Effect of elevated CO2 and high temperature on seed-set and grain quality of rice.

Abstract

Hybrid vigour may help overcome the negative effects of climate change in rice. A popular rice hybrid (IR75217H), a heat-tolerant check (N22), and a mega-variety (IR64) were tested for tolerance of seed-set and grain quality to high-temperature stress at anthesis at ambient and elevated [CO(2)]. Under an ambient air temperature of 29 °C (tissue temperature 28.3 °C), elevated [CO(2)] increased vegetative and reproductive growth, including seed yield in all three genotypes. Seed-set was reduced by high temperature in all three genotypes, with the hybrid and IR64 equally affected and twice as sensitive as the tolerant cultivar N22. No interaction occurred between temperature and [CO(2)] for seed-set. The hybrid had significantly more anthesed spikelets at all temperatures than IR64 and at 29 °C this resulted in a large yield advantage. At 35 °C (tissue temperature 32.9 °C) the hybrid had a higher seed yield than IR64 due to the higher spikelet number, but at 38 °C (tissue temperature 34-35 °C) there was no yield advantage. Grain gel consistency in the hybrid and IR64 was reduced by high temperatures only at elevated [CO(2)], while the percentage of broken grains increased from 10% at 29 °C to 35% at 38 °C in the hybrid. It is concluded that seed-set of hybrids is susceptible to short episodes of high temperature during anthesis, but that at intermediate tissue temperatures of 32.9 °C higher spikelet number (yield potential) of the hybrid can compensate to some extent. If the heat tolerance from N22 or other tolerant donors could be transferred into hybrids, yield could be maintained under the higher temperatures predicted with climate change.

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RESEARCH PAPERRESEARCH PAPER
In Posidonia oceanica cadmium induces changes in DNA
methylation and chromatin patterninggrain quality of rice
Maria Greco, Adriana Chiappetta, Leonardo Bruno and Maria Beatrice Bitonti*P. Madan1,2,*, S. V. K. Jagadish2,3,*, P. Q. Craufurd2,4, M. Fitzgerald5, T. Lafarge6,7and T. R. Wheeler2,†
Department of Ecology, University of Calabria, Laboratory of Plant Cyto-physiology, Ponte Pietro Bucci, I-87036 Arcavacata di Rende,
Cosenza, Italy
3Plant Breeding, Genetics, and Biotechnology Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines
4Resilient Dryland Systems, ICRISAT, Patancheru, AP 502324, India
5Grain Quality, Nutrition, and Postharvest Center, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines
6Crop and Environmental Sciences Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines
7CIRAD, UMR AGAP, F-34398, Montpellier, France
* To whom correspondence should be addressed. E-mail: b.bitonti@unical.it
Received 29 May 2011; Revised 8 July 2011; Accepted 18 August 2011
Abstract
* These authors have contributed equally to this work.
yTo whom correspondence should be addressed. e-mail: t.r.wheeler@reading.ac.uk
In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent
epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with
its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in
actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 mM) and high (50 mM) doses of Cd,
through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach,
respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase,
was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron
microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de
novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of
interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd
perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are
linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin.
Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.
for seed-set. The hybrid had significantly more anthesed spikelets at all temperatures than IR64 and at 29 �C this
resulted in a large yield advantage. At 35 �C (tissue temperature 32.9 �C) the hybrid had a higher seed yield than IR64
due to the higher spikelet number, but at 38 �C (tissue temperature 34–35 �C) there was no yield advantage. Grain gel
consistency in the hybrid and IR64 was reduced by high temperatures only at elevated [CO2], while the percentage of
broken grains increased from 10% at 29 �C to 35% at 38 �C in the hybrid. It is concluded that seed-set of hybrids is
susceptible to short episodes of high temperature during anthesis, but that at intermediate tissue temperatures of
32.9 �C higher spikelet number (yield potential) of the hybrid can compensate to some extent. If the heat tolerance
from N22 or other tolerant donors could be transferred into hybrids, yield could be maintained under the higher
temperatures predicted with climate change.
Key words: 5-Methylcytosine-antibody, cadmium-stress condition, chromatin reconfiguration, CHROMOMETHYLASE,
DNA-methylation, Methylation- Sensitive Amplification Polymorphism (MSAP), Posidonia oceanica (L.) Delile.
