Journal of Experimental Botany, Vol. 61, No. 1, pp. 191–202, 2010
doi:10.1093/jxb/erp294Advance Access publication 9 October, 2009
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
Physiological and molecular changes in Oryza meridionalis
Ng., a heat-tolerant species of wild rice
Andrew P. Scafaro1, Paul A. Haynes2and Brian J. Atwell1,*
1Department of Biological Sciences, Macquarie University, NSW 2109, Australia
2Department of Chemistry and Biomolecular Sciences, Macquarie University, NSW 2109, Australia
Received 7 May 2009; Revised 10 August 2009; Accepted 8 September 2009
Oryza meridionalis Ng. is a wild relative of Oryza sativa L. found throughout northern Australia where temperatures
regularly exceed 35 ?C in the monsoon growing season. Heat tolerance in O. meridionalis was established
by comparing leaf elongation and photosynthetic rates at 45 ?C with plants maintained at 27 ?C. By comparison with
O. sativa ssp. japonica cv. Amaroo, O. meridionalis was heat tolerant. Elongation rates of the third leaf of
O. meridionalis declined by 47% over 24 h at 45 ?C compared with a 91% decrease for O. sativa. Net photosynthesis
was significantly higher in O. sativa at 27 ?C whereas the two species had the same assimilation rates at 45 ?C. The
leaf proteome and expression levels of individual heat-responsive genes provided insight into the heat response of
O. meridionalis. After 24 h of heat exposure, many enzymes involved in the Calvin Cycle were more abundant, while
mRNA of their genes generally decreased. Ferredoxin-NADP(H) oxidoreductase, a key enzyme in photosynthetic
electron transport had both reduced abundance and gene expression, suggesting light reactions were highly
susceptible to heat stress. Rubisco activase was strongly up-regulated after 24 h of heat, with the large isoform
having the largest relative increase in protein abundance and a significant increase in gene expression. The
protective proteins Cpn60, Hsp90, and Hsp70 all increased in both protein abundance and gene expression. A
thiamine biosynthesis protein (THI1), previously shown to act protectively against stress, increased in abundance
during heat, even as thiamine levels fell in O. meridionalis.
Key words: Calvin Cycle, dark reaction, ferredoxin-NADP(H) oxidoreductase, heat shock protein, heat stress, leaf elongation, O.
meridionalis, Rubisco activase, thiamine biosynthesis protein (THI1).
The Intergovernmental Panel on Climate Change is predict-
ing a likelihood of more intense, more frequent, and longer
lasting heat waves (IPCC, 2007). Although rice is a pan-
tropical grass and therefore relatively well adapted to high
temperatures in comparison with other cereals such as
wheat (Triticum aestivum L.), peak temperatures will in-
crease over this century, providing an abiotic stress to which
cultivated rice might not be adapted. The ability of modern
rice (Oryza sativa L.) to be cultivated in hotter climatic
regimes may be limited by its narrow gene pool as
domesticated rice has only about 10–20% of the genetic
diversity found in wild progenitors (Zhu et al., 2007).
The pre-eminent method currently practised for improving
abiotic stress tolerance in rice cultivars is to source germ-
plasm for desirable traits. Recent attempts to do so have
been successful, with backcrossing of O. sativa ssp. japonica
and indica leading to substantial improvements in resistance
to many abiotic stresses (Ali et al., 2006; Lafitte et al., 2006;
Cheng et al., 2007). Although this approach has been
productive, it has been limited to O. sativa. With more than
* To whom correspondence should be addressed: E-mail: firstname.lastname@example.org
Abbreviations: Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; RCAI, Rubisco activase large isoform; RCAII, Rubisco activase small isoform; FNR,
ferredoxin-NADP(H) oxidoreductase; THI1, thiamine biosynthesis protein; HSP, heat shock protein; dnaK, heat shock protein 70 gene; GroEL, chaperone 60 gene;
PGK, phosphoglycerate kinase gene; PRK, phosphoribulokinase gene; GDC-P, glycine dehydrogenase gene; TK, transketolase; tkt, transketolase gene; SBPase,
sedoheptulose-1,7-bisphosphatase; sbp, sedoheptulose-1,7-bisphosphatase gene; RuBP, ribulose-1,5-bisphosphate.
