Growth Under Elevated Atmospheric CO2 Concentration Accelerates Leaf

8/11/2019 Growth Under Elevated Atmospheric CO2 Concentration Accelerates Leaf

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Journal of Plant Physiology 169 (2012) 13921400

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Journal ofPlant Physiology

j ournal homepage: www.elsevier .de/ jp lph

Growth under elevated atmospheric CO2concentration accelerates leafsenescence in sunflower (Helianthus annuus L.) plants

Lourdes de la Mata, Purificacin Cabello, Purificacin de la Haba, Elosa Agera

Departamento de Botnica, Ecologa y Fisiologa Vegetal, rea de Fisiologa Vegetal, Facultad de Ciencias, Universidad de Crdoba, Campus de Rabanales, Edificio Celestino Mutis(C4),3a planta, E-14071 Crdoba, Spain

a r t i c l e i n f o

Article history:Received 5 February 2012Received in revised form 24 April 2012

Accepted 21May2012

Keywords:Elevated CO2Hexoses

Oxidative status

Photosynthetic pigments

Senescence

Sunflower

a b s t r a c t

Some morphogenetic and metabolic processes were sensitive to a high atmospheric CO2 concentration

during sunflower primary leafontogeny. Young leaves ofsunflower plants growing under elevated CO2concentration exhibited increased growth, as reflected by the high specific leafmass referred to as dry

weight in young leaves (16days). The content ofphotosynthetic pigments decreased with leafdevelop-

ment, especially in plants grownunder elevatedCO2concentrations, suggesting that high CO2accelerates

chlorophyll degradation, and also possibly leaf senescence. Elevated CO2 concentration increased the

oxidative stress in sunflower plants by increasing H2O2 levels and decreasing activity of antioxidant

enzymes such as catalase and ascorbate peroxidase. The loss of plant defenses probably increases the

concentration ofreactive oxygen species in the chloroplast, decreasing the photosynthetic pigment con-

tent as a result. Elevated CO2concentrationwas found to boost photosynthetic CO2fixation, especially in

young leaves. High CO2 also increased the starch and soluble sugar contents (glucose and fructose) and

the C/N ratio during sunflower primary leafdevelopment. At the beginning ofsenescence, we observed

a strong increase in the hexoses to sucrose ratio that was especially marked at high CO2 concentration.

These results indicate that elevatedCO2concentration could promote leafsenescence in sunflower plants

by affecting the soluble sugar levels, the C/N ratio and the oxidative status during leafontogeny. Itis likely

that systemic signals produced in plants grownwith elevated CO2 , lead to early senescence and a higher

oxidation state ofthe cells ofthese plant leaves. 2012 Elsevier GmbH. All rights reserved.

Introduction

Continuous emissionsof CO2from theburning of fossil fuels are

expected to raise global atmospheric CO2 concentrations. Human

activities not only affect CO2 concentrations, but also alter the

global nitrogen cycle by increasing the inputs of fixed forms of

nitrogen,mainly through extensiveuse of chemical fertilizers. The

Intergovernmental Panel on Climate Change (IPCC) has predicted

that the CO2 concentration may increase by 660790L L1 from

2060 to2090(IPCC, 2007). This is expected to raise global temper-

atures due to the CO2 capacity to absorb infrared light (Schneider,1989; Taylor andMacCracken, 1990). Therefore, continuous emis-

sionsof this gasat high levelsarebelieved to causeclimate change.

One of the most obvious effects of climate change is its effect

Abbreviations: APX,ascorbate peroxidase;DW,dryweight;ROS, reactiveoxygenspecies; RuBP, ribulose-1,5-bisphosphate; rubisco, ribulose-1,5-bisphophate car-

boxylase/oxygenase; SLM,specificleafmass;XET,xyloglucan endotransglycosidase. Corresponding author. Tel.: +34957218367;fax: +34957211069.

E-mailaddresses:[email protected](L.de laMata), [email protected] (P.Cabello),[email protected] (P. de la Haba), [email protected] (E. Agera).

on living beings, especially on plants, which have been found to

exhibit alterations potentially affecting some steps of their growth

cycle. Studies on various plant species have suggested that climate

changes will affect the development, growth and productivity of

plants through alterations in their biochemical, physiological and

morphogenetic processes (Bazzaz andFajer, 1992).

