Environmental and Experimental Botany 99 (2014) 75–85

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Environmental and Experimental Botany

journal homepage: www.elsevier.com/locate/envexpbot

Photosynthesis and protein metabolism associated with elevated

CO2 -mitigation of heat stress damages in tall fescue
Jingjin Yu a,b,1 , Zhimin Yang a,b,1 , David Jespersen b , Bingru Huang a,b,∗
a
College of Agro-Grassland Science, Nanjing Agricultural University, Nanjing 210095, PR China
b
Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 08901, USA

a r t i c l e i n f o a b s t r a c t

Article history: Heat stress is a primary factor limiting the growth of cool-season (C3 ) perennial grass species during sum-
Received 26 August 2013 mer months. Elevated CO2 may alleviate heat stress damage in C3 plants. The objective of this study was
Received in revised form to investigate mechanisms underlying elevated CO2 -mitigation of adverse effects due to heat stress in C3
16 September 2013
perennial grass species by examining effects of elevated CO2 on major photosynthetic components and
Accepted 17 September 2013
proteins for tall fescue (Festuca arundinacea) subjected to heat stress. Plants of tall fescue (cv. ‘Rembrandt’)
were grown under ambient CO2 (400 ␮mol mol−1 ) or elevated CO2 (800 ␮mol mol−1 ) and subjected to
Keywords:
ambient temperature (25/20 ◦ C day/night) or heat stress (35/30 ◦ C day/night). Elevated CO2 enhanced
Carbon dioxide
High temperature
photosynthetic rate under both ambient temperature and heat stress in tall fescue. The improved pho-
Perennial grass tosynthesis under elevated CO2 was associated with the increase in the abundance of proteins involved
Proteomics in photosynthetic light reactions (chlorophyll a–b binding protein), electron transport carrier molecule
(ferredoxin), and ATP generation enzyme (adolase), as well as higher carbon assimilation efficiency and
carboxylation enzyme activities of the Calvin cycle [higher carbon:nitrogen ratio (C:N), maximal rate
of photosynthetic electron transport (Jmax ), Rubisco activity and Rubisco activation]. Elevated CO2 also
induced the accumulation of proteins involved in antioxidant metabolism (ascorbate peroxidase and 2-
Cys peroxiredoxin). Elevated CO2 induced stomatal closure and chlorophyll content decline under both
ambient temperature and heat stress, which could have limited the positive effects of elevated CO2 on the
photosynthetic capacity. It would be useful to select cultivars of C3 perennial grass species with decreased
stomatal sensitivity to elevated CO2 to achieve maximal benefits of elevated CO2 on photosynthesis and
whole-plant growth. Our results suggested that the increased photosynthetic efficiency and activities, as
well as protein abundance involved in photosynthesis and antioxidant metabolism could play important
roles in elevated CO2 -mitigation of heat stress damage in C3 perennial grass species.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction to increasing temperatures, and heat-inhibition of photosynthesis

Elevated temperature and CO2 concentration can have sig-
nificant impact on plant growth. During this century, global
temperatures are predicted to rise by 2–5 ◦ C (IPCC, 2007). The
optimal temperature for shoot and root growth of cool-season C3
plant species is 18–24 ◦ C but during summer months the ambi-
ent temperature can be 10 ◦ C higher or more than the temperature
requirement driving optimal growth in cool-season plants, which
leads to various cellular and physiological damages (DiPaola and
Beard, 1992). Photosynthesis is one of the most sensitive responses
∗ Corresponding author at: Department of Plant Biology and Pathology, Rutgers
University, New Brunswick, NJ 08901, USA. Tel.: +86 2584396359;
fax: +1 7329329441.
E-mail address: huang@aesop.Rutgers.edu (B. Huang).
1
These authors contributed equally to this work.
contributes to the decline in the availability of carbohydrates for the
supply of energy and carbon skeletons for plant growth (Bencze
et al., 2005; Crafts-Brandner and Salvucci, 2000; Liu and Huang,
2008; Salvucci and Crafts-Brandner, 2004). Along with increas-
ing temperature, atmospheric CO2 concentration has increased by
100 ␮mol mol−1 since the beginning of the industrialized era and
the concentration is predicted to continue rising at a rate of approx-
imately 2 ␮mol mol−1 per year (IPCC, 2007). Changes in these global
climate factors will likely lead to plant exposure to combined ele-
vated temperature and CO2 , which may have interactive effects on
plant growth.
Previous research has shown that elevated CO2 promotes plant
growth under optimal growing temperatures in various plant
species, particularly C3 species (Hamerlynck et al., 2000; Kirkham,
2011; Prasad et al., 2002, 2009; Qaderi et al., 2006). The physio-
logical and biochemical effects of elevated CO2 on various plant
species grown under optimal growth temperatures have been well