ª 2009 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/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
20 known species within the Oryza genus, a large source of
genetic material remains to be exploited (Brar and Khush,
Oryza meridionalis Ng. is a wild rice species likely to
have abiotic stress tolerance, as it shows high levels of
genetic diversity between geographically isolated acces-
sions, probably as a result of selective pressure on isolated
gene pools (Juliano et al., 2005). Oryza meridionalis was
first recognized as a species in 1981, found in northern
Australia (Ng et al., 1981) and subsequently in West
Papua, Indonesia (Lu and Silitonga, 1999). Phylogenetic
analysis of Adh1 and Adh2 genes, as well as mitochondrial
and chloroplast microsatellites support O. meridionalis as
a divergent lineage within the rice genome (Ge et al., 1999;
Nishikawa et al., 2005), similar to that of O. sativa. The
extent of phenotypic diversity in O. meridionalis has not
been described: however, it shares the AA genome with
O. sativa, making O. meridionalis a good candidate for
genetic improvement of cultivated rice through introgres-
sion of new genetic material. For example, the introgres-
sion of two genes, yld1.1 and yld2.1 from the progenitor of
modern rice, O. rufipogon, improved grain yield by 18%
and 17%, respectively, in an elite hybrid breed of O. sativa
(Xiao et al., 1998). This improvement occurred without the
detection of any deleterious impacts on other desirable
traits. Previous transgenic manipulations targeting osmotic
solute production, transcription factors, oxidative stress
detoxification and ion transport have provided increased
resistance in many species to stress such as heat (Wang
et al., 2003b). The genetic bottleneck in domesticated rice,
in conjunction with the probability that its wild relatives
have a diverse array of stress-related genes, opens the
possibility of rapid genetic improvement through breeding
Genomic analysis through the use of expression se-
quence tags and cDNA libraries from stressed plant tissue
has been used effectively in identifying the response of
plants to stress and discovering the identity of genes
involved (Sreenivasulu et al., 2007). An alternative tech-
nique previously used for determining the molecular
responses of rice to stress is proteomic analysis (Salekdeh
et al., 2002; Komatsu et al., 2003; Komatsu and Tanaka,
2004; Lee et al., 2007). The proteomic approach is
important to the understanding of the abiotic stress
response because many key proteins are regulated transla-
tionally and post-translationally (Greenbaum et al., 2003;
Lee et al., 2004).
Heat tolerance in the wild rice O. meridionalis was
established by comparison of seedling growth and photo-
synthetic rates at optimal and high temperatures, using O.
sativa. ssp. japonica (cv. Amaroo) as a domesticated control
cultivar. Based on these findings, proteomic analysis using
two-dimensional gel electrophoresis coupled with nanoLC-
MS/MS established which proteins might play key roles in
the thermotolerance of O. meridionalis. Finally, semi-
quantitative RT-PCR on the genes of interest was used to test
whether increased protein abundance was transcriptionally
Materials and methods
Seeds of Oryza meridionalis Ng. were collected from a wild
accession located in the Cape York Peninsula of Australia
(15?41#57## S, 145?02#48## E). Seedlings were grown in a mixture
(1:1:1 by vol.) of silty loam, clay, and organic potting mix. This
soil mixture was used throughout all the experiments. A 2 cm layer
of vermiculite was placed across the soil surfaces to reduce
evaporation and to maintain soil moisture. All experiments were
carried out on 22-d-old seedlings that were grown in 500 ml
polyvinyl pots in growth chambers (Thermoline Scientific Equip-
ment, Australia) with an illumination of approximately 500 lmol
m?2s?1and a temperature of 27/22 ?C (day/night) with a 12 h
photoperiod. Heat-treated plants were held at 45 ?C continuously
for 24 h, commencing 2 h into a light period. A 12 h dark period
followed 10–22 h into the heat treatment, and leaf data (excepting
continuous growth measurements) were collected 2 h into the
subsequent light period. Seedlings were well watered and fertilized
weekly with a commercial liquid fertilizer.
Seedling growth was determined by measuring elongation of the
third leaf blade, using a HR4000 Linear Variable Displacement
Transducer (LVDT) with data logged every 6 min by the software
program VuGrowth ver. 1.0 (Applied Measurement, Oakleigh,
Vic). Seedlings grown in pots were transferred to a growth
chamber containing the LVDT unit one day after the emergence
of the third leaf blade. For control experiments, seedlings were
grown at a constant 27 ?C for 46 h with four of the eight
measuring stations randomly assigned to each of the two species.
Heat experiments were conducted as above with an increase to
45 ?C in the period 22–46 h. Both control and heat treatments
were repeated in four independent experiments.
Gas exchange and water measurements
Net photosynthetic rates (NPR), respiration rates, stomatal
conductance (gs), intercellular CO2(Ci), transpiration rates (T),
and leaf temperatures (Tleaf) were determined using a LI-6400 (Li-
Cor, NE, USA) portable gas exchange system. All gas exchange
measurements were made within growth chambers on seedlings
subjected to previously mentioned temperature and light regimes
and a CO2concentration of 380 lmol mol?1. Cuvette temperatures
were adjusted to 27 ?C or 45 ?C to match growth chamber
temperatures for the two treatments. Net photosynthetic rates were
measured in the first 3 h of the light period with gas chamber
illumination at saturating levels of 1500 lmol m?2s?1. To
minimize the possibility of dry air reducing stomatal conductance,
10 ml of H2O was added to the soda lime canister prior to use and,
subsequently, sample chamber relative humidity was maintained
above 30% at 45 ?C. Respiration rates of seedlings subjected to
greater than a 9 h dark period were measured in the absence of
Leaf water potential (Wleaf) was measured using a pressure
bomb. The third leaf was cut from plants close to their base and
immediately placed in the pressure chamber for measurement of
balancing pressure. Relative water content (RWC) was derived
from the formula (fresh mass–dry mass)/(turgid mass–dry mass).