Introduction
In the Mediterranean coastal ecosystem, the endemic
seagrass Posidonia oceanica (L.) Delile plays a relevant role
by ensuring primary production, water oxygenation and
provides niches for some animals, besides counteracting
coastal erosion through its widespread meadows (Ott, 1980;
Piazzi et al., 1999; Alcoverro et al., 2001). There is also
considerable evidence that P. oceanica plants are able to
absorb and accumulate metals from sediments (Sanchiz
et al., 1990; Pergent-Martini, 1998; Maserti et al., 2005) thus
influencing metal bioavailability in the marine ecosystem.
For this reason, this seagrass is widely considered to be
a metal bioindicator species (Maserti et al., 1988; Pergent
et al., 1995; Lafabrie et al., 2007). Cd is one of most
widespread heavy metals in both terrestrial and marine
environments.
rice have tested conventionally bred japonica (Kim et al.,
Although not essential for plant growth, in terrestrial
plants, Cd is readily absorbed by roots and translocated into
aerial organs while, in acquatic plants, it is directly taken up
by leaves. In plants, Cd absorption induces complex changes
at the genetic, biochemical and physiological levels which
ultimately account for its toxicity (Valle and Ulmer, 1972;
Sanitz di Toppi and Gabrielli, 1999; Benavides et al., 2005;
Weber et al., 2006; Liu et al., 2008). The most obvious
symptom of Cd toxicity is a reduction in plant growth due to
an inhibition of photosynthesis, respiration, and nitrogen
metabolism, as well as a reduction in water and mineral
uptake (Ouzonidou et al., 1997; Perfus-Barbeoch et al., 2000;
Shukla et al., 2003; Sobkowiak and Deckert, 2003).
At the genetic level, in both animals and plants, Cd
can induce chromosomal aberrations, abnormalities in
may, therefore, be greater for hybrids than for conventional
ª 2011 The Author(s).
ª 2012 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-
nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Effect of elevated CO2and high temperature on seed-set and
1Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi 110012, India
2Plant Environment Laboratory, University of Reading, Cutbush Lane, Shinfield, Reading RG2 9AF, UK
Received 26 September 2011; Revised 13 February 2012; Accepted 20 February 2012
Abstract
Hybrid vigour may help overcome the negative effects of climate change in rice. A popular rice hybrid (IR75217H),
a heat-tolerant check (N22), and a mega-variety (IR64) were tested for tolerance of seed-set and grain quality to
high-temperature stress at anthesis at ambient and elevated [CO2]. Under an ambient air temperature of 29 �C
(tissue temperature 28.3 �C), elevated [CO2] increased vegetative and reproductive growth, including seed yield in all
three genotypes. Seed-set was reduced by high temperature in all three genotypes, with the hybrid and IR64 equally
affected and twice as sensitive as the tolerant cultivar N22. No interaction occurred between temperature and [CO2]
Key words: Elevated CO2, flowering, grain quality, high temperature, rice, spikelet fertility.
Introduction
Atmospheric CO2 concentration [CO2] could increase to
almost 700 ppm by the end of the century (IPCC, 2007). At
the plant level, a higher [CO2] increases photosynthesis,
growth, development, and yield of a wide range of
cultivated crops, including rice (Long et al., 2004, 2006;
Ainsworth and Long, 2005; Ainsworth et al., 2008). To
date, the majority of experiments with elevated [CO2] and
2001, 2003a, b; Sasaki et al., 2005, 2007; Shimono et al.,
2007; Yang et al., 2007) and indica cultivars (Weerakoon
et al., 2005; De Costa et al., 2007). However, modern hybrid
rice cultivars exhibit higher seedling vigour, rate of tillering,
relatively higher growth rate, and higher yield potential
than conventional inbred rice cultivars (Ling et al., 1994;
Xie et al., 1996). Yield enhancement under elevated [CO2]
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-
Journal of Experimental Botany, Vol. 63, No. 10, pp. 3843–3852, 2012
doi:10.1093/jxb/ers077 Advance Access publication 20 March, 2012

Page 2

rice cultivars. Recently, the response of two hybrid rice
cultivars to CO2enrichment was tested under in situ Free
Air CO2 Enrichment (FACE) systems (Liu et al., 2008;
Yang et al., 2009). A three-line indica hybrid, Shanyou 63,
grown at 570 ppm CO2increased yield by 34% (Liu et al.,
2008). Similarly, an inter-specific rice hybrid, Liangyoupei-
jiu, grown at approximately 580 ppm CO2had 24% and
20% higher grain yield and biomass, respectively, than
plants grown at ambient [CO2] (Yang et al., 2009).