0098-8472/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.envexpbot.2013.09.007
76 J. Yu et al. / Environmental and Experimental Botany 99 (2014) 75–85
documented (Bunce, 2001; Griffin and Seemann, 1996; Stitt and
Krapp, 1999; Woodward, 2002). The enhancement of plant growth
by elevated CO2 under optimal temperature conditions has been
associated with promotion of photosynthesis and inhibition of dark
respiration and photorespiration rates, as well as increased carbo-
hydrate accumulation (Leakey et al., 2006; Gonzàlez-Meler et al.,
1996; Reddy et al., 2010). However, a limited number of studies
have examined the interactive effects of elevated CO2 and heat
stress or the beneficial effects of elevated CO2 on plant growth in
relation to abiotic stress tolerance (Hamilton et al., 2008; Prasad
et al., 2002, 2009). Few available studies have demonstrated that
elevated CO2 can mitigate the adverse effects of heat stress on pho-
tosynthesis, water use, and overall plant growth in different plant
species (Idso and Idso, 1994; Hamilton et al., 2008; Prasad et al.,
2009), including C3 perennial grass species (Yu et al., 2012a,b), but
the mechanisms of enhanced heat tolerance on photosynthesis due
to elevated CO2 are unclear.
Proteomic profiling combining two-dimensional polyacrylam-
ide gel electrophoresis (2-D PAGE) in combination with mass
spectrometry (MS) is a powerful approach to identify and quan-
tify stress-responsive proteins in various plant species (Abbasi
and Komatsu, 2004; Hashimoto and Komatsu, 2007; Lee et al.,
2007; Xu and Huang, 2008; Xu et al., 2010; Zhao et al., 2011).
Heat stress alone alters protein metabolism, causing up-regulation
or down-regulation of proteins involved in different metabolic
processes, such as energy metabolism (photosynthesis and respira-
tion), antioxidant stress defense, protein synthesis and storage, and
protein stability (Ferreira et al., 2006). Elevated CO2 also induces
changes in protein expression, as shown by Bae and Sicher (2004)
who identified six proteins responding to elevated CO2 concen-
tration under optimal temperatures in Arabidopsis, among which
three proteins were involved in plant growth and development or
stress defense, one protein (23 kDa subunit) of the oxygen evolv-
ing complex (OEC23) involved in photosynthesis and two proteins
with unknown functions. The authors concluded that elevated CO2
did not singly have a significant impact on protein expression in
Arabidopsis because only a few proteins were responsive to CO2
enrichment. Others have found potentially important changes in
proteins related to plant physiological response to abiotic stress
as discussed below. Several proteins involved in photosynthesis,
such as Rubisco activase (van Oosten and Besford, 1995), carbonic
anhydrase (Majeau and Coleman, 1996; Peet et al., 1986) and the
chlorophyll a–b binding protein (Moore et al., 1998; van Oosten
et al., 1994) decreased at the transcript level in response to ele-
vated CO2 compared to those under ambient CO2 under optimal
growth temperatures. However, few studies have investigated ele-
vated CO2 -induced changes in proteins, particularly those involved
in photosynthetic metabolism and stress defense under heat stress
conditions which may be associated with CO2 -mitigation of heat
stress.
Tall fescue is a widely utilized C3 forage and turfgrass through-
out cool temperate regions and is sensitive to heat stress, whereas
tall fescue heat tolerance could be improved by doubling ambi-
ent CO2 concentration (Yu et al., 2012a,b). Elevated CO2 mitigated
the adverse effects of heat and drought stress on tall fescue
by enhancing plant water status, cellular membrane stability,
and photosynthetic capacity, as well as suppressing carbon con-
sumption through lowering respiration rates (Yu et al., 2012a).
Doubling ambient CO2 concentration also enhanced accumulation
of metabolites involved in photosynthesis, respiration, and amino
acid metabolism under heat stress (Yu et al., 2012b). Understanding
changes to photosynthetic components and proteins in response to
CO2 increase and interaction with heat stress tolerance of tall fescue
will provide further insights into the mechanisms underlying heat
stress mitigation by elevated CO2 in cool-season perennial grass
species. Therefore, the objective of this study was to investigate
CO2 -responsive photosynthetic components and proteins associ-
ated with the positive effects of elevated CO2 concentration on heat
tolerance in tall fescue. The ultimate aim was to reveal metabolic
processes and molecular mechanisms underlying elevated CO2
enhancement of heat tolerance in C3 perennial grass species.
2. Materials and methods
2.1. Plant materials and growth conditions
Sod plugs of tall fescue (cv. ‘Rembrandt’) plants were col-
lected from the turfgrass research farm at Rutgers University in
Adelphia, NJ, and transplanted into plastic pots (10 cm diame-
ter and 60 cm long) filled with a mixture of fine sand and soil
(1:1, v/v). During this establishment period, plants were watered
every other day and fertilized once a week with half-strength
Hoagland’s solution (Hoagland and Arnon, 1950). Plant leaves were
trimmed once per week to maintain a copy height of 5–6 cm. Plants
were maintained in a greenhouse with an average temperature
of 21/16 ◦ C (day/night) and 810 ␮mol m−2 s−1 photosynthetically
active radiation (PAR) in natural sun light, and 65% relative humid-
ity for 50 d (May–June 2011) to fully establish the canopy and
root system. After establishment, plants were moved to growth
chambers with the temperature set at 25/18 ◦ C (day/night), 70%
relative humidity, PAR of 650 ␮mol m−2 s−1 and a 12-h photope-
riod to allow for acclimation of controlled-environment conditions
for 7 d before elevated CO2 and temperature treatments were
imposed.
2.2. Experimental design and treatments
The experiment consisted of two factors, CO2 concentration
and temperature, each with two levels, which were arranged in
a factorial design with four replicates per treatment. The CO2 treat-
ments included ambient CO2 (400 ± 10 ␮mol mol−1 ) and elevated
CO2 (800 ± 10 ␮mol mol−1 ). Plants were grown at the two CO2
levels in the growth chambers for 40 d before imposition of tem-
perature treatments. Temperature treatments were controlled at
two levels: 25/20 ◦ C (day/night, optimal temperature control) and
35/30 ◦ C (day/night, heat stress) for 28 d. Plants were well-watered
to maintain soil water content at field capacity (28%, v/v) and were
fertilized once a week with half-strength Hoagland’s solution dur-
ing the CO2 and temperature treatment period.
The CO2 and temperature treatment set-up in growth cham-
bers followed the same designed as described in Yu et al. (2012a,b).
Four growth chambers were maintained at the ambient CO2 level