All gas-exchange and water measurements were made on four
plants per pot, with three pots representing each treatment.
Leaf blades were collected from both control and heat-treated
plants (see ‘Plant material’ section) and immediately ground in
liquid nitrogen. Leaf powder was washed with TCA and acetone
and subjected to phenol/SDS extraction as described by Wang
(2003a). The protein extract was resuspended in 200 ll of
192 | Scafaro et al.
rehydration buffer [7 M urea, 2 M thiourea, 4% CHAPS (w/v), 30
mM DTT, trace amount of bromophenol blue]. Approximately
250 mg of protein quantified by the Bradford assay (Bio-Rad
Protein Assay) was loaded on 11 cm, 4–7 ReadyStrip IPG strips
(Bio-Rad) with rehydration following the manufacturer’s instruc-
tions. Focusing occurred using a PROTEAN IEF Cell (Bio-Rad)
at 20 ?C with a total of 37 200 focusing hours (200 V for 1 h, 1000
V for 1 h, 4000 V for 3 h, and 8000 V for 3 h). After focusing, IPG
strips were placed in re-equilibration buffer (50 mM TRIS pH 8.8,
6 M urea, 30% glycerol, 2% SDS, trace amount of bromophenol
blue) for 30 min then run in the second dimension on 8–16%
gradient Criterion pre-cast gels (Bio-Rad). All gels were stained
with Lava purple (FLUOROtechnics, Sydney, Australia) as di-
rected by the manufacturer’s guidelines and fluorescently imaged
on a Typhoon 9200 scanner (Amersham Pharmacia Biotech).
Harvesting of leaf tissue, sample preparation, and gel electropho-
resis experiments were replicated in three independent experiments
so that each control and heat-stress sample was biologically and
experimentally replicated three times.
Image analysis and protein identification
Spots that were matched across all gels were labelled accordingly
and abundances measured as integrated density using ImageJ
software (Abramoff et al., 2004). Percentage relative integrated
density values were calculated to normalize against loading differ-
ences and the values used to determine heat-induced differentially
Following image analysis, 48 spots that had a heat-induced
change in relative integrated density values greater than 1.8-fold
(up or down) were selected for further analysis. The fold change
values in both Tables 2 and 3 are expressed as abundance (heat)/
abundance (control) so in Table 3 smaller values indicate a more
significant change. Additional spots of two highly abundant
proteins, with a fold change that was consistent across the
replicates, but slightly below the 1.8-fold threshold, were also
analysed (spots 148 and 132) and included in Table 2.
Spots of interest were excised and washed for 5 min with 100
mM NH4HCO3, followed by destaining with 50% acetonitrile (v/v)
in 50 mM NH4HCO3for 10 min twice. Reduction and alkylation
required 10 mM DTT in 100 mM NH4HCO3, applied at 37 ?C for
60 min followed by 55 mM iodacetamide in 100 mM NH4HCO3
for 45 min. Gels were digested with 20 ll of 12.5 ng ll?1trypsin
(Promega) in 50 mM NH4HCO3on ice for 30 min then incubation
at 37 ?C overnight. Peptides were extracted by washing gel pieces
with 30 ll of 50% acetonitrile and 2% formic acid (v/v) for 20 min
on three occasions. Vacuum centrifugation was used to concen-
trate the peptide solution to 10 ll prior to loading of samples onto
a ThermoFinnigan LCQ-Deca ion trap mass spectrometer for
peptide identification through nano liquid chromatography on line
with tandem mass spectrometry (nanoLC-MS/MS) as described by
Medina et al. (2005). Tandem mass spectra were searched using the
XTandem algorithm run under the GPM-XE interface (Craig and
Beavis, 2003; Fenyo and Beavis, 2003). The default XTandem
search parameters were used and sequences searched against
a database of rice (O. sativa) protein sequences (March 2008
version), representing the complete rice genome, downloaded from
Leaf blades of seedlings were instantly frozen in liquid nitrogen
after 24 h of treatment and stored at –80 ?C until RNA was
extracted using a RNeasy Plant Mini Kit (Qiagen) following the
manufacturer’s instructions. RNA was converted to cDNA using
a SuperScript VILO cDNA synthesis kit (Invitrogen) with 2.1 lg
of RNA used as a template. PCR was performed using a GoTaq
Green Master Mix (Promega), with a 25 ll reaction solution
containing 2 ll of cDNA and gene-specific forward and reverse
primers. Primers were designed using protein sequences identified
by nanoLC-MS/MS. Protein sequences were BLAST searched
through NCBI and the corresponding rice cDNA sequences used
as the template for primer design. The primers are given in
Supplementary Table S1 at JXB online. For PCR, an initial step
of 95 ?C for 2 min was followed by 22, 26 or 30 cycles of 95 ?C for
30 s, 52 ?C for 30 s, and a final step of 72 ?C for 20 s. PCR
products were visualized on a 2.5% agarose gel stained with Gel
Red (Biotium) and imaged with GeneSnap version 6.00.26
software (Syngene, Frederick, MD). Band intensities were analysed
using ImageJ software and normalized by comparison with actin.