However, the response of hybrids to a combination of
elevated [CO2] and high temperature has not been studied.
Studies on both high day temperatures (Jagadish et al.,
2010a, b) and high night temperatures (Peng et al., 2004;
Nagarajan et al., 2010; Welch et al., 2010) have demon-
strated negative effects on rice spikelet fertility and yields.
High day temperatures beyond the critical threshold during
sensitive developmental stages like gametogenesis and
flowering leads to low seed-set (Prasad et al., 2006; Jagadish
et al., 2007, 2008, 2010a, b, 2011). The impact of high night
temperatures leading to either increased respiration or other
mechanisms which lower rice yields has not been studied in
detail. Moreover, [CO2] is relatively high during the night,
so a further increase could lead to smaller beneficial effects
and a fall in the domain of diminishing returns on plant
processes (Pinter et al., 2000). Recently, it was shown that
the major part of the night had 500–700ppm of CO2even
under ambient level in a soybean FACE system (Bunce,
2011). Further, most FACE sites including the DUKE
forest face (http://face.env.duke.edu/description.cfm), aspen
FACE (http://aspenface.mtu.edu/), soy FACE (http://soyface.
illinois.edu/technology.htm) and others don’t fumigate CO2
at night. Hence the ideal and most practical combination of
high day temperature and elevated CO2 conditions at
sensitive flowering stage was tested in this study.
Although elevated [CO2] per se should increase pro-
ductivity and yield in rice, the increasing frequency and
intensity of short-duration high temperature events (>33 ?C)
pose a serious threat to agricultural production, especially
in cereals such as wheat (Modarresi et al., 2010) and rice
(Wassmann et al., 2009). The threat is highest when high
temperatures coincide with flowering (Yoshida et al., 1981;
Matsui et al., 1997; Prasad et al., 2006; Jagadish et al., 2007,
2008, 2010a, b; Rang et al., 2011) and grain-filling
(Fitzgerald and Resurreccion, 2009). These effects may be
exacerbated by higher tissue/canopy temperature associated
with stomatal response to elevated [CO2] (Vara Prasad
et al., 2006; Long and Ort, 2010). Although the effects of
[CO2] and high temperature over the entire life-cycle have
been studied in rice (Baker et al., 1992; Ziska et al., 1996;
Matsui et al., 1997; Baker, 2004; De Costa et al., 2006;
Cheng et al., 2009), the effect of more realistic impacts of
short episodes of high temperature at flowering at different
[CO2] has not been studied.
Furthermore, studies related to [CO2] and temperature
(FACE or controlled environments) have been restricted to
phenological and yield parameters; their effects on spikelet
fertility and grain quality are not known. Acceptable grain
quality is essential for any new cultivar to be accepted by
consumers and thereby adopted by farmers. The main traits
that define market price are the proportion of chalk
and broken grains in the sample (Cooper et al., 2008).
Chalkiness causes grains to break, and this increases with
higher temperature during grain-filling (Lisle et al., 2000;
Fitzgerald and Resurreccion, 2009). Amylose concentration
and gelatinization temperature contribute to texture and
cooking time; with high temperature, amylose concentration
declines in many rice varieties (Chen et al., 2008) and
gelatinization temperature increases (Cuevas et al., 2010).
The objective of this paper was to determine the
responses of a rice hybrid, a standard indica and a heat-
tolerant aus cultivar to a short episode of elevated
temperature (5 d) under ambient and elevated [CO2] at
flowering on (i) seed-set and seed number and (ii) rice grain
characteristics and quality. The hypotheses to be tested were
that (i) seed-set is reduced by high tissue temperature with
no interaction between tissue temperature and [CO2]; (ii)
high [CO2] increases spikelet and seed number at all
temperatures; (iii) there is no difference in the response of
seed-set, seed yield or grain characteristics and quality traits
of the hybrid and indica cultivar to high temperature and
[CO2]; and (iv) short episodes of high temperature at
anthesis at ambient and high [CO2] have no effect on grain
characteristics and quality traits.