with two of them at elevated temperature and the other two at
optimal temperature. The ambient CO2 treatment was conducted
in four growth chambers from February to April in 2012. Four
growth chambers were set to the elevated CO2 level, with two
at elevated temperature and two at the optimal temperature. The
elevated CO2 treatment was conducted from May to July in 2012.
Plants were re-randomized among the growth chambers once per
week to minimize confounding effects of possible environmental
variations between different chambers. The concentration of CO2
inside each growth chamber was maintained with an automated,
open-chamber CO2 control system connected to a gas tank con-
taining 100% CO2 (Airgas, Inc., Radnor, PA) (Yu et al., 2012a,b).
The CO2 levels were continuously monitored through an infrared
gas analyzer (Li-820, LI-COR, Lincoln, NE) and controlled using an
automatic system consisting of a programmable logic controller
unit, solenoid valves, and a laptop computer with monitoring soft-
ware accurate to within 10 ␮mol mol−1 of the target levels (400 and
800 ␮mol mol−1 ).
 J. Yu et al. / Environmental and Experimental Botany 99 (2014) 75–85
2.3. Measurements of leaf gas exchange
Leaf net photosynthetic rate (Pn ), intercellular CO2 concentra-
tion (Ci ), and stomatal conductance (gs ) were measured on 20 s
full-expanded leaf from the top of the plant from each pot with
a portable infrared gas analyzer (Li-6400, LICOR, Inc., Lincoln,
NE, USA). Leaves were placed in a leaf chamber with a built-in
red and blue light source of the Li-6400 with the light level of
800 ␮mol photon m−2 s−1 . The response of Pn to intercellular CO2
concentration was examined by varying the ambient CO2 concen-
tration in 12 steps (50, 100, 150, 200, 300, 400, 600, 800, 1000, 1300,
1500 ␮mol mol−1 ) at 28 d of the treatment. The response of Pn to
intercellular CO2 concentration was analyzed by non-linear regres-
sion (Miao et al., 2008) to obtain the maximal rate of photosynthetic
electron transport (Jmax ).
2.4. Analysis of chlorophyll content, nitrogen content, carbon
content
For the determination of leaf chlorophyll content (Chl), fresh
leaves (0.2 g) were detached from plants and then soaked in
dimethyl sulfoxide in the dark for 72 h. The density values of Chla
and Chlb were obtained at the respective wavelengths of 663 and
645 nm by a spectrophotometer (SpectronicGenesys 2; Spectronic
Instruments, Rochester, NY, USA). Chl was calculated as described
in Arnon (1949). Chl measurements were taken every 7 d following
the induction of temperature treatments.
Fresh samples of leaves (0.2 g) collected at 28 d of the treat-
ment were lyophilized and ground to a powder. Leaf carbon content
and nitrogen content were analyzed using an elemental analyzer
(CE440, Exeter Analytical, Inc. North Chelmsford, MA). The ratio of
C:N was then calculated.
2.5. Photosynthetic enzyme extraction and activity assays
Rubisco extraction and activity assays were conducted using
methods described by Hu et al. (2010) with modifications. Samples
of fresh leaves (0.2 g) were detached from plants and then immedi-
ately frozen in liquid nitrogen and kept at −80 ◦ C prior to extraction.
For extraction, the leaf tissue was ground in liquid nitrogen with
3 mL extraction buffer containing 50 mM Hepes-KOH (pH = 7.5 at
25 ◦ C), 10 mM MgCl2 , 2 mM EDTA, 10 mM DTT, 10% glycerol (v/v), 1%
bovine serum albumin (w/v) and 1% Triton X-100 (v/v). The super-
natant was isolated by centrifugation at 14,000 × g for 10 min at 4 ◦ C
and used immediately for Rubisco activity assays. Rubisco activ-
ity was measured by adding RuBP to the assay solution [100 mM
Bicine (pH = 8.0), 25 mM KHCO3 , 20 mM MgCl2 , 3.5 mM ATP, 5 mM
phosphocreatine, 5 units glyceraldehyde-3-phosphate dehydroge-
nase, 5 units 3-phosphoglyceric phosphokinase, 17.5 units creatine
phosphokinase and 0.25 mM NADH] and absorbance measured
at 340 nm with a spectrophotometer (Helios Alpha, Thermospec-
tronic, Rochester, NY). Initial activity was measured immediately
after adding RuBP and total activity was measured after incubating
samples at 25 ◦ C for 5 min. Rubisco activation state was determined
by the ratio of initial to total activity.
2.6. Protein extraction and quantification
Protein content was determined using the method of Bradford
(1976). Leaf samples consisting of both mature and young leaves
were collected from each pot at the end of the treatment (28 d)
and immediately frozen in liquid nitrogen. Samples were then
ground into a fine powder and stored at −80 ◦ C until analysis.
Proteins were extracted using the trichloroacetic acid/acetone
method, as described in Xu and Huang (2008). Leaf samples
(∼0.5 g) were homogenized on ice using a mortar and pestle
77
in 10 mL of precipitation solution (10% trichloroacetic acid and
0.07% 2-mercaptoethanol in acetone) for 10 min and incubated
at −20 ◦ C for 2 h. The protein pellet was collected by cen-
trifugation and washed with cold acetone containing 0.07%
2-mercaptoethanol until the supernatant was colorless. The pel-
let was then dried using a vacuum-dryer and suspended in a
resolubilization solution consisting of 8 M urea, 2 M thiourea, 2% 3-
[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 1%
dithiothreitol, and 1% immobilized pH gradient (IPG) buffer. The
solution was centrifuged at 21,000 × g for 20 min to remove insol-
uble material and the resulting supernatant was collected. 0.5 mL
of a commercial dye reagent (Bio-Rad Laboratories, Hercules, CA,
USA) was mixed with a 10 ␮L aliquot of protein extract and the
absorbance was then measured spectrophotometrically at 595 nm
5 min after the reaction. Bovine serum albumin was used to create a
standard curve to determine the protein concentration of extracts.
2.7. Protein separation and identification
Two-dimensional electrophoresis (2-DE) was utilized for pro-
tein separation and quantification (Klose and Kobalz, 1995). An
Ettan IPGPhor II apparatus (GE Healthcare, Waukesha, WI, USA) was
used for isoelectric focusing (IEF) for the first dimension. Aliquots
of the extracts containing 300 ␮g of protein were subjected to IEF
using immobilized pH gradient strips (pH 3.0–10.0, linear gradient,
13 cm). A rehydration buffer (8 M urea, 2 M thiourea, 2% CHAPS, 1%
dithiothreitol, 1% pharmalyte, and 0.002% bromophenol blue) was
added for a total volume of 250 ␮L. The IEF program included a
14 h active rehydration step at 50 V, 1 h at 500 V, 1 h at 1000 V, 1 h
at 5000 V, and then 8000 V until 80 kVh were reached. Following
IEF, the strips were equilibrated on shaker at room temperature
for 15 min in an equilibration buffer consisting of 50 mM Tris–HCl
pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and 1% dithiothreitol, then
transferred to a similar equilibration buffer containing 2.5% iodoac-