RNA was extracted from triplicate, biological (pot) replicates to
Determination of thiamine concentration
Thiamine was extracted from leaf blades, converted to thiochrome
and fluorescence measured by a method similar to that of Ohta
(1993). Leaf blades were ground in liquid nitrogen and 0.6 g added
to 10 ml of 0.1 M HCl, 40% methanol (Buffer A). Samples were
vortexed and incubated at 60 ?C for 30 min and centrifuged at
4000 g for 15 min, supernatant was filtered (0.45 lm) and an equal
volume added to 0.1% potassium hexacyanoferrate (III) in 15%
NaOH (Buffer B). This 50:50 mixture was diluted a further 8-fold
again in equal volumes of Buffer A and Buffer B, before
fluorescence measurements were taken to overcome a quenching
effect that, at higher concentrations, resulted in an underestima-
tion of thiamine levels (see Supplementary Fig. S3 at JXB online).
To confirm the accuracy of the assay, a sample was spiked with
a known quantity of thiamine, resulting in a 99.5% recovery (see
Supplementary Fig. S4 at JXB online). Fluorescence was measured
(Ex. 375 nm, Em. 455 nm, and 10 nm bandpass) on a PerkinElmer
LS 55 luminescence spectrometer. Thiamine concentration was
determined by comparing results with a standard curve between
0.02 ng ll?1and 0.625 ng ll?1(R2¼0.9999).
When applying statistics a one-way analysis of variance was used
with a 5% LSD test in cases of multiple comparisons. The
statistical analysis was carried out using SPSS statistical analysis
software (Ver. 16.0.1, SPSS Inc). Values are based on the means
6SE of 3–4 experimental replicates.
Growth, photosynthesis and water relations in
response to temperature
Third leaves of O. sativa elongated faster than O. meridio-
nalis at 27 ?C, however, when exposed to 45 ?C, O.
meridionalis elongated faster than O. sativa (Fig. 1). After
4 h of heat, LER of O. sativa was halved whereas leaf
growth was not affected in O. meridionalis. Furthermore,
24 h at 45 ?C caused a 91% decrease in the LER of O. sativa
but only a 47% decline in O. meridionalis (Table 1). There
was a noticeable increase in the growth rates of both species
towards the end of the 24 h heat treatment, possibly
connected to the second light photoperiod which began
22 h into the heat treatment.
Although 45 ?C is a severe temperature for an herbaceous
plant, neither species showed physical leaf symptoms of
temperature stress such as wilting, necrosis or loss of
pigmentation after heat exposure, which was consistent with
sustained leaf elongation following heat application. In
Heat tolerance of O. meridionalis | 193
particular, the more heat-sensitive O. sativa had a LER
equal to O. meridionalis after 8 h of recovery (Fig. 1).
There was a significant difference in the net photosyn-
thetic rate between O. sativa and O. meridionalis at 27 ?C
but not at 45 ?C (Table 1) and, therefore, the impact of heat
on net photosynthesis was greater for O. sativa (53% fall)
than for O. meridionalis (42% fall). There was a significant
increase in the dark respiration rates of both species at 45
?C with a slightly greater increase recorded in O. sativa.
There was no difference between the species in transpira-
tion rates, RWC or Wleaf at 45 ?C (Table 1). While
transpiration rates increased to the same extent in both
species when exposed to 45 ?C, Wleaf increased simulta-
neously, which could only occur if soil was able to maintain
water supply and hydraulic function remained unimpaired.
Thus, the 45 ?C treatment imposed heat stress without any
leaf water deficit.
Identification of proteins associated with heat stress
In total, 392 individual spots were matched and labelled
across all replicates and treatments (Fig. 2; see Supplemen-
tary Fig. S1 at JXB online). Spots that did not have
a unidirectional change across all replicates were excluded
from further analysis. Protein identification was made of 50
spots by nanoLC-MS/MS. This includes all spots which
showed an expression level change of greater than 1.8-fold
between the two conditions. Table 2 contains identification
and abundance measurements for 23 spots that showed
greater than 1.8-fold increases under heat stress, along with
two additional high-abundance spots which were consis-
tently increased, but by less than 1.8-fold. Table 3 contains
identification and abundance measurements for 25 spots
showing a greater than 1.8-fold decrease in abundance due
to heat stress.
Due to the overlap of spots one to five, this cluster was
considered as a single unit for the purpose of image analysis
and mass spectrometry. Similarly, spots six to eight were
treated as a single spot cluster rather than individual spots
(see Supplementary Fig. S2 at JXB online). Many proteins
were identified in multiple spots, which is not uncommon
and suggestive of alternative RNA splicing and post-trans-
lational modifications such as glycosylation (Rodrı ´guez-
Pin ˜eiro et al., 2007).