Materials and methods
The experiment was carried out between April and September 2007
using controlled environment facilities at the Plant Environment
Laboratory, Department of Agriculture, University of Reading,
UK (51?27# N, 00?56# W). Plants were raised inside growth
cabinets under optimum temperature and photoperiod conditions,
and transferred to adjacent growth cabinets to impose high-
temperature treatments at flowering.
Plant growth and maintenance
Plants were grown in a medium without soil exactly as previously
described (Jagadish et al., 2007; 2008). Two rice varieties (Oryza
sativa L.), the aus type N22 and the indica cv. IR64 and one
hybrid, IR75217H (IRRI138–IR68897A3IR60819-34-2 R), were
tested. For each, five seeds were sown in each pot at a depth of
2–2.5 cm and thinned to one plant per pot at the three-leaf stage.
Plants were maintained under fully watered conditions with
a complete nutrient solution containing 100 mg l?1inorganic
nitrogen throughout the crop growth cycle. The nutrient solution
was acidified to pH 5 to avoid Fe deficiency (Yoshida et al., 1976).
Plants were sprayed twice with foliar feed (Miracle-Gro, The
Scotts Company, UK Ltd.) at 3.75 g l?1at 7 d intervals until
panicle emergence. There were no major pest or disease problems.
Growth chambers
The three varieties were grown in controlled environment cham-
bers (internal size 1.431.431.5 m). Aspirated temperature and
relative humidity (RH) were measured every 10 s using copper–
constantan thermocouples and a data logger (Delta T Devices,
Burwell, Cambridge, UK) and averaged over 10 min for the
entire crop growth period. An optimum day/night temperature
(2960.57 ?C/2160.34 ?C) and RH (6061.45%/8061.21%) with
a short inductive photoperiod of 11 h (Summerfield et al., 1992)
from 08.00 h to 19.00 h with a thermo period of 13 h (07.00 h to
20.00 h) was imposed. A photosynthetic photon flux density of
3844 | Madan et al.

Page 3

650 lmol m�2s�1was maintained at the floor of the chamber
using a combination of cool white fluorescent tubes and in-
candescent lamps. Lamps were balanced to ensure uniform flux
densities throughout the cabinet. A centrally placed fan circulated
air uniformly throughout the chamber.
CO2and temperature treatments
The [CO2] in the chambers was controlled by a 12-channel
measurement and control system using ADC WA 526 IRGA
(Infra-Red Gas Analyser) manufactured by ADC, Unit 35,
Hoddesdon Industrial Centre, Pindar Road, Hoddesdon, Herts.,
UK. The system sampled each chamber in turns and delivered
(pure) CO2to each chamber via 12 solenoid valves, according to
a calculation based on the difference between the reading and the
set point. The sampling ‘dwell’ on each chamber was 40 s, so it
sampled each chamber once every 5.3 min (8 chambers340 s) and
CO2was delivered after recalculating the valve opening based on
the reading and the set point. Two independent sets of plants were
maintained at ambient (380 ppm) or elevated (760 ppm) [CO2] for
the entire growth period except during anthesis, when one of the
two sets was exposed to either 35 ?C or 38 ?C for 5 d. The
transition time from day-time maximum to night-time minimum
was 5 h. A square wave heat treatment was applied to overcome
the potentially confounding effects of gradually increasing temper-
ature (Jagadish et al., 2007, 2008, 2010a, b). There were therefore
six combinations of temperature and [CO2]: (i) ambient tempera-
ture and CO2(29 ?C and 380 ppm CO2), (ii) ambient temperature
and elevated CO2(29 ?C and 760 ppm CO2), (iii) 35 ?C at anthesis
and 380 ppm CO2, (iv) 35 ?C at anthesis and 760 ppm CO2, (v)
38 ?C at anthesis and 380 ppm CO2, and (vi) 38 ?C at anthesis and
760 ppm CO2. Apart from the change in conditions during the
treatment period, all other aspects relating to the environmental
conditions in the growth chamber were identical to those described
earlier. For the high-temperature treatments, at the initiation of
anthesis (identified by the protruding anthers), four replicate plants
from both ambient and elevated [CO2] were transferred into
growth chambers maintained at either 35 ?C or 38 ?C between
09.00 h and 15.00 h to impose treatments (iii) to (vi), while another
set of four plants was left undisturbed in the chambers to impose
treatments (i) and (ii). The experimental design for the analysis of
temperature and [CO2] treatments was therefore six treatments in
two replicated blocks (growth chambers) each with four replicate
plants (pots) per growth cabinet, i.e. n¼8 observations per
treatment.