etamide instead of 1% dithiothreitol for an additional 20 min. The
second dimension electrophoresis was performed using a Hoefer
SE 600 Ruby electrophoresis apparatus (GE Healthcare, Waukesha,
WI, USA) and a 12.5% sodium dodecyl sulfate-polyacrylamide gel.
Gels were run at 5 mA per gel for 30 min followed by an increase
to 20 mA per gel for 5 h. The gels were stained using a colloidal
Coomassie brilliant blue (G-250) solution and scanned using a Per-
sonal Densitometer SI (63-0016-46, GE Healthcare, Waukesha, WI,
USA).
Gel image analysis was performed by Progenesis Same Spots
software (Nonlinear Dynamics, Durham, NC, USA). Automatic spot
alignment was used to align gels with additional manual edit-
ing included as needed. The spot volumes were normalized as a
percentage of the total volume of all spots on the gel to correct
the variability due to loading or staining and give relative protein
quantities. Selected protein spots were manually excised from gels
and subjected to a trypsin digestion. The resulting peptides were
analyzed by matrix-assisted laser desorption/ionization (MALDI)
and time of flight mass spectrometry (TOF-MS) (Suckau et al.,
2003). The TOF-MS method determines the mass-to-charge ratio
of an ion via a time measurement. Using a local MASCOT search
engine (V1.9, Matrix Science, Boston, MA, USA) on a group-based
phosphorylation scoring (GPS) server (V. 3.5, Applied Biosystems,
Framingham, MA, USA) data were searched against the National
Center for Biotechnology Information (NCBI) database. Proteins
containing at least two peptides with a confidence interval value
>95% were considered identified.
2.8. Statistical analysis
Data were analyzed using statistics software (SAS 9.0; SAS Insti-
tute Inc., Cary, NC). ANOVA was used to determine differences
78 J. Yu et al. / Environmental and Experimental Botany 99 (2014) 75–85
Fig. 1. Leaf net photosynthetic rate (Pn ) (A), intercellular CO2 concentration (Ci )
(B) and leaf stomatal conductance (gs ) (C) of tall fescue at two CO2 levels (400 and
800 ␮mol mol−1 ) under two temperature conditions (25 and 35 ◦ C) at 7, 14, 21, and
28 d of treatment. Circles represent elevated CO2 treatments and triangles represent
ambient CO2 treatments. Black icon represents optimal temperature treatments and
white circles represent heat stress treatments. Vertical bars indicate LSD values
(P ≤ 0.05) for the comparison of treatments.
among treatments in Pn , Ci , gs , Jmax , Chl, nitrogen content, carbon
content, Rubisco activity, activation state, and protein abundance.
When a particular F test was significant, means were tested with
Fisher’s protected least significant difference (LSD) at a confidence
level of 0.05.
3. Results
3.1. Leaf net photosynthetic rate (Pn ), intercellular CO2
concentration (Ci ), and stomatal conductance (gs ) as affected by
elevated CO2 and heat stress
Heat stress caused significant decreases in Pn under both
ambient and elevated CO2 concentrations over the 28-d treat-
ment period (Fig. 1A). Heat-induced reduction in Pn was more
pronounced (52%) under ambient CO2 concentration compared to
that (39%) under elevated CO2 concentration averaged over the 28-
d treatment period. Elevated CO2 treatment led to a significantly
higher Pn compared to the ambient CO2 treatment under both opti-
mal temperature and heat stress, but to a greater extent (67%) under
heat stress than under the optimal temperature (31%) averaged
over the 28-d treatment period (Fig. 1A).
Leaf Ci exhibited decline with heat stress under either ambient
or elevated CO2 concentration during most of the 28-d treatment
period (Fig. 1B). The decline in Ci due to heat stress was to a simi-
lar extent under ambient and elevated CO2 concentration. Elevated
CO2 concentration significantly increased Ci regardless of temper-
ature over the entire treatment period (Fig. 1B). The Ci was 1.97
times higher in leaves exposed to elevated CO2 than those under
ambient CO2 concentration.
Stomatal conductance (gs ) was significantly lower under heat
stress than under ambient temperature for both CO2 treatments
(Fig. 1C). Heat-induced decline in gs was greater under ambient CO2
(70%) than that under elevated CO2 concentration (51%) (Fig. 1C).
Elevated CO2 concentration also resulted in significant lower gs
under ambient temperature, and the decline was an average of
52% over the entire 28-d treatment period (Fig. 1C). No significant
changes in gs were observed with elevated CO2 under heat stress
compared to ambient CO2 under heat stress, as gs declined to a very
low level regardless of CO2 treatment.
3.2. Rubisco enzyme activity, activation state, and electron
transport-driving regeneration of RuBP (Jmax )
Heat stress caused a significant decline in Rubisco activity
at both ambient and elevated CO2 concentration during the 28-
d treatment period (Fig. 2A). The reduction in Rubisco activity
induced by heat stress was 36% and 33% under ambient CO2 and ele-
vated CO2 concentration, respectively, over the 28-d of treatment
period. Rubisco activity increased under elevated CO2 compared to
that under ambient CO2 concentration at 14 and 21 d of treatment
under both optimal temperature and heat stress. No significant
interactive effects of elevated CO2 and temperature on Rubisco
activity were detected during the entire experimental period.
Heat stress led to a significant decline in Rubisco activation state
at 21 and 28 d of treatment under elevated CO2 and at 28 d under
ambient CO2 concentration (Fig. 2B). Rubisco activation state was
increased by elevated CO2 during most of the treatment period
under optimal temperature and at 7 and 14 d under heat stress
(Fig. 2B). The beneficial effects of elevated CO2 concentration on
Rubisco activation state were more pronounced under optimal
temperature than under heat stress.
The response of Pn to intercellular CO2 concentration was
depicted in Fig. 3A, which was used to calculate Jmax . Heat stress
caused significant reduction in Jmax under both elevated and ambi-
ent CO2 concentration and the decline was less severe (11%) under
elevated CO2 than that under ambient CO2 concentration (30%)
(Fig. 3B). Elevated CO2 did not have effects on Jmax at optimal tem-
perature, but resulted in a 34% high Jmax under heat stress when
compared to ambient CO2 .
3.3. Leaf chlorophyll (Chl) content, nitrogen (N) content, carbon
(C) content and C:N ratio
Leaf Chl content increased within the first 21 d of heat stress
and then declined as heat stress was prolonged to 28 d under ambi-
ent CO2 concentration (Fig. 4). Under elevated CO2 condition, heat
stress induced the reduction of Chl after 7 d of treatment. Elevated
CO2 also caused a significant decrease in Chl regardless of temper-
ature, but the extent of CO2 -induced Chl decline under heat stress
 J. Yu et al. / Environmental and Experimental Botany 99 (2014) 75–85 79