Senescence is a stage of the plant growth cycle that involves

strong metabolic and structural changes. Markers associated with

leaf senescence in sunflower plants have shown that senescence

initiates and progresses in primary leaves aged between 28 and

42days (Cabello et al., 2006). Senescence typically involves ces-sation of photosynthesis and degeneration of cellular structures,

withstronglosses of chlorophyll (Oughametal., 2008), carotenoids

and proteins and a great increase of lipid peroxidation (Srivalli

and Khanna-Chopra, 2004; Agera et al., 2010). Senescence is not

only a degenerative process, but also a recycling process bywhich

nutrients are translocated from senescing cells to young leaves,

developing seeds or storage tissues (Gan and Amasino, 1997).

Leaf senescence is therefore an active, highly regulated and pro-

grammed degeneration process, required for plant survival and

controlled by multiple developmental and environmental signals

(Lim et al., 2003). Senescence induction anddevelopment are both

0176-1617/$ seefrontmatter 2012 Elsevier GmbH. All rights reserved.

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L. de laMata et al. / Journal of Plant Physiology169 (2012) 13921400 1393

seemingly governed by intrinsic and extrinsic factors that act by

acceleratingor delayingtheprocess. Somestudies haveshown that

leaf senescence is regulatednot onlybychanges inhormone levels,

photosynthetic performance, carbohydrate contents and specific

signals, but also by reactive oxygen species (ROS) (Hensel et al.,

1993; Quirinoet al.,2000;Orendi et al.,2001; Buchanan-Wollaston

et al., 2003a).

Senescence can start prematurely by the effects of exposure to

environmental stress or nutrient deprivation (Quirino et al., 2000;

Lim et al., 2003, 2007; Wingler et al., 2009; Agera et al., 2010).

Leaf senescence in sunflowerplantsis acceleratedby nitrogendefi-

ciency (Agera et al., 2010) and also by increased light exposure

during growth (De la Mata et al., 2012). Nitrogen deficiency and

growth at high irradiance can result in sugar accumulation, which

may induce leaf senescence through hexose-dependent signaling

(Agera et al., 2010; De la Mata et al., 2012). The combined effect

of sugar accumulation and certain environmental conditions may

increase the sugar sensitivity of plants. However, senescence may

also be regulated by pathways that are independent of sugar sig-

naling (Wingler et al., 2006; Van Doorn, 2008).

Elevated CO2 concentrations may enhance potential net

photosynthesis of C3plants because ribulose-1,5-bisphophate car-

boxylase/oxygenase (rubisco), an enzyme involved in both CO2fixation and photorespiration, is not CO2 saturated at the currentconcentration (Drake et al., 1997). Thus, an increase in ambient

CO2 raises the leaf internal CO2 concentration and the CO2/O2ratio at the rubisco site, favoring carboxylation over oxygenation

in ribulose-1,5-bisphosphate (RuBP). Therefore, elevated CO2con-

centrations can reduce photorespiration and thus cellular H2O2production associated with glycolatemetabolism (Pritchard et al.,

2000).

The impact of elevated CO2 concentrations on the oxidative

status of leaves has been examined in various plant species

(Cheeseman, 2006; Qiu et al., 2008), in which it seems to cause a

decreaseintheactivityof someantioxidantenzymesandalso inthe

concentration of some antioxidants (Wustman et al., 2001), lead-

ing to an increase of ROS levels in most plants (Erice et al., 2007).

ROS are continuously formed as by-products of various metabolicpathwaysin differentcellularcompartments (FoyerandHarbinson,

1994; Apel and Hirt, 2004). Under physiological steady-state con-

ditions, these molecules are scavenged by different antioxidant

defense components (Alscher et al., 1997). However, the balance

between ROS production and scavenging may be perturbed by

adverse environmental factors that increase the intracellular lev-

els of ROS (Polle, 2001; Vanacker et al., 2006). In plants, ROS

are detoxifiedvia enzymatic and non-enzymatic mechanisms; the

enzymatic mechanisms involve superoxide dismutase, catalase,

ascorbate peroxidase (APX) and other antioxidant enzymes such

as glutathione reductase (Mittler, 2002). These enzymes play a key

role in controlling the level of oxygen free radicals (Irigoyen et al.,

1992) andalso in the regulation of various processes including leaf

senescence (Prochzkov and Wilhelmov, 2007). The failure ofdefense metabolites and enzymes to detoxify ROS affects biolog-

ical structures and processes, including DNA nicking, amino acid

andproteinoxidation,andlipidperoxidation(Asada,1999; Johnson

et al., 2003), with the consequent generation of breakdown prod-

ucts such asmalondialdehyde (Esterbauer, 1982).

