An attempt to interpret a biochemical mechanism of C4 photosynthetic thermo-tolerance under sudden heat shock on detached leaf in elevated CO2 grown maize
An attempt to interpret a biochemical mechanism of C4 photosynthetic thermo- tolerance under sudden heat shock on detached leaf in elevated CO2 grown maize
Mingnan Qu 0 1 2
James A. Bunce 0 2
Richard C. Sicher 0 2
Xiaocen Zhu 0 1 2
Bo Gao 0 2
Genyun Chen 0 1 2
0 Natural Science Foundation of China (31700201) and Sailing Project, Shanghai Municipal Science and Technology Commission , China, 17YF1421800
1 CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese academy of Sciences , Shanghai, China, 2 USDA-ARS , Crop Systems and Global Change Laboratory , Beltsville, MD , United States of America, 3 Centralab Institute of Basic Medical Science, Chinese Academy of Medical Sciences , Beijing , China
2 Editor: Shaojun Dai, Northeast Forestry University , CHINA
Detached leaves at top canopy structures always experience higher solar irradiance and leaf temperature under natural conditions. The ability of tolerance to high temperature represents thermotolerance potential of whole-plants, but was less of concern. In this study, we used a heat-tolerant (B76) and a heat-susceptible (B106) maize inbred line to assess the possible mitigation of sudden heat shock (SHS) effects on photosynthesis (PN) and C4 assimilation pathway by elevated [CO2]. Two maize lines were grown in field-based open top chambers (OTCs) at ambient and elevated (+180 ppm) [CO2]. Top-expanded leaves for 30 days after emergence were suddenly exposed to a 45ÊC SHS for 2 hours in midday during measurements. Analysis on thermostability of cellular membrane showed there was 20% greater electrolyte leakage in response to the SHS in B106 compared to B76, in agreement with prior studies. Elevated [CO2] protected PN from SHS in B76 but not B106. The responses of PN to SHS among the two lines and grown CO2 treatments were closely correlated with measured decreases of NADP-ME enzyme activity and also to its reduced transcript abundance. The SHS treatments induced starch depletion, the accumulation of hexoses and also disrupted the TCA cycle as well as the C4 assimilation pathway in the both lines. Elevated [CO2] reversed SHS effects on citrate and related TCA cycle metabolites in B106 but the effects of elevated [CO2] were small in B76. These findings suggested that heat stress tolerance is a complex trait, and it is difficult to identify biochemical, physiological or molecular markers that accurately and consistently predict heat stress tolerance.
Data Availability Statement; All relevant data are within the paper
The daily, seasonal, and annual mean temperatures experienced by plants have increased as a
result of human-caused increase in atmospheric CO2 concentration [
]. Accumulative studies
have examined interactive effects of temperature and CO2 on both C3 and C4 plants. The
interactive effects cover varies of physiological and biochemical aspects, for instance, stomata
driven water use efficiency [2±5], leaf morphology [
], photorespiration [
], photosystem II
] and Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) activase [
]. In contrast, the interactions between elevated CO2 and sudden heat shock (SHS) have
been examined in only a few studies [
]. In the field conditions, it has been proved that this
type of abnormally extreme weather events also may decrease crop growth and reduce
harvestable yields [
]. Studies on the effects of heat stress in C4 plants have been well reported [e.g.,
4±7, 11, 13]. In general, C4 plants have better adaption to warmer climates than C3 plants, this
may be due to the fact that C4 possess special pathway of photosynthetic-carbon metabolism
(PCM). However, it remains unclear if elevated [CO2] can reprogram the PCM in response to
SHS in C4 plants, such as maize. Therefore, understanding physiological, biochemical and
molecular processes in maize caused by elevated [CO2] undeniably facilitates the prediction of
plant responses to future climate [
Previous studies have shown that the decreases of net photosynthesis (PN) after SHS in
maize cannot be fully explained by a stomatal limitation because Ci values were sufficiently
high to prevent CO2 from inhibiting rates of PN [
]. Therefore, it was likely that high
temperature effects on PN were the result of impaired metabolic processes in the leaf. Prior evidence
suggested that a deactivation of Rubisco is an early event in the inhibition of PN in response to
high temperature [
]. Additionally, Rubisco activase, which is a chloroplast protein that is
essential for maintaining Rubisco in an active state, may be inhibited by high temperature
]. Consequently, these evidences suggest that high temperatures inhibit the Calvin
cycle and reduce the rate of synthesis of ribulose-1,5-bisphosphate, the substrate for Rubisco.