Fig. 2. Leaf Rubisco activity (initial activity) (A) and Rubisco activation state (B)
of tall fescue at two CO2 levels (400 and 800 ␮mol mol−1 ) under two temperature
conditions (25 and 35 ◦ C) at 7, 14, 21, and 28 d of treatment. Vertical bars indicate
LSD values (P ≤ 0.05) for the comparison of treatments.
Fig. 3. The response of net photosynthetic rate (Pn ) to intercellular CO2 concentra-
tions at two CO2 levels (400 and 800 ␮mol mol−1 ) under two temperature conditions
(25 and 35 ◦ C) at 28 d of treatment (A) and leaf electron transport-driving regener-
ation of RuBP (Jmax ) of tall fescue at two CO2 levels (400 and 800 ␮mol mol−1 ) under
two temperature conditions (25 and 35 ◦ C) at 28 d of treatment (B). Vertical bars
indicate LSD values (P ≤ 0.05) for the comparison of treatments.

(42%) was more pronounced than that under optimal temperature

(16%). three categories: energy metabolism, cell structure and unknown
Leaf N content decreased by 22% under heat stress for plants functions (Fig. 7A). Proteins affected by elevated CO2 under heat
exposed to elevated CO2 concentration (Fig. 5A). Under ambient stress were mainly involved in energy metabolism, stress defense,
CO2 concentration, leaf N content increased by 11% under heat cell structure, cell growth/division and some with unknown
stress. Elevated CO2 concentration caused a significant reduction functions (Fig. 7B). A total of 4 proteins were up-regulated (spot
in N content under both optimal temperature and heat stress. Leaf 354, 339, 79, 180) and 6 proteins were down-regulated (spot
C content was not affected by either temperature or CO2 concentra-
tion (Fig. 5B). The ratio of C:N was increased by elevated CO2 under
both temperatures. Under elevated CO2 level, the ratio of C:N was
significantly higher under heat stress than under optimal tempera-
ture, but the trend was opposite under ambient CO2 concentration,
with heat stress plants having a lower C:N ratio compared to the
optimal temperature (Fig. 5C).

3.4. Proteomic responses to elevated CO2 under ambient

temperature and heat stress

Over 400 spots were detected in each 2-D gel (Fig. 6). The
overall abundance of 38 spots was altered by elevated CO2
concentration. Out of the 38 spots, 31 were successfully identi-
fied by MALDI-TOF-MS, which exhibited differential expression
(up-regulation or down-regulation) in response to elevated CO2
concentration under optimal temperature or heat stress. Those
proteins were categorized into five functional categories according
to the criteria proposed by Bevan et al. (1998): energy metabolism,
Fig. 4. Leaf chlorophyll content (Chl) in leaves of tall fescue at two CO2 levels (400
cell growth/division, cell structure, stress defense, and unclear
and 800 ␮mol mol−1 ) under two temperature conditions (25 and 35 ◦ C) at 7, 14, 21,
classification (Table 1 and Fig. 7A and B). Proteins affected by ele- and 28 d of treatment. Vertical bars indicate LSD values (P ≤ 0.05) for the comparison
vated CO2 under ambient temperature were mainly classified into of treatments.
80 J. Yu et al. / Environmental and Experimental Botany 99 (2014) 75–85

Table 1
Differentially expressed proteins identified by mass spectrometry in plants exposed to two temperature regimes (25 and 30 ◦ C) and two CO2 levels (400 and 800 ␮mol mol−1 ).

Protein spot number Protein ID [source] MW pI Accession no. 25 ◦ C- 35 ◦ C-

800 ␮mol mol−1 /25 ◦ C- 800 ␮mol mol−1 /35 ◦ C-
400 ␮mol mol−1 400 ␮mol mol−1

Functional category 09: cell structure

180 Actin [Setaria italica] 37 5.6 gi|9965319 ↑* ns

Functional category 03: cell growth/division

503 Histone H2B [Volvox carteri f. nagariensis] 17 9.8 gi|302829292 ns ↑*

Functional category 11: disease/defense

425 Ascorbate peroxidase (APX) [Hordeum vulgare 27 5.1 gi|15808779 ns ↑*
subsp. vulgare]
480 2-Cys peroxiredoxin BAS1 chloroplastic 23 5.7 gi|2829687 ns ↑*
[Triticum aestivum]