Main tiller panicles were tagged and seed-set was measured on
these panicles. Any spikelets that underwent anthesis (i.e. with
a visible anther) outside the 5 d temperature treatment period were
marked with acrylic paint and were not included in further
analyses (Jagadish et al., 2008). The majority of the unmarked
spikelets on the main tiller exposed to the stress treatments were
located toward the top of the panicle and had finished flowering
within the experimental treatment period of 5 d (Table 1). In all
three gentoypes, across each treatment more than 100 spikelets
were used for determining the seed-set, except in IR64 under 29 ?C
(n¼92) and 35 ?C (n¼89) under ambient [CO2]. Around 100
spikelets are considered as a threshold level for estimating seed-set
under high temperature stress in rice.
Spikelet tissue temperature for IR64 was measured by placing
copper–constantan micro-thermocouples inside the spikelets of
three independent plants in all treatments. The data on spikelet
tissue temperatures were recorded by a data logger (Delta
T Devices, Burwell, Cambridge, UK) every 10 s and averaged over
10 min for the entire period (Table 2).
Analyses
Yield parameters and phenology: Spikelets on the main tiller
panicles were separated into unmarked (anthesed during tempera-
ture treatments) and marked (anthesed under control conditions)
and seed-set was calculated from the number of unmarked filled
spikelets as a proportion of the total number of unmarked
spikelets. Plants from all six treatments were harvested as replicate
samples and separated into root, stems, and leaves. Plant parts
were dried separately at 60 ?C until a constant weight was
obtained. The root–shoot ratio and total plant biomass were
calculated from the components. The total number of tillers per
plant was recorded while the tillers with a productive panicle (i.e.
bearing filled grains) were considered for the determination of
panicle number.
Grain quality: Filled grains from the main-tiller spikelets used
to calculate seed-set were dried at 38 ?C in a forced-air oven for
8–10 d until they attained a constant dry weight. Individual replicate
samples for each genotype/treatment were bulked to give sufficient
material for the analysis of quality traits. From the bulked sample,
two replicate sub-samples of seeds from each treatment and entry
were separated and analysed for amylose content, gel consistency,
chalkiness (%), and length, width, and percentage of whole grain at
the Grain Quality and Nutrition Center, IRRI, Philippines.
Physical characteristics of the grain were measured using a 1625
Grain Inspector (Foss, Denmark). In order to measure amylose
Table 1. Average number of spikelets on the main tiller exposed
to stress treatments (unmarked) at flowering and the remaining
number of spikelets flowering under ambient conditions (marked)
CO2
(ppm)
Temperature
(?C)
Genotype/
hybrid
No. of
unmarked
spikelets
No. of
marked
spikeletsa
38029 IR64
IR75217H
N22
IR64
IR75217H
N22
IR64
IR75217H
N22
IR64
IR75217H
N22
IR64
IR75217H
N22
IR64
IR75217H
N22
94.065.28
169.369.79
116.464.18
78.662.65
172.065.67
93.763.25
93.764.81
156.664.27
98.562.89
129.965.71
209.6612.2
118.764.53
100.365.20
168.165.50
104.962.94
111.962.53
170.165.66
104.462.74
*
*
*
34.063.0
53.262.94
23.462.31
25.962.47
71.163.17
26.461.94
*
*
*
31.362.43
68.562.90
28.162.00
28.161.70
77.362.71
30.661.77
35
38
760 29
35
38
aIndicates that spikelets that flowered on the main panicle under
ambient conditions were not marked.