On the other hand, it has been argued that the C4 cycle is more sensitive to water stress than
the Calvin cycle [
], and the same could be true for high temperature stress. The
inactivation of PN by high temperatures may also be related to membrane damage within the
chloroplast and at various other sites in the cell. For example, increased electrolyte leakage was
observed following high temperature treatments, suggesting that the cellular membrane was
disrupted by heat stress .
Metabolite analysis is an effective method for elucidating mechanisms of abiotic stress
tolerance, including heat stress [
]. Mayer et al. [
] reported an increase in the abundance of
γaminobutyric acid (GABA), β-alanine, alanine, and proline in cowpea (Vigna unguiculata) as a
result of heat shock. Song et al. [
] also reported that several metabolites in leaves of Kentucky
bluegrass (Poa pratensis) were accumulated shortly after heat stress treatments. These previous
metabolite studies were exclusively performed using ambient CO2 and very few studies have
investigated the changes of metabolite accumulation in response to SHS under elevated [CO2]
conditions. Elevated [CO2] treatments can have mitigating effects on the response of plant
growth to drought stress or nutritional deficiencies [
]. Therefore, it would be valuable to
know if elevated [CO2] could mitigate the effects of SHS on plant metabolism. The hypothesis
in this study was given that elevated [CO2] would mitigate SHS effects on maize seedlings, and
that the effects would differ in lines with contrasting heat stress tolerance.
Materials and methods
Materials and experimental set-up
Two maize (Zea mays L.) inbred lines, i.e., B76 (heat tolerant) and B106 (heat susceptible),
were used in this study. These two maize genotypes differed with respect to thermo-tolerance
based on the polymorphism of several phenotypic markers [
]. Seeds of B76 (PI 550483)
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and B106 (PI 594049) were obtained from U.S. Germplasm Resources Information Network
The experiment was conducted in field-based, open top chambers (OTCs) to examine the
heat tolerance of maize cultivars grown under ambient and elevated [CO2]. The experimental
site was located at the Beltsville Agricultural Research Center, USDA-ARS (39Ê 00' N, 76Ê 56'
W), Beltsville, MD, USA. Seeds of both inbred lines were sown in six OTCs measuring 2 m
long by 1.5 m wide by 2 m high starting from May 24th in 2013. Each chamber was spaced 2 m
apart to minimize shading, and individual plants were thinned at 7 days after emergence
(DAE) to 15 cm distance. The soil in each OTC was kept moist by applying water once weekly
to field capacity. Plants in OTCs were exposed to ambient air or ambient air plus 180 ppm
CO2 as described elsewhere [
]. There were three chambers per CO2 treatment, and all
chambers were planted with both maize inbred lines. Mean and maximum air temperatures were
23.8 and 37.6 oC, respectively, during the period when experiments were performed. Mean
daytime CO2 concentrations were 394 and 566 ppm, in the ambient and elevated OTC
Heat stress treatments and gas exchange measurements
Sudden heat shock (SHS) treatments were applied to individual leaves between 10:00 am and
12:00 pm on six clear sunny days in 2013. The treatments were applied when the sixth leaf was
fully expanded about 30 days after emergence (DAE). Leaf gas exchange rates were measured
on the marked sections of the leaves using a CIRAS-1 Portable Photosynthesis System (PP
system, Amesbury, MA). After initial leaf gas exchange measurements, marked sections of intact
leaves were placed in water-jacketed leaf cuvettes with an internal radiator and fan. Air
temperatures within the cuvettes were raised to 45 oC by circulating heated water from a
temperature controlled bath through the cuvette. Air from the OTC was continuously flushed through
each cuvette. Instaneous measurements of net photosynthetic rates (PN) and stomatal
conductance (gs) were carried out on the same sections of leaves after the heat treatments ended at
12:00 pm. Leaf samples collected immediately after gas exchange measurements were used to
determine electrolyte leakage, or frozen in liquid nitrogen for further analysis.