Photosynthetic metabolism proteins
Many proteins of O. meridionalis associated with the dark
reaction of photosynthesis increased in abundance during
Table 1. Impact of temperature on leaf elongation, gas exchange and leaf thiamine concentrations
All measurements were taken 24 h into treatment. LER, leaf elongation rate; NPR, net photosynthetic rate; gs, stomatal conductance; Ci,
intercellular CO2; T, transpiration rate; Wleaf, leaf water potential; RWC, relative water content; Tleaf, leaf temperature. Values are means 6SE,
n¼3–4. Superscript letters indicate significant differences (LSD multiple comparison test, P <0.05).
Oryza sativa Oryza meridionalis
27 ?C 45 ?C 27 ?C 45 ?C
LER (mm h?1)
NPR (lmol CO2m?2s?1)
Dark respiration rate (lmol CO2m?2s?1)
gs(mol H2O m?2s?1)
T (mmol H2O m?2s?1)
Thiamine concentration (lg g?1FW)
Fig. 1. Leaf elongation rates (LER) of third leaves beginning 1
d after emergence in O. sativa ssp. japonica (cv. Amaroo)
(squares) and O. meridionalis (circles). Seedlings were grown in
a growth chamber at 27 ?C with a 12 h photoperiod and
illumination of 300 lmol m?2s?1. Measurements were taken by
a Linear Variable Displacement Transducer (LVDT) placed within
the chamber for 24 h at control, 27 ?C (closed symbols) and heat,
45 ?C (open symbols), followed by an 8 h recovery period (shaded
grey) where heat-treated plants where returned to 27 ?C. The first
light period commenced 2 h before heat was applied and
continued 10 h into the heat treatment; the second light period
commenced 22 h into the heat period. An asterisk denotes 24 h of
heat treatment when protein, gene and photosynthetic observa-
tions were made. Values are means 6SE, n¼4.
194 | Scafaro et al.
Fig. 2. Two-dimensional electrophoresis gels of 22-d-old O. meridionalis seedlings grown under either control, 27 ?C (A) or 45 ?C, 24 h
heat treatment (B). Approximately 250 lg of leaf blade protein was initially run on pH 4–7 IPG strips followed by SDS-PAGE
electrophoresis using 8–16 gradient Criterion gels. Labelled spots are those that were found in all replicates. The noticeable protein spot
(spot 110) found at between 50 kDa and 60 kDa and a pI of just over 6.5 was identified as the Rubisco large subunit.
Heat tolerance of O. meridionalis | 195
heat stress (Fig. 3A). The large isoform of Rubisco activase
(RCAI) increased in relative abundance more than any
other protein, while the small isoform of Rubisco activase
(RCAII) was found in multiple spots that both increased
and decreased (Tables 2, 3). In rice, there are two isoforms
of Rubisco activase, a 45 kDa large isoform and a 41 kDa
small isoform (To et al., 1999). The expression level of the
mRNA encoding RCAI increased substantially after 24 h of
heat while RCAII gene expression declined (Fig. 4).
Enzymes involved in the Calvin Cycle; chloroplastic
phosphoglycerate kinase, transketolase, phosphoribuloki-
nase (PRK), and chloroplastic sedoheptulose-1,7-bisphos-
phatase consistently increased in abundance with heat.
However, expression of the genes encoding all of these
Glycine dehydrogenase (GDC-P) increased substantially
with heat. Glycine metabolism is an essential part of
photorespiration (Douce et al., 2001). Previously, reduced
expression of GDC-P in chilled rice has been viewed as
a loss of photorespiratory function (Yan et al., 2006). An
increase in GDP-C, therefore, suggests an up-regulation of
photorespiration by O. meridionalis during heat exposure.
The photosynthetic light reaction protein ferredoxin-
NADP(H) oxidoreductase (FNR) displayed a substantial
decline in abundance over the heat period (Fig. 3B). Three
of the spots that decreased in volume with heat were
identified as FNR, including spot 224 which decreased to
the greatest extent (Table 3). The mRNA levels for this gene
Heat-induced protective proteins
The protective proteins chaperone 60 (Cpn60), heat shock
protein 70 (HSP70), and heat shock protein 90 (HSP90) had
increased levels of protein and gene expression following
heat treatment (Figs 3C, 4). Proteins homologous to Cpn60
increased in multiple spots. A protein homologous to
a germin-like protein was found in lower amounts with
heat. Although germin-like proteins are protective proteins
they seem to be associated with pathogen response in plants
rather than abiotic stress such as heat (Byron, 2002; Miche
et al., 2006; Zimmermann et al., 2006; Elvira et al., 2008).
Soluble germin-like protein in barley has previously been
found in reduced amounts when plants were subjected to
heat (Vallelian-Bindschedler et al., 1998).