TheeffectsofelevatedCO2 concentrationsonplant productivity

have been extensively studied. Overall, plants tend to increase

growth and to produce greater amounts of biomass in the pres-

ence of elevated CO2 concentrations. Also, the C3 photosynthetic

pathway exhibits a greater relative increase than does the C4pathway under these conditions. Comparatively less research

has been conducted on the effects of CO2 on plant development,

with occasionally dissimilar results (Bazzaz, 1990; Patterson

and Flint, 1990). Elevated CO2 concentrations were found to

boost the expression of storage proteins, but also to upregulate

endo-xyloglucan transferase andxyloglucan endotransglycosidase

(XET) (Cosgrove, 1997), both of which are involved in the incor-

poration of newly secreted xyloglucans into cell walls (Nishitani

and Tominaga, 1992; Fry et al., 1992; Wu and Cosgrove, 2000).

This expression is correlated with the upregulation of genes

coding for various elements of the cytoskeleton associated with

growth, such as thealphaandbeta subunits of tubulin, andvarious

actin-depolymerizing factors. Many physiological studies indicate

that expression of these genes may contribute to increased leaf

size at elevated CO2concentrations (Ferris et al., 2001).

The aim of this work was to examine the possible role of an

elevated atmospheric CO2 concentration on the induction of sun-

flower primary leaf senescence and theeffects on biochemical and

physiological processes during leaf ontogeny.

Materials and methods

Plant material and growth

Seeds of sunflower (Helianthus annuus L.) from the isogeniccultivar HA-89 (Semillas Cargill SA, Seville, Spain) were surface-

sterilized in 1% (v/v)hypochlorite solution for15min.After rinsing

in distilled water, the seeds were imbibed for 3h and then sown

in plastic trays containing a 1:1 (v/v) mixture of perlite and ver-

miculite. Seeds were germinated and plants grown in a growth

chamber with a 16h photoperiod (400molm2 s1 of photo-

synthetically active radiation supplied by cool white fluorescent

lamps supplemented by incandescent bulbs) and a day/night

regime of 25/19 C and 70/80% relative humidity. Plants were

irrigated daily with a nutrient solution containing 10mM KNO3(Hewitt, 1966).

Plants were grown under these conditions for 8days and then

transferred to different controlled-environment cabinets (Sanyo

Gallenkam Fitotron, Leicester, UK) fitted with an ADC 2000 CO2gas monitor. The plants were kept under ambient CO2 levels

(400LL1) orelevatedCO2concentration(800L L1) undercon-stant conditions of photonic flux (400molm2 s1), temperature

(25/19 C) and relative humidity (70/80%) for another 34days.

High-purity CO2was supplied from a compressed gascylinder (Air

Liquid, Seville, Spain). Samples of primary leavesaged 16, 22, 32 or

42dayswere collected2 h after thestartof the photoperiod.Whole

leaves were excised and pooled in two groups: one was used to

measure leaf area and specific leaf mass (SLM)dry weight (DW),

andtheotherwas immediately frozen in liquid nitrogenandstored

at 80 C. The frozen plant material was ground in a mortar pre-

cooled with liquid N2 and the resulting powder distributed into

small vials that were stored at 80 C until enzyme activity and

metabolite determinations.

The net CO2 fixation rate, transpiration rate and stomatal con-

ductance were measured on attached leaves, using a CRS068portable infrared gas analyzer (IRGA) with the software CIRAS-2.

Measurementswere made on primary leaf samples from different

plants in each treatment.