Relative leaf injury measurements
Relative leaf injury (RI) was measured by quantifying electrolyte leakage before and after heat
stress treatments, as described previously [
Quantitative transcript abundance
Changes of transcript abundance (qPCR) in maize leaves were determined as described
]. Two maize leaf dics (0.6 cm diameter, and approximately 0.5 g fresh weight) were
ground using liquid N2 in a sterile mortar and pestle, and total RNA was extracted using
TRIzol1 reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). After
quantification with a NanoDrop spectrophotometer (model 2000c, Thermo-Fisher Scientific
Inc., Waltham, MA), first strand cDNA was synthesized with 2 μg of total RNA (OD260 nm/
OD280 nm > 1.95) using oligo(dT) 20 primers and SuperScript III RNase H reverse
transcriptase from Invitrogen. The resultant cDNA was diluted 10-fold and was used as a template for
real-time quantitative polymerase chain reaction (qPCR). Amplifications were performed with
a model Mx3005P qPCR System plus Brilliant SYBR1 Green qPCR Master Mix (Stratagene,
La Jolla, CA).
Primers and functional annotations for C4 related photosynthetic enzyme genes are listed
in Table 1. Assays were performed with three biological samples from each treatment, and
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Product length (bp)
measurements were replicated three times. Maize ACTIN1 gene was used as an expression
control and relative transcript abundance was calculated by 2-ΔΔCT×100 according to Pfaffl
C4 photosynthetic enzyme assays
Five leaf discs (about 3.14 cm2) were removed from the lamina of leaves in field experiments as
described above. Leaf materials were rapidly transferred to labeled envelopes and immediately
immersed in liquid N2 to quench metabolism. All samples were stored at −80ÊC prior to
analysis. Two leaf discs from each plant were extracted with 0.6 ml ice cold extraction buffer
consisting of 50 mM Tris±HCl (pH 7.50), 10 mM MgCl2, 1 mM EDTA, 1% (w/v) PVP-40, 5 mM
Na+-pyruvate and 10% glycerol. Immediately prior to extraction, freshly prepared 1 μM
leupeptin and 5 mM dithiothreitol were added to the buffer solution. Samples were extracted at
0ÊC with a ground glass tissue homogenizer and the homogenates were transferred to 2 ml
plastic centrifuge tubes and spun for 3 min at 12,000 × g in an Eppendorf model 5415D
microfuge. The supernatant was transferred to a 1.5 mL Eppendorf tube and assayed immediately or
stored briefly in liquid N2.
Enzyme activity measurements were performed spectrophotometrically at 25ÊC as
described by Maroco et al. [
]. Activities of NADP-malate dehydrogenase (MDH) were
measured in 1 ml solution containing 50mM Tris-HCl (PH 8.0), 1 mM EDTA, 100 mM oxalacetic
acid, 10 mM NAHPH and 0.025 ml leaf extract. PEP carboxylase (PEPCase) activities were
measured in 1 ml solution containing 50 mM Tris±HCl (pH 8.0), 5 mM NaHCO3, 5 mM
MgCl2, 0.14 mM NADH, 10 mM PEP (tricyclohexlamine salt), 1 unit Malate dehydrogenase
and 0.025 ml sample as described in Ziska et al. (1999). Activities of NADP-malic enzyme
(NADP-ME) were measured in 1 ml solution containing 50 mM Tris-HCl (pH 8.0), 5mM
EDTA, 500 mM MgCl2, 100 mM malic acid, 250 mM dithioerythritol, 20 mM NADP+ and
0.025 ml sample. All measurements were performed using a Shimadzu model 2101
spectrophotometer operated in the kinetic mode. Enzyme activities were calculated from the rate of
the changes in optical wavelength at 340 nm.
Freeze-dried leaf tissue (~30 mg total) for each treatment was added to a 2.0 ml Eppendorf tube
containing a 3.2 mm ceramic bead and ~100 μl fine garnet powder. Maize leaf tissue was
homogenized in a Tissue Lyzer ball mill at 30 cycles s-1. A 50 μl mixture of 2.5 mM
α-aminobutyric acid, 2.0 mg ribitol and 1.4 ml ice-cold 70% methanol was injected into each sample and
vortexed vigorously. The suspended plant tissues were heated to 45ÊC for 15 minutes in a water
bath and then the extracts were microcentrifuged for 5 minutes at 12000 x g as described above.
