growth under elevated atmospheric co2 concentration accelerates leaf
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8/11/2019 Growth Under Elevated Atmospheric CO2 Concentration Accelerates Leaf
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Journal of Plant Physiology 169 (2012) 13921400
Contents lists available at SciVerse ScienceDirect
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:b42matsl@uco.es(L.de laMata), bv1cahap@uco.es (P.Cabello),bv1hahep@uco.es (P. de la Haba), vg1agbue@uco.es (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.
http://dx.doi.org/10.1016/j.jplph.2012.05.024
http://localhost/var/www/apps/conversion/tmp/scratch_7/dx.doi.org/10.1016/j.jplph.2012.05.024http://localhost/var/www/apps/conversion/tmp/scratch_7/dx.doi.org/10.1016/j.jplph.2012.05.024http://www.sciencedirect.com/science/journal/01761617http://www.elsevier.de/jplphmailto:b42matsl@uco.esmailto:bv1cahap@uco.esmailto:bv1hahep@uco.esmailto:vg1agbue@uco.eshttp://localhost/var/www/apps/conversion/tmp/scratch_7/dx.doi.org/10.1016/j.jplph.2012.05.024http://localhost/var/www/apps/conversion/tmp/scratch_7/dx.doi.org/10.1016/j.jplph.2012.05.024mailto:vg1agbue@uco.esmailto:bv1hahep@uco.esmailto:bv1cahap@uco.esmailto:b42matsl@uco.eshttp://www.elsevier.de/jplphhttp://www.sciencedirect.com/science/journal/01761617http://localhost/var/www/apps/conversion/tmp/scratch_7/dx.doi.org/10.1016/j.jplph.2012.05.024 -
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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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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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L. de laMata et al. / Journal of Plant Physiology169 (2012) 13921400 1395
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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L. de laMata et al. / Journal of Plant Physiology169 (2012) 13921400 1397
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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