Functional category 02: energy

173 Phosphoglycerate kinase (PGK), chloroplastic 50 6.6 gi|129915 ↓* ns
339 Chloroplast PS type III chlorophyll a/b-binding 12 9.2 gi|125656346 ↑* ns
protein [Brassica juncea]
181 GAPDH B, chloroplastic [Oryza sativa (japonica 48 7.6 gi|120663 ↓* ns
group)]
337 Chlorophyll a-b binding protein of LHC II type 29 5.0 gi|115793 ↓*
ns
III, chloroplastic [Oryza sativa]
262 Chloroplast fructose-bisphosphate aldolase 29 5.0 gi|223018643 ↓* ns
[Triticum aestivum]
309 Rubisco large subunit [Sporobolus f.] 51 6.2 gi|164565109 ↓* ns
354 Light-harvesting complex I [Hordeum vulgare] 24 8.1 gi|544700 ↑* ns
79 ATP synthase CF1 beta subunit [Lolium perenne] 54 5.2 gi|159106871 ↑* ns
544 Ribulose-1,5-bisphosphate carboxylase small 19 8.6 gi|6573202 ns ↓*
subunit [Avena clauda]
141 Succinate dehydrogenase (SDH) 1-1 70 5.9 gi|297797713 ns ↑*
[Arabidopsis lyrata subsp. lyrata]
317 Fructose-bisphosphate aldolase [Secale cereale] 39 6.4 gi|226316439 ns ↑*
469 Chlorophyll a-b binding protein [Zea mays] 28 6.9 gi|226504520 ns ↑*
574 Rubisco large subunit [Castellia tuberculosa] 52 6.1 gi|144583542 ns ↓*
62 Rubisco large subunit [Zeugites pittieri] 43 6.4 gi|54303900 ns ↓*
123 Transketolase, chloroplastic [Oryza sativa 73 5.5 gi|75140229 ns ↓*
(japonica group)]
368 Ferredoxin [Zea mays] 41 7.6 gi|162458489 ns ↑*
461 Possible: putative carbonic anhydrase [Triticum 28 8.4 gi|290875537 ns ↓*
turgidum subsp. durum × Secale cereale]
576 Rubisco large subunit [Axonopus compressus] 50 6.5 gi|144583498 ns ↓*
305 Rubisco activase 1, chloroplast precursor, 48 6.1 gi|255566442 ns ↓*
putative [Gossypium hirsutum]
176 NADP-thioredoxin reductase C precursor 52 5.4 gi|164598928 ns ↓*
[Hordeum vulgare]
222 Rubisco large subunit, partial (chloroplast) 51 6.2 gi|164565109 ns ↓*
[Sporobolus f.]

Functional category 12: unclear classification

32 Hypothetical protein SELMODRAFT 441675 282 6.9 gi|302780199 ns ↓*
[Selaginella moellendorffii]
142 Possible: hypothetical protein 7 8.7 gi|242091027 ns ↑*
SORBIDRAFT 09g024840 [Sorghum bicolor]
138 Hypothetical protein OsJ 23002 [Oryza sativa 72 6.8 gi|222636391 ns ↑*
(Japonica Group)]
178 Hypothetical protein SORBIDRAFT 05g022470 59 5.3 gi|242071459 ns ↓*
[Sorghum bicolor]
306 Unknown ↓* ns
436 Unknown ns ↓*

MW (kDa), molecular weight; pI, isoelectrical point; Accession no., a unique identifier given to a protein sequence in sequence databases; gi, gene identification number; not
significant; ↑/↓, up/down-regulated when comparing elevated CO2 to ambient CO2 levels within a given temperature treatment.
*
Treatment effect significant, P < 0.05.

