, 27% in grains) which compares well to earlier findings in cereals (McKee and Woodward, 1994; Manderscheid et al., 1995; Fangmeier et al., 1997).

Several hypotheses have been proposed to ex-plain the reduced tissue nitrogen concentrations

under CO2 enrichment (Conroy and Hocking,

1993). The most likely candidates to explain these findings are: (1) an optimisation of the

photosyn-thetic apparatus in plants grown under high CO2

concentrations, by which less nitrogen is invested in RubisCO and more nitrogen is allocated to

RuBP-regeneration and to Pi-regeneration (Sage

et al., 1989; Webber et al., 1994; Moore et al., 1999) though this type of acclimation does not always take place and is less obvious in plants well supplied with nitrogen (Theobald et al., 1998; Stitt and Krapp, 1999), and (2) a reduction of the photosynthetic carbon oxidation (PCO) pathway, and thereby a reduction of the requirement for

PCO enzymes, under altered CO2/O2partial


et al., 1999), and of PCO enzymes (Fangmeier and Ja¨ger, 1998) has often been observed in plant

leaves grown at elevated CO2.

The phenological development of the barley crops before grain filling was not responsive to

CO2 enrichment. Dates of plant emergence, of

terminal spikelet formation, of flag leaf appear-ance and of anthesis were identical at either treat-ment. Nevertheless, flag leaf senescence was

significantly affected by CO2 enrichment.

En-hancement of flag leaf senescence was detectable from both estimations of chlorophyll degradation and of the contents of soluble proteins. The latter estimations suggest an earlier protein breakdown and re-allocation away from the leaves. Among the three plastid proteins assessed in our study, the amounts of the LSU of RubisCO declined first. Contents of Cyt b559 clearly declined later than LSU. SSU kept rather high amounts until the end of the harvest period. On JD 205, i.e. 27 days after anthesis, still 31% of maximum SSU

contents were detected at ambient CO2. The lack

of coordination in the decline of RubisCO sub-units has also been observed with flag leaves of field-grown barley crops (Humbeck et al., 1996).

CO2 enrichment accelerated the decline of both

LSU and SSU contents by approximately four days. In contrast, the decline of Cyt b559

ap-peared to be somewhat delayed under CO2

enrich-ment. This might be related to a possible

acclimation effect of CO2enrichment on the

pho-tosynthetic apparatus. However, the data are to scarce to draw further conclusions.

Earlier senescence under CO2 enrichment has

been shown in many other studies with annual C3

crops. Miller et al. (1997) exposed tobacco to 350

or 950 mmol mol−1 CO2 and followed leaf CO2

exchange over the course of leaf development. The authors observed an earlier achievement of

maximum photosynthesis rates at elevated CO2

concentrations, but also a faster progress of devel-opment and an earlier beginning of senescence. From their data, Miller et al. (1997) conclude that

faster leaf development under CO2 enrichment

might explain the often observed photosynthetic

acclimation to elevated CO2. This statement is in

part supported by findings from wheat exposure

to CO2 enrichment (Sicher and Bunce, 1998)

where also an earlier flag leaf senescence was observed. The authors state that premature


A.Fangmeier et al./En6ironmental and Experimental Botany44 (2000) 151 – 164 162

senescence contributed to decreased

photosyn-thetic rates at elevated CO2concentrations. There

is further experimental evidence for a faster de-crease of photosynthetic properties and of en-zymes of the photosynthetic apparatus as a result

of CO2 exposure. Sicher and Bunce (1997) found

decreased RubisCO contents and photosynthesis rates, and an acceleration of senescence, in wheat

and barley leaves grown in elevated CO2, and

Garcia et al. (1998) observed a decline in flag leaf photosynthesis and an earlier senescence at final stages of wheat crop development under free air

CO2 enrichment.

We do not believe that a general enhancement of leaf development and, thus, an earlier onset of leaf senescence can be used as a simple explana-tion of photosynthetic down-regulaexplana-tion due to

CO2 enrichment. Functional and molecular

changes, and the redistribution of nutrients, oc-curring during senescence are too complex for such a simple explanation. Rather, one must search for a signal triggering senescence which is

affected due to growth at elevated CO2


In monocarpic annual species like barley or wheat, a rather rapid transition takes place from a

vegetative ‘green‘ plant acquiring CO2 from the

atmosphere and other nutrients from the soil, to a ‘generative‘ plant which does not acquire soil nutrients at significant amounts any longer be-cause of the breakdown of the root system (Fangmeier et al. 1996), but only redistributes these from vegetative tissues to the generative organs, i.e. to the grains, and which gradually reduces carbon acquisition from the atmosphere.