Table 2. Spikelet tissue temperature measured using micro-
thermocouples from replicated growth chambers in IR64 under
three different air temperatures and two CO2concentrations;
numbers are 6SD
CO2
(ppm)
Spikelet tissue temperature (?C)
29 3538
380
760
D
28.460.42
28.260.44
–0.18
32.660.45
32.961.11
0.28
34.060.69
35.060.67
0.97
Elevated temperature and CO2 affect rice yield and quality | 3845

Page 4

and gel consistency, polished grains were ground to pass through
a 0.5 mm sieve in a cyclone mill (Udy Cyclone Sample Mill 3010-
030, Fort Collins, CO). Amylose concentration and gel consistency
were measured as previously described (Juliano, 1971; Tran et al.,
2011).
Statistical analysis
All the yield parameters, phenological traits, and grain quality
components were analysed as a completely randomized design with
growth chambers as blocks and four pots as replicates using
Genstat Version 11 (Rothamsted Experimental Station, UK).
Comparison of regressions was also carried out with the same
software.
Results
Spikelet tissue temperature was lower than the air temper-
ature at 29, 35, and 38 ?C by 0.67, 2.28, and 3.48 ?C, on
average, respectively. These effects were similar to those
observed in the same growth chambers for rice cultivar
Azucena (Jagadish et al., 2007). On average, higher [CO2]
reduced spikelet tissue temperature by 0.18 ?C at 29 ?C,
but increased spikelet temperature by 0.28 ?C and 0.97 ?C
at 35 ?C and 38 ?C, respectively (Table 2).
Growth and development
The overall effects of [CO2] on the growth and development
of the three genotypes were as expected based on other
[CO2] studies and so are only summarized here. The
biomass of IR64 increased from 34.5 g plant?1at 380 ppm
to 51.4 g plant?1at 760 ppm CO2at 29 ?C, an increase of
49%. Comparable figures for the hybrid were 57.9 and 82.2 g
plant?1(an increase of 42%). The short 5 d episodes of high
temperature did not affect overall growth or development
(see Supplementary Table S1 at JXB online). Elevated [CO2]
influenced the phenology and days to flowering and harvest
were delayed in all three entries at high [CO2]. The biggest
delay in days to flowering was recorded with the hybrid (+7 d)
and the least with N22 (+3 d). However, the influence of
elevated CO2on the grain-filling phase was smaller, with
both IR64 and the hybrid having an increase by a day while
it was 3 d with N22, compared with ambient [CO2].
Anthesis
In all three genotypes, irrespective of the temperature
regimes or CO2levels, the majority of the spikelets on the
main tiller panicle completed flowering in 5 d (Table 1). The
total number of spikelets on the main tiller panicle differed
significantly with variety, temperature, and [CO2] (P <0.001)
with a significant interaction between [CO2] and variety
(P <0.05) and temperature and variety (P <0.001). In both
IR64 and N22 the number of spikelets on the main
tiller panicle were not significantly affected by increasing
temperatures (P >0.05). However, the hybrid recorded
a significant increase in the spikelet numbers with higher
temperatures compared with the control under both ambi-
ent and elevated [CO2] (P <0.001). The hybrid had more
Fig. 1. Seed-set and number of spikelets on the main tiller in IR64
(a), N22 (b), and a hybrid, IR75217H (c), exposed to high-
temperature stress for five consecutive flowering days in combina-
tion with either ambient or elevated CO2concentration. Solid
symbols represent seed-set and open symbols the number of
spikelets. Bars indicate 6SE.
3846 | Madan et al.

Page 5

(P <0.001) spikelets opening on the main tiller than N22 or
IR64 at all temperatures and [CO2]: 176 to 236 compared
with 110 to 142 in IR64 at 380 ppm and 760 ppm,
respectively (Fig. 1). Higher temperature reduced the
number of anthesing spikelets (P <0.05) in all three
genotypes, with the effect being more prominent under
elevated [CO2], especially in IR64 (P <0.001) and the hybrid
(P <0.05). Spikelet numbers were generally greater with
higher [CO2], notably at 29 ?C in IR64 and the hybrid. By
contrast, the number of spikelets anthesing in N22 was not
influenced by higher [CO2].