A thiamine biosynthesis protein homologous to THI1
found in Arabidopsis (Arabidopsis thaliana), increased in
abundance with heat (Table 2; Fig. 3C) while the mRNA
Table 2. Protein spots with increased abundance upon heat stress
Spot no. Fold-changea
Accession nob. Putative proteinc
1277.5 BAA97583Rubisco activase, large isoform 27/–15151.4
6 to 8
Rubisco activase small isoform
Rubisco activase small isoform
Phosphoglycerate kinase, chloroplast
Chaperonin-60, chloroplast alpha subunit
Chaperonin-60, chloroplast beta subunit
Chaperonin-60, chloroplast beta subunit
Chloroplast heat shock protein 70
Endosperm lumenal binding protein (HSP70)
Thiamine biosynthesis protein (THI1)
ATP synthase CF1 beta subunit
Hypothetical protein of the AAA family
1 to 51.8
aThe fold-change values were derived from heat/control spot volumes.
bNCBI listed accession numbers of proteins in downloaded rice database matched by Xtandem algorithm to nanoLC-MS/MS spectra.
cMS matched proteins were BLAST searched (Altschul et al., 1997) and high scoring homologues provided annotation for putative O. sativa
196 | Scafaro et al.
level dropped slightly (Fig. 4B). Results showed a small but
significant decline in the amount of thiamine present in O.
meridionalis after exposure to heat (Table 1). This is in
contrast to the increase in expression of the THI1 homo-
logue protein involved in thiamine synthesis.
The major component of Rubisco, easily identified as it
accounted for an average of 22% of all protein in each gel,
did not show substantial or consistent change across
replicates. However, certain isoforms of the Rubisco large
subunit (spots 250, 254, 255) were substantially reduced
with heat (Table 3). The ATPase beta subunit was identified
in multiple spots that both increased and decreased with
heat. Of the other proteins identified as having a greater
than 1.8-fold reduction under high temperatures, many
were proteins of unknown function, while others were
proteins of broad metabolic function (spots 213, 243, 205,
219, 242, 220, 204, 198).
Many of the heat-induced proteins found in the wild rice
species O. meridionalis have previously been shown to
increase in O. sativa seedlings exposed to 42 ?C, as
determined by two-dimensional gel electrophoresis by Lee
et al. (2007). For example, transketolase, HSP70, Cpn60,
protein reported here all became more abundant over
a similar 24 h period in O. sativa. Similar to the findings
for O. meridionalis, there was a reduced abundance of FNR
in O. sativa. By contrast, PRK declined in O. sativa during
the 24 h heat stress, whereas it increased substantially with
heat in O. meridionalis.
More than any other functional group, photosynthesis-
related proteins were differentially expressed in O. meridio-
nalis during heat stress, as is the case in cold-stressed rice
seedlings (Yan et al., 2006) suggesting a specific connection
between photosynthetic enzymes and temperature stress in
A complex expression profile of Rubisco activase was
observed in O. meridionalis under heat stress. The increased
overall abundance of Rubisco activase with heat was caused
by specific subunits of the multimeric protein. Rubisco
activase is a member of the AAA+protein family, as are
most chaperones. Through ATP hydrolysis, Rubisco acti-
vase regenerates Rubisco that has been deactivated by
bound non-substrate sugar phosphates, or RuBP bound to
uncarbamylated active sites (Spreitzer and Salvucci, 2002;
Table 3. Protein spots with decreased abundance upon heat stress
Accession no.Putative proteinPeptide
2500.254 CAG34174 Rubisco, large subunit 16/–38 52.8
Rubisco, large subunit
Rubisco, large subunit
Rubisco activase small isoform
Rubisco activase small isoform
Rubisco activase small isoform
Rubisco activase small isoform
Rubisco, large subunit
Photosystem II 10 kDa polypeptide
Mitochondrial F1-ATPase beta subunit
atpB gene product
ATP synthase subunit beta
S-adenosylmethionine synthetase, type 1
Germin-like protein 1
Germin-like protein 1
aAs in Table 1, fold-change values were derived from heat/control spot volumes.
Heat tolerance of O. meridionalis | 197
Portis, 2003). Rubisco activase seems to be heat-labile in
many plant species, limiting photosynthetic capacity during
heat stress (Law and Crafts-Brandner, 1999; Salvucci and
Crafts-Brandner, 2004; Kurek et al., 2007).
Specifically, RCAI was almost undetectable at 27 ?C but
the protein increased in abundance at 45 ?C through
transcriptional up-regulation: preferential expression of the
RCAI isoform therefore occurs at high temperatures in
O. meridionalis. The high levels of RCAI observed are
consistent with the heat tolerance of Rubisco activase
isoforms in spinach (Spinacea oleracea L.), where the
optimum temperature for ATP hydrolysis was 45 ?C for
the large isoform compared with 32 ?C for the small
isoform (Crafts-Brandner et al., 1997). Although the
thermotolerance of RCAI has not previously been estab-
lished in rice, under non-stressed conditions, transgenic rice
over-expressing RCAI show greater photosynthetic capac-
ity, through improvements in both dark and light reactions
(Wu et al., 2007).