As stated above, CO2 enrichment reduces the

nitrogen demand of green tissues by several mech-anisms, causing an increase in nitrogen use effi-ciency. In our study (and in hardly any previous

experiment with C3 plants), growth at elevated

CO2 concentrations resulted in higher biomass

(Poorter et al., 1996) and, finally, in greater yield, irrespective of possible down-regulation of the photosynthetic apparatus at leaf level. For the whole plant, yield increase of the barley crops occurred due to increased tillering. Because of

increased nitrogen use efficiency under CO2

en-richment, nitrogen acquisition of the crops did

not keep pace with carbon gain. Rather, crop nitrogen uptake was dependent on nitrogen

sup-ply but did not respond to CO2 treatment. In

cereals, nearly all the nitrogen resources required for grain filling originate from vegetative tissues and there is hardly any further uptake of nitrogen from the soil after anthesis (Van Kraalingen, 1990; Fangmeier et al., 1999). Thus, the grains in

plants grown at elevated CO2 concentrations,

al-though they had 48% more biomass (at 140 kg

ha−1 of N fertilization), had to cope with the

same amount of nitrogen in vegetative pools as

the grains from ambient CO2. There is no reason

to assume that CO2 enrichment reduces the

nitro-gen demand of grains, since none of the mecha-nisms reducing nitrogen demand in green tissues is working in grains. Rather, for optimal grain vitality and (ecological) quality, a certain amount of grain proteins is required and monocarpic plants in particular have been selected to achieve this protein content in order to ensure survival of the population.

Thus, we believe that the higher nitrogen sink capacity of the growing population of grains

un-der CO2 enrichment works as a trigger to induce

nitrogen release from the leaves and can explain the earlier senescence of barley flag leaves ob-served in our study. Nitrogen deficiency has been proved to induce senescence in previous studies (Smart, 1994; Noode´n et al., 1997). In this case,

CO2 enrichment works as a tool to induce

nitro-gen deficiency as ’seen‘ by the grains in late developmental stages, though it does not cause a true deficiency in green tissues, in spite of lowered concentrations, at earlier developmental stages when the leaves act as carbon sources.


5. Conclusions

Our findings explain the acceleration of

senes-cence in annual monocarpic C3plants under CO2

enrichment as a process driven by the differing

effects of CO2on nutrients, in particular nitrogen,

in vegetative and generative tissues. We postulate

that CO2 enhances flag leaf senescence in barley

crops (and probably leaf senescence in

mono-carpic C3 species in general) by a sequence of

several processes induced by CO2: (1) CO2reduces

the nitrogen demand of green tissues by alter-ations of the photosynthetic apparatus, i.e. lower RubisCO contents, lower contents of enzymes of the PCO-cycle. Thus, nitrogen uptake does not keep pace with carbon acquisition during

vegeta-tive growth. (2) CO2 increases the number of

diaspores. Thus, the sink size for nutrients during seed ripening is enlarged. (3) During grain filling (or seed ripening in general) there is a higher demand for nutrient redistribution to the seeds

since CO2 enrichment does not reduce the

nitro-gen demand of the diaspores. Thus, a nitronitro-gen deficiency is induced during the switch from vege-tative to generative growth. (4) This deficiency acts as a trigger to induce leaf senescence in order to release nutrients from vegetative tissues accord-ing to the nutrient salvage function of senescence, although the demand of the seeds can not be accomplished due to restricted nutrient pools.


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Fig. 1. (A) Daily mean air temperature and cumulative dailyglobal radiation, and (B) COsix OTC chambers used to grow spring barley crops in the2 concentrations (daily means) ingrowth period, 1997
Fig. 1. (A) Daily mean air temperature and cumulative dailyglobal radiation, and (B) COsix OTC chambers used to grow spring barley crops in the2 concentrations (daily means) ingrowth period, 1997 p.4
Table 1Yield and biomass of barley crops exposed to CO2 enrichment at two levels of nitrogen supply

Table 1Yield

and biomass of barley crops exposed to CO2 enrichment at two levels of nitrogen supply p.6
Fig. 2. Chlorophyll breakdown (assessed as SPAD values)during flag leaf senescence in spring barley crops exposed tocate OTC
Fig. 2. Chlorophyll breakdown (assessed as SPAD values)during flag leaf senescence in spring barley crops exposed tocate OTC p.7
Table 2Nitrogen acquisition of barley crops exposed to CO2 enrichment at two levels of nitrogen supply

Table 2Nitrogen

acquisition of barley crops exposed to CO2 enrichment at two levels of nitrogen supply p.8
Fig. 3. Concentrations of total soluble protein during flag leafsenescence in spring barley crops exposed to ambient orfrom four flag leaves randomly selected from three replicateelevated CO2 and fertilized with 140 kg N ha−1
Fig. 3. Concentrations of total soluble protein during flag leafsenescence in spring barley crops exposed to ambient orfrom four flag leaves randomly selected from three replicateelevated CO2 and fertilized with 140 kg N ha−1 p.9
Fig. 4. Contents of different proteins (relative units per cm2 leaf area) during flag leaf senescence in spring barley crops exposed toambient or elevated CO2 and fertilized with 140 kg N ha−1
Fig. 4. Contents of different proteins (relative units per cm2 leaf area) during flag leaf senescence in spring barley crops exposed toambient or elevated CO2 and fertilized with 140 kg N ha−1 p.10
Fig. 5. Immunoblot analyses of plastid proteins during flag leaf senescence in spring barley crops exposed to ambient or elevatedCO2 and fertilized with 140 kg N ha−1
Fig. 5. Immunoblot analyses of plastid proteins during flag leaf senescence in spring barley crops exposed to ambient or elevatedCO2 and fertilized with 140 kg N ha−1 p.11


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