Seed-set
Seed-set in the three genotypes was reduced by temperature
(P <0.001) and [CO2] (P <0.05), with a significant interac-
tion only between the temperature and genotype (P <0.001)
(Fig. 1). There was no effect of [CO2]3temperature in-
teraction, nor did the growth cabinets have any significant
effect on percentage seed-set (P >0.55). Temperature had
a large effect on seed-set in all genotypes, with seed-set
declining from between 75% and 80% at 29 ?C to <20% in
the hybrid at 38 ?C. N22 was the most tolerant genotype,
achieving 57% seed-set at 38 ?C. The interaction between
genotype and temperature mainly reflected the response of
IR64 when seed-set was higher at 29 ?C and lower at 38 ?C.
A comparison of regressions for seed-set against tissue
temperature and [CO2] by genotype showed effects of
temperature (P <0.001) alone and in interaction with
genotype, but no effect of [CO2] (P >0.05). Hence, across
[CO2] treatments, responses to temperature and [CO2] were
best described by three separate lines with different intercepts
and slopes (Table 3). N22 was the most tolerant of
temperature, with a slope of –4.3560.21 (SE) while IR64
(slope –9.7860.33) and the hybrid (slope –8.8060.30), were
approximately twice as sensitive to temperature as N22
(Table 3).
Seed yield and seed number
Seed yield per main tiller was highly correlated with seed
number (r¼0.98) and with seed-set (r¼0.71) (Fig. 2). Seed
yield and seed number were significantly affected by
temperature(P
<0.001),[CO2]
(P <0.01) in both IR64 and the hybrid. In N22, both seed
yield and seed number were affected by temperature
(P <0.001) but not [CO2] or their interaction (P >0.40). At
29 ?C and 380 ppm CO2, the hybrid gave a yield advantage
of about 1 g per panicle (c. 65%) over N22 and IR64.
However, at the highest temperature (38 ?C), this yield
advantage was lost and N22 yielded more than IR64 and
the hybrid. High [CO2] increased seed yield at 29 ?C,
especially in IR64 and the hybrid, but this advantage was
also completely lost at 35 ?C and 38 ?C. Individual kernal
weight increased with increasing temperature (P <0.01) and
withelevated [CO2](P
<0.001)
interaction (P >0.70). The ANOVA from a comparison of
regressions for seed yield against temperature, [CO2] and
genotype revealed that the seed yield of N22 at different
tissue temperatures and [CO2] could be described by a single
line (slope –0.105). By contrast, in IR64 and the hybrid, the
response to temperature varied with [CO2] and individual
regressions were better (Table 3). Thus, the sensitivity
of seed yield to temperature at ambient [CO2] increased
from –0.10560.01 g ?C�1in N22 to –0.16260.02 in IR64
and –0.2960.03 in the hybrid. At higher [CO2], the values
were –0.31160.04 g ?C�1and –0.4460.07 in IR64 and the
hybrid, respectively (Table 3).
andtheirinteraction
withno significant
Grain quality parameters
Amylose concentration was affected by temperature and
[CO2] and their interaction (P <0.05), but the differences
were only within 0.5–1%. At 380 ppm [CO2], amylose
concentration decreased very slightly with increasing tem-
perature, particularly in IR64 (Fig. 3). For the other two
Table 3. Regression parameters for seed-set and seed-yield using tissue temperatures in two rice genotypes and a hybrid; values are
6SE
CO2
N22
Intercept
IR64
Intercept
Hybrid
InterceptSlopeSlopeSlope
Seed-set
380 ppm
Single line
780 ppm
Temperature (T)
CO2(parallel lines)
T3CO2(separate lines)
Seed-yield
380 ppm
Single line
780 ppm
Temperature (T)
CO2(parallel lines)
T3CO2(separate lines)
–
209.5666.80
–
P <0.001
P >0.244
–
–4.35260.211
–
–
362.40610.7
–
P <0.001
P >0.450
–
–9.77660.333
–
–
326.6469.60
–
P <0.001
P >0.270
–
–8.79860.299
–
–
4.61860.333
–
P <0.001
P >0.970
–
–0.10560.01
–
6.09160.534
–
11.19661.289
P <0.001
P <0.001
P <0.001
–0.16260.017
–
–0.31160.041
10.78260.987
–
15.87262.387
P <0.001
P <0.05
P <0.01
–0.29060.030
–
–0.44060.073
Elevated temperature and CO2 affect rice yield and quality | 3847