The expression profile of RCAII was complex. RCAII
was found in multiple protein spots that both increased and
decreased in abundance while rcaII expression levels de-
creased after exposure to heat. In pea (Pisum sativum L.)
and spinach, which also express large and small Rubisco
activase polypeptides, the small form is believed to be the
more labile at higher temperatures (Crafts-Brandner et al.,
1997; Salvucci et al., 2001). This is attributed to observa-
tions that the small form denatured and formed insoluble
aggregates at high temperature. If this were the case in rice,
identification of multiple spots of RCAII could be expected
after application of heat. However, in O. meridionalis, there
were no new RCAII spots detected in heat-treated tissue.
The protein is therefore not being degraded but, instead,
changing conformation or shifting to new isoforms in
a consistent manner. Expression of distinctive activase
forms at high temperatures, which have not been attributed
to loss of function and aggregation, have been previously
noted in cotton (Gossypium hirsutum L.), spinach, and
wheat (Law et al., 2001; Law and Crafts-Brandner, 2001;
Rokka et al., 2001). Similar post-translational protein
modifications in tomato (Solanum lycopersicum) and oilseed
rape (Brassica napus L. Reston) have been observed, with
a series of spots corresponding to isoforms of a given
protein (Agrawal and Thelen, 2006; Hattrup et al., 2007).
The overall increase in Rubisco activase was not matched
by an increase in Rubisco which did not consistently
change. This implies an increase in the activase/Rubisco
ratio. Previous analysis of cotton and tobacco leaves found
Fig. 3. Comparison of percentage spot volume on 2-DE gels between 27 ?C (light shade) and 45 ?C (dark shade) treated O. meridionalis
seedlings. (A) Proteins associated with the dark reaction of photosynthesis including the Calvin Cycle enzymes, phosphoglycerate
kinase, chloroplast precursor (PGK), transketolase, chloroplast precursor (TK), phosphoribulokinase (PRK), sedoheptulose-1,7-
bisphosphatase, chloroplast precursor (SBPase), the photorespiration enzyme glycine dehydrogenase (GDC-P), and Rubisco activase,
large isoform (RCAI). (B) The light reaction of photosynthesis represented by ferredoxin-NADP(H) oxidoreductase (FNR). (C) Protective
proteins chaperone 60 (Cpn60), heat shock proteins 70 and 90 (HSP70, HSP90), and the thiamine biosynthesis protein THI1. Seedlings
were grown in growth chambers at 27/22 ?C with a 12 h photoperiod and illumination of 500 lmol m?2s?1. Seedlings were either
harvested under control conditions or exposed to 45 ?C for a 24 h period prior to harvesting 2 h into a light period. Spots were analysed
using ImageJ software with integrated density used as a determinant of spot volume. Values are mean 6SD, n¼3.
198 | Scafaro et al.
that an increase in the activase/Rubisco ratio leads to
comparatively higher Rubisco activation states at temper-
atures up to 42 ?C (Crafts-Brandner and Salvucci, 2000).
Furthermore, by maintaining higher activation states,
photosynthetic rates were less inhibited by heat stress.
Unlike O. sativa (Lee et al., 2007), in the heat-tolerant O.
meridionalis there is an increase in multiple components of
the Calvin Cycle including a consistent increase across all
replicates of PRK, the enzyme responsible for the final step
in RuBP regeneration. Alone these results indicate up-
regulation of the Calvin Cycle when O. meridionalis was
heat-treated. However, this would require sustained energy
output from electron transport to provide the substrates
required for the reduction phases of the Calvin Cycle.
Crafts-Brandner and Law (2000), Cen and Sage (2005), and
Kubien and Sage (2008) showed that the electron transport
pathway of photosynthesis is highly susceptible at severe
temperatures above 40 ?C. In O. meridionalis, there is
a decrease in both the gene expression and protein levels of
FNR which catalyses the last enzymatic step of the non-
cyclic photosynthetic light reaction responsible for the
reduction of NADP+in the PSI complex (Hurley et al.,
2002). It is therefore likely that, although O. meridionalis
had increased abundances of Calvin Cycle enzymes, in-
hibition of electron transport at such a severe temperature
would inhibit both the light and dark reactions of photo-
synthesis. This may explain the significant reduction in the
photosynthetic rate of O. meridionalis (and O. sativa) after
heat exposure. Analysis by Hajirezaei et al. (2002) found
growth rate and CO2assimilation were reduced in tobacco
plants with reduced levels of FNR. However, it was noted
in their study that levels of Rubisco activase and trans-
ketolase were not altered by the impact of lower FNR
levels. This is consistent with the reduced FNR but in-
creased Rubisco activase and transketolase abundances in
Gene expression and protein abundance of the protective
chaperone HSP70, HSP90,and Cpn60 increase in O. mer-
idionalis upon heat stress. Cpn60 is a form of chaperone
found in mitochondria and chloroplasts of plants and
believed to support protein folding (Wang et al., 2004).
HSP70 and HSP90 have been associated with an array of
protective functions including protein refolding, transporta-
tion, and protein signalling pathways (Wang et al., 2004).
Mutational studies of Cpn60 demonstrate that both the
alpha and beta subunits are necessary for effective chloro-
plast function and are thus important in heat tolerance
(Apuya et al., 2001; Ishikawa et al., 2003). Similarly the
HSP70 family is directly correlated with thermotolerance in
plants (Lee and Scho ¨ffl, 1996; Sung and Guy, 2003).