173, 181, 262, 309, 306, 337) by elevated CO2 under ambient
temperature. Elevated CO2 up-regulated 9 proteins (spot 141,
138, 142, 317, 368, 425, 469, 480, 503) and down-regulated 12
proteins (spot 544, 461, 436, 305, 222, 176, 178, 123, 574, 32,
62, 576) under heat stress (Fig. 8). The biological functions of
up-regulated and down-regulated by elevated CO2 are discussed
below, with an emphasis on the proteins regulated by elevated
CO2 that could contribute to CO2 -mitigation of heat damages in tall
fescue.
4. Discussion
As discussed in the introduction, the majority of previous work
has examined the effects of heat stress on photosynthesis under
ambient CO2 conditions or effects of elevated CO2 under optimal
temperatures and few studies have investigated the interactive
effects of heat stress and elevated CO2 . It is well documented that
heat stress can be detrimental to plant growth, with one of the
major effects being the inhibition of Pn and carbon assimilation
 J. Yu et al. / Environmental and Experimental Botany 99 (2014) 75–85
Fig. 5. Leaf nitrogen content (A), leaf carbon content (B) and ratio of C:N (C) in leaves
of tall fescue at two CO2 levels (400 and 800 ␮mol mol−1 ) under two temperature
conditions (25 and 35 ◦ C) at 28 d of treatment. Vertical bars indicate LSD values
(P ≤ 0.05) for the comparison of treatments.
under ambient CO2 concentrations (Bencze et al., 2005; Crafts-
Brandner and Salvucci, 2000; Liu and Huang, 2008; Rachmilevitch
et al., 2006; Salvucci and Crafts-Brandner, 2004). Efficient carbon
fixation and allocation is critically important for plant adaptation
to heat stress (Rachmilevitch et al., 2006). In this study, the reduc-
tion in Pn after prolonged periods of heat tress (28 d) was associated
with the reduction in leaf Chl content for light harvesting, Rubisco
activity and activation for carboxylation, and Jmax for electron trans-
port that drives the regeneration of RuBP. Leaf chlorosis is a typical
symptom of heat stress in cool-season grasses, which can be due
to heat-suppression of chlorophyll synthesis and/or accelerated
chlorophyll degradation (DiPaola and Beard, 1992). The decrease
in Rubisco activation could be due to the reduced ability of Rubisco
activase to promote activation and increased Rubisco deactivation
(Crafts-Brandner and Salvucci, 2000; Salvucci and Crafts-Brandner,
81
Fig. 6. Representative Coomassie-stained 2D polyacrylamide gels of separated pro-
teins in leaves of tall fescue grown under ambient temperature (25 ◦ C) (A) and heat
stress (35 ◦ C) (B). Numbers correspond to spot identification numbers in Table 1.
2004; Wise et al., 2004). The lower Jmax under heat stress conditions
was in agreement with those of Borjigidai et al. (2006) who found
Jmax decreased with increasing temperature in rice (Oryza sativa).
The reduction in Jmax by heat stress under ambient CO2 suggested
that RuBP regeneration was limited, and may have restricted the
supply of RuBP to the Calvin cycle for carbon assimilation in plants
suffering from heat stress.
Elevated CO2 alleviated the decline in Pn caused by heat stress
in tall fescue in this study. Our previous study observed that ele-
vated CO2 mitigated the negative effects of combined heat stress
and drought stress on photosynthesis in tall fescue (Yu et al.,
2012a). Wise et al. (2004) also reported that elevated CO2 allevi-
ated the inhibition of heat stress on photosynthesis in Pima cotton
(Gossypium barbadense). The increase in Pn by elevated CO2 for
plants grown under optimal temperatures has been associated with
CO2 suppression of photorespiration in other plant species, which
was decreased by up to 50% under elevated CO2 concentration
(Sharkey, 1988). Accordingly, elevated CO2 may enhance the diver-
sion of energy (ATP and NADP) from photorespiratory metabolism
to photosynthetic carbon assimilation (Sujatha et al., 2008). Under
optimal temperatures, the improved Pn was also related to the
effects on carboxylation (Bernacchi et al., 2005; Farquhar et al.,
1980). In this study, the improved Pn in tall fescue by elevated
CO2 under heat stress was mainly associated with increased accu-
mulation of intercellular carbon concentration, the maximal rate
82 J. Yu et al. / Environmental and Experimental Botany 99 (2014) 75–85
Fig. 7. Functional categorization of CO2 responsive proteins identified in leaves of
tall fescue grown under ambient temperature (25 ◦ C) (A) and heat stress (35 ◦ C) (B)
at 28 d.
of regeneration of RuBP (Jmax ), as well as carboxylation activities
(Rubisco activity and activation state).
Stomatal movement controlling gas exchange for photosynthe-
sis is sensitive to changes in CO2 concentration and temperature.
Stomatal conductance was reduced with elevated CO2 under the
ambient temperature in this study. Similar results have been
reported in other plant species (Faria et al., 1996; Kirkham, 2011).
Stomatal closure could potentially limit the positive responses of
photosynthesis to CO2 under normal temperatures. Faria et al.
(1996) observed that elevated CO2 led to the reduction in gs in
Quercus suber L. seedlings, but under heat stress stomata had a
lesser decline in gs when compared to ambient CO2 levels. Elevated
CO2 did not have significant effects on stomatal conductance under
heat stress in tall fescue in this study, suggesting that the effects of
elevated CO2 on Pn were not related to changes in stomatal con-
ductance under heat stress since closure of stomata was already
induced by heat stress.
Chlorophylls are primary pigments for light harvesting in pho-
tosynthesis (Taiz and Zeiger, 2010). Chlorophyll content declined
with elevated CO2 , despite of the increase in Pn in tall fescue in
this study. Wullschleger et al. (1992) also found photosynthesis
was increased by elevated CO2 but Chl content declined in yellow-
poplar (Liriodendron tulipifera L.) and white oak (Quercus alba L.)
seedlings. The decline in chlorophyll content could have limited
light harvesting capacity for full photosynthetic responses to ele-
vated CO2 . However, plants grown at elevated CO2 levels could
have improved light-harvesting efficiency over plants under ambi-
ent CO2 . The decline in Chl content under elevated CO2 could be
related to the decline in leaf N content in our study. Similarly,
Hunt et al. (1996) also observed that doubling ambient CO2 led to
lower tissue N concentration in a C3 perennial grass (Pascopyrum
smithii). Zhang and Dang (2006) found that elevated CO2 caused
lower leaf total N content than ambient CO2 concentration despite
of the increase in photosynthetic rate after 2.5-month treatment
in white birch seedlings (Betula papyrifera Marsh.). The increase
in Pn in leaves with lower leaf Chl and N content at elevated CO2
conditions suggested that elevated CO2 could promote photosyn-
thetic efficiency, which was reflected by increases in the ratio of
C:N, although photosynthetic capacity could be compromised.
Elevated CO2 also altered the abundance of proteins involved
in major steps of photosynthesis, including light harvesting and
energy transfer (Chlorophyll a-b binding protein (CBP) of light-
harvesting complex II, electron transport (ferredoxin), carbon
fixation (carbonic anhydrase, CA), carboxylation (Rubisco and