Protein, pigment and H2O2determinations

Frozenmaterialwashomogenizedwith coldextractionmedium

(4mLg1) consisting of50mMHepes-KOH(pH7), 5mMMgCl2and

1mM EDTA, and analyzed with the Bio-Rad protein assay accord-

ingto Bradford (1976). Pigmentsweredetermined inplant extracts

according to Cabello et al. (1998). For H2O2 determination, 1 g

leaf material was ground with 10mLcool acetone in a cold room

andpassed throughWhatman filterpaper.Hydrogenperoxidewas


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1394 L.de laMata et al. / Journal of Plant Physiology 169 (2012) 13921400

Fig. 1. Changes in specific leaf mass (SLM) referred to dryweight(DW),leaf area and soluble protein during sunflower primary leaf development.Plants were grown under

different atmospheric CO2 concentrations: 400L L1 (closed circles) and 800L L1 (open circles). Data aremeansSD of duplicate determinations from three separate

experiments. Asterisks indicate statistically significantdifferences among the CO2 treatments at theindicatedtimes according to Studentst-test (P


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0

2

4

6

8

10

12

14

16 22 32 42

(mgg1)

Days

Chlorophylla

A

0

1

2

3

4

5

16 22 32 42

(mgg1)

Days

Chlorophyllb

B

0

5

10

15

20

16 22 32 42

(mgg1)

Days

Totalchlorophyll

C

1

2

3

4

5

16 22 32 42

(mgg1)

Days

Carotenoids

D

*

*

*

*

**

*

*

**

*

*

* *

*

*

Fig. 2. Changes in thepigments levels during sunflower primary leaf development. Plantswere grown under different atmospheric CO2 concentrations: 400L L1 (closed

circles) and800L L1 (open circles).Data aremeansSDof duplicatedeterminationsfrom threeseparate experiments.Asterisksindicatestatistically significantdifferences

among the CO2 treatments at theindicatedtimes according to Studentst-test (P


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0

1

2

3

4

5

6

7

8

9

16 22 32 42

(molCO2m2s1)

Days

CO2Fixaon

0

0,5

1

1,5

2

2,5

3

3,5

4

16 22 32 42

(m

molH2Om2s1)

Days

Transpiraon

B

0

100

200

300

400

16 22 32 42

(mmolH2Om2s1)

Days

StomatalConductance

C

A**

*

*

*

*

*

*

Fig. 3. CO2 fixation rate, transpiration rate and stomatal conductance during sun-

flower primary leaf development. Plants were grown under different atmospheric

CO2 concentrations: 400L L1 (closed circles) and 800L L1 (open circles). Data

aremeansSDof duplicatedeterminations fromthreeseparate experiments.Aster-

isks indicate statistically significant differences among the CO2 treatments at the

indicated timesaccording to Students t-test (P


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0

50

100

150

200

250

300

16 22 32 42

(mgg1DW)

Days

Glucose

0

50

100

150

200

250

16 22 32 42

(mgg1DW)

Days

Fructose

0

20

40

60

80

100

120

140

160

16 22 32 42

(mgg1DW)

Days

Sucrose

0

50

100

150

200

250

300

16 22 32 42

(mgg1DW)

Days

Starch

0

1

2

3

4

5

6

16 22 32 42

(Glucose+Fructose)/Sucrose

Days

(mgg1DW)

A B

C D

E

*

*

*

*

*

*

*

*

*

* *

Fig. 4. Changes in thecontents of glucose, fructose,sucrose,starch andin thehexosesto sucrose ratio duringsunflowerprimary leaf development.Plants were grownunder

different atmospheric CO2 concentrations: 400L L1 (closed circles) and 800L L1 (open circles). Data are meansSD of duplicate determinations from three separate

experiments. Asterisks indicate statistically significant differences among the CO2 treatments at theindicated times according to Studentst-test (P


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0

5

10

15

20

25

16 22 32 42

Days

Carbon/Nitrogen

rao

0

200

400

600

800

16 22 32 42

Days

Carbon(mgg1DW)

0

20

40

60

80

16 22 32 42

Days

Nitrogen(mgg1DW)

A

B

C

*

*

*

*

*

*

*

Fig.5. Changes inthe contentsof carbonandnitrogenand theC/N ratio duringsun-

flower primary leaf development. Plants were grown under different atmospheric

CO2 concentrations: 400L L1 (closed circles) and 800L L1 (open circles). Data

aremeansSDof duplicatedeterminations fromthreeseparate experiments.Aster-

isks indicate statistically significant differences among the CO2 treatments at the

indicated timesaccording to Students t-test (P


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Growth Under Elevated Atmospheric CO2 Concentration Accelerates Leaf

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