Of particular interest is the dual increase in RCAI and
Cpn60 in O. meridionalis upon heat stress. An interaction
between Rubisco activase and the Cpn60-b subunit is likely,
as the chaperone has been shown to bind to Rubisco
activase during heat stress in what is thought to be
a protective role (Salvucci, 2008).
A THI1 homologue was found in greater abundance with
heat in O. meridionalis. Similarly, in the thermotolerant
species Populus euphratica, THI1 increased 3-fold during
the first 6 h of exposure to 42 ?C (Ferreira et al., 2006). In
Arabidopsis, thi1 gene expression increased in roots sub-
jected to hypoxia and in roots and rosettes subjected to high
salt concentrations (Ribeiro et al., 2005). The most likely
mode of action for THI1 would be an increased abundance
of its known product, thiamine. In support of this, the
application of thiamine to rice resulted in protection against
a wide range of pathogens, with mutational studies
Fig. 4. Semi-quantitative RT-PCR temperature comparisons for
selected O. meridionalis genes. RNA was initially extracted from
the leaf tissue of seedlings exposed to either control (27 ?C) or 24
h of high temperature (45 ?C). (A) PCR products were run on
a 2.5% agarose gel and stained with Gel Red (Biotium).
Abbreviated gene names are; Rubisco activase large (RcaI) and
small (RcaII) isoform genes, phosphoglycerate kinase (pgk), trans-
ketolase (tkt), phosphoribulokinase (prk), sedoheptulose-1,7-
bisphosphatase (sbp), glycine dehydrogenase (gdc-p), ferredoxin-
NADP(H) oxidoreductase (fnr), Heat shock protein 90 (hsp90),
chaperone 60 (GroEL), heat shock protein 70 (dnaK), and thiamine
biosynthesis protein (thi1). All genes underwent 26 cycles of PCR
except RcaI, RcaII, and GroEL that underwent 22 cycles and dnaK
which required 30 cycles. (B) Spot intensities of bands were
compared and relative gene expression after 24 h at 45 ?C (heat/
control) was calculated with actin used as an internal standard.
Values are mean 6SD, n¼3.
Heat tolerance of O. meridionalis | 199
attributing this to interaction between the thiamine and
the salicylic-acid pathway (Ahn et al., 2005). Recently,
abiotic stress through the application of polyethylene glycol,
NaCl, and H2O2in maize (Zea mays L.) lead to an increase
in leaf thiamine concentration, again supporting a direct
role of thiamine in the stress response (Rapala-Kozik et al.,
By contrast, the concentration of thiamine in leaves of O.
meridionalis fell significantly during heat stress, in spite of
the enzyme THI1, which is responsible for its synthesis,
increasing substantially. Findings in Arabidopsis and yeast
suggest that THI1 might fulfil a function distinct from
thiamine biosynthesis during heat stress. Specifically, Arabi-
dopsis THI1 protein and THI4 found in yeast appear to be
involved in the protection and repair of damaged mitochon-
drial DNA (Machado et al., 1997; Chabregas et al., 2001).
In yeast, a THI4 mutant is more susceptible to oxidative
stress under high temperatures even though the cultures
were supplemented with thiamine (Medina-Silva et al.,
2006). Alternatively, if thiamine were degraded faster at
high temperatures, an increase in THI1 may simply be
indicative of an increase in thiamine turnover.
The higher growth rate of O. meridionalis at 45 ?C
compared with O. sativa ssp. japonica, as well as a lesser
impact of heat on photosynthesis, indicated tolerance of O.
meridionalis to the extreme heat typical of its natural range.
Rubisco activase and the regulation of the large and small
isoforms found in rice are a striking aspect of the heat stress
response of O. meridionalis. The Rubisco activase large
isoform, in particular, is selectively up-regulated in response
to heat. Multiple enzymes of the Calvin Cycle increased in
abundance with heat. A fall in FNR, an important
component of the light reaction, implies a susceptibility of
electron transport at 45 ?C for O. meridionalis. The con-
sistent increase in expression of a THI1 homologue at high
temperatures was notable because both THI1, an enzyme
involved in thiamine biosynthesis, and thiamine have been
linked to the heat stress response in plants. Interestingly,
thiamine levels fell in heat-stressed O. meridionalis even
though the abundance of THI1 increased.
Supplementary data are available at JXB online.
Supplementary Table S1. Primers used in semi-quantita-
Supplementary Fig. S1. 2-DE triplicate gels.
Supplementary Fig. S2. 2-DE spot clusters.
Supplementary Fig. S3. Serial dilution of leaf thiamine
Supplementary Fig. S4. Accuracy of thiamine quantifica-
The authors would like to thank Artur Sawicki, Moham-
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Robert Willows, Juliet Suich, Phyllis Farmer, and Thomas
Roberts. PH acknowledges support from the NSW Office of
Science and Medical Research in the form of a Biofirst
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