Rubisco activase), and ATP generation (fructose-bisphosphate
adolase) in tall fescue exposed to heat stress. The chlorophyll
a–b binding proteins of light-harvesting complex II help form the
chlorophyll-protein complex embedded in the thylakoid mem-
brane for light energy transfer to chlorophyll a molecules in the
reaction center of photosystem II (Boekema et al., 1999a,b). Leaves
exposed to elevated CO2 had higher abundance of CBP than plants
grown at ambient CO2 . Ferredoxin acts as electron carrier in
electronic transport chain of photosynthesis, as well as electron
donor for many proteins (Arnon, 1969; Buchanan and Evans, 1969;
Buchanan et al., 1967). Elevated CO2 also led to an increase in ferre-
doxin abundance under heat stress. These results suggested that
elevated CO2 may enhance light-energy and electron transfer abil-
ity under heat stress, which could contribute to the enhanced Pn in
tall fescue exposed to heat stress.
Carbonic anhydrase (CA) is an enzyme catalyzing the conversion
of CO2 and water into bicarbonate. The abundance of CA decreased
with elevated CO2 under heat stress, which could help to main-
tain higher intercellular CO2 concentration and facilitate carbon
fixation. The content of CA also decreased under elevated CO2 in
cotton (Gossypium hirsutum) and Chlorella vulgaris (Chang, 1975;
Hogetsu and Miyachi, 1979). Rubisco large subunit, Rubisco small
subunit, and Rubisco activase were all down-regulated by ele-
vated CO2 . van Oosten and Besford (1995) also reported that the
abundance of Rubisco large subunit and Rubisco activase declined
after 22-d exposure to elevated CO2 in mature leaves of tomato
(Lycopersicon esculentum). While the protein content decreased,
the activity of Rubisco and Rubisco activase increased under ele-
vated CO2 , suggesting an enhanced efficiency of photosynthetic
enzymes at elevated CO2 concentration. Fructose-bisphosphate
aldolase in chloroplasts is involved in sugar phosphate metabolism
of carbon assimilation, which is a primary enzyme catalyzing
fructose-1-6-bisphosphate into glyceraldehyde 3-phosphate (G3P)
getting dihydroxyacetone phosphate (DHAP) and ATP (Abbasi and
Komatsu, 2004). Aldolase could be up-regulated by various envi-
ronmental stresses, such as cold, drought, salt and ABA stresses
(Abbasi and Komatsu, 2004). In this study, aldolase abundance
increased with elevated CO2 under heat stress, which could facili-
tate the generation of ATP for plants exposed to high temperatures.
Elevated CO2 also caused changes in the expression of pro-
teins involved in respiration under heat stress. Phosphoglycerate
kinase (PGK) is a major enzyme involved in glycolysis catalyz-
ing a phosphoryl-group from 1,3-bis-phosphoglycerate to ADP to
produce 3-phosphoglycerate and ATP (Bernstein et al., 1997). The
abundance of PGK deceased with elevated CO2 under heat stress
 J. Yu et al. / Environmental and Experimental Botany 99 (2014) 75–85
Fig. 8. Selected differentially expressed protein spots in leaves of tall fescue grown under heat stress (35 ◦ C) at two CO2 levels (400 and 800 ␮mol mol−1 ) showing examples
in changes in protein accumulation. Numbers correspond to spot identification numbers in Table 1.
in this study. Geiger et al. (1999) also found PGK activity declined
under elevated CO2 concentration in tobacco (Nicotiana tabacum).
Succinate dehydrogenase (SDH) is an enzyme participating in both
the citric acid cycle and electronic transport chain (Willeford et al.,
1989). The abundance of SDH increased with elevated CO2 under
heat stress. These results indicated that elevated CO2 had differen-
tial effects on glycolysis, electron transport, and carbon oxidation
in respiratory metabolism under heat stress.
Some proteins associated with the antioxidant defense system
were affected by elevated CO2 under heat stress. Ascorbate per-
oxidase (APX) and 2-Cys peroxiredoxin (BAS1) were up-regulated
by elevated CO2 under heat stress conditions, but not under the
non-stress optimal temperature. APX and BAS1 are enzymes which
scavenge reactive oxygen species in order to reduce oxidative dam-
ages caused by various environmental stresses (He and Huang,
2010). Jiang and Huang (2001) reported that APX activity was
decreased by 18 d of heat stress in both tall fescue and Ken-
tucky bluegrass. Cui et al. (2006) found APX activity was reduced
in heat sensitive tall fescue but increased in heat tolerant culti-
var. Antioxidant activity declined under stressful conditions, but
increased by elevated CO2 concentration under stress (Pritchard
et al., 2000; Schwanz et al., 1996). The increase in BAS1 was also
beneficial for plants to resist oxidative damage and protect pho-
tosynthetic apparatus (Baier and Dietz, 1997). The increase of the
abundance of APX and BAS1 in response to elevated CO2 under high
temperature could contribute to strengthened oxidative defense
systems.
83
In addition to proteins related with photosynthesis, respira-
tion and antioxidant system, other proteins such as actin, were
also up-regulated due to the elevated CO2 concentration under the
ambient temperature in the present study. Actin is an important
protein influencing cell cytoskeleton associating with cytoplasmic
streaming, cell shape determination, cell division and elongation,
organelle movement, extension growth and cell signaling (Costa
et al., 1998). Environmental stresses cause reduction in the abun-
dance of actin, restricting cell growth (Costa et al., 1998). The
accumulation of actin that resulted from elevated CO2 indicated
positive effects of elevated CO2 on cell growth and development in
tall fescue.
In summary, this study provided evidence for the beneficial
effects of elevated CO2 on the mitigation of heat stress damage
to photosynthesis in tall fescue. The improved photosynthesis by
elevated CO2 was associated with the increase in the abundance
of proteins in photosynthetic light reactions (CBP), electron trans-
port (ferredoxin), and ATP generation (adolase), as well as higher
carbon assimilation efficiency and carboxylation enzyme activities
of the Calvin cycle (higher C:N ratio, Jmax, Rubisco activity and
Rubisco activation). Elevated CO2 also induced the accumulation
of proteins involved in antioxidant metabolism (APX and BAS1),
which could also contribute to CO2 -mitigation of heat stress in tall
fescue. Elevated CO2 induced stomatal closure and chlorophyll con-
tent decline under ambient temperatures, which could have limited
the positive effects of elevated CO2 on photosynthetic capacity. It
would be useful to select cultivars of C3 perennial grass species with
84 J. Yu et al. / Environmental and Experimental Botany 99 (2014) 75–85
less sensitive stomata to elevated CO2 and increase nitrogen fertil-
ity to achieve the full benefits of CO2 effects on photosynthesis and
whole-plant growth in changing climate conditions.
Acknowledgements
The authors wish to thank Youth Technology Innovation Project
from Nanjing Agricultural University (KJ2013021) and Center for
Turfgrass Science at Rutgers University for funding support. Thanks
also go to Patrick Burgess for critical review of the manuscript.
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