Supernatants were gently transferred into 15 ml fresh conical, plastic centrifugation tubes. The
pellets were washed once with 70% methanol as described above and the supernatants were
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combined. The washed pellets were air-dried overnight and used for the determination of starch
as previously described [
A total of 10 organic acids and soluble carbohydrates was measured by gas chromatography
coupled to mass spectrometry (GC-MS) as described by Roessner et al. [
samples were separated by gas chromatography and the resultant ions were detected with a mass
selective detector (model 7125, Agilent technologies, Wilmington, DE). Total ion
chromatograms were quantified using peak identification and calibration parameters within the Agilent
MSD CHEMSTATION software program. Independent standard curves were prepared for each set
of extractions with known mixtures of organic acids and soluble carbohydrates. Ribitol was
added during extraction functioned as the internal standard. Compounds included in the
organic acid fraction were citrate, aconitate, malate, fumarate and succinate. Compounds in
the soluble carbohydrate fraction were fructose, glucose, sucrose, maltose, and starch.
Each gas exchange analysis in OTC experiment is the mean of 11 independent measurements.
For PEPCase assays, membrane integrity and qPCR, data was derived from 3 independent
replicates for each maize cultivar. One-way analysis of variance (ANOVA) via software SPSS 10.0
(SPSS Inc., USA) was applied to identify significant differences between heat stress and CO2
treatments for specific maize cultivars or for specific daily time point. Three-way ANOVA
using R software (3.3.0 version) was used to test the significant effects of maize cultivars,
grown CO2 and heat stress treatments, and their interactions on physiological traits and
Results and analysis
Differential response of relative leaf injury to heat stress in two maize cultivars
Mean temperatures of non-heat treated leaves were about 31ÊC when measured at 10:00 am
and this increased about 1.5ÊC during the 2 h period that sudden heat shock (SHS) was applied
(Fig 1). The increases in leaf temperature between 10:00 am and 12:00 pm can be attributed to
natural, diurnal temperature fluctuations in the field. Temperatures across heat treated and
non-heat treated leaves did not differ significantly between the CO2 treatments at any time
Differences in relative leaf injury (RI) among the two maize inbred lines due to SHS are
shown in Fig 2 and Table 2. The highest values of RI due to SHS were observed for B106 inbred
line, with 70% regarding elevated [CO2]±nonheat treated leaves. In contrast, RI values of B76
inbred line were significantly unaffected by SHS across [CO2] treatments. In contrast to SHS
treatments, CO2 treatments did not significantly alter RI in B76.
Responses of leaf gas exchange to heat stress and varying CO2
Effects of heat stress, CO2 and cultivars, and their interactions on PN were significant (Table 2).
Rates of PN for both maize inbred lines were between 24.7 and 30.9 μmol m- 2 s-1 for nonheat
treated leaves in either CO2 treatment (Fig 3A). Rates of PN in B76 decreased about 75% under
ambient CO2 following 2 h SHS compared with nonheat treated leaves. In terms of B76 grown
under elevated [CO2], SHS dependent decrease of PN is about 41% compared with nonheat
treatments. SHS inhibited PN in B106 about 35% and 51% in the ambient and elevated [CO2]
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Fig 1. Time courses of leaf temperature in response to combinations of grown CO2 and sudden heat shock
treatments. There are four combinations: grown ambient CO2 with heat treatments (Amb[CO2]-Heat), grown elevated CO2
with heat treatments (Elv[CO2]-Heat), grown ambient CO2 without heat treatments (Amb[CO2]-Nonheat), grown elevated
CO2 without heat treatments (Elv[CO2]-Nonheat). The abbreviations for combinations of CO2 and heat treatments were
same as following figures. Red regions represent 2 hours sudden heat shock treatments. One-way ANOVA was applied to
analyze statistical significance levels of leaf temperature among the four combinations for each time point, while symbol
ª***º represent P-value <0.001.
Values of gs for B76, immediately after SHS treatment at elevated CO2 were about 60%
lower than that of elevated [CO2] with nonheat treatments (Fig 3B). In comparison with
nonheat treatments, the reduction of gs due to SHS for B106 was no more than 35% across CO2
treatments. As expected, enhanced CO2 treatments decreased gs of both cultivars majorly due
to CO2 induced stomatal closure.
Responses of C4 enzyme activities to heat stress and CO2 enrichment
The enzymes, PEPCase, NADP-ME and NADP-MDH, function in the C4 dicarboxylic acid
cycle and catalyze important photosynthetic reactions in maize. Activities of all three C4
enzymes in both maize inbred lines significantly decreased in response to SHS treatments (Fig
4; Table 2). Activities of NADP-ME in the ambient [CO2] treatment in B76 were decreased
88% by SHS compared with nonheat treatments, and this is the greatest reduction of enzyme
activity due to SHS. Conversely, activities of NADP-MDH in ambient [CO2] treatment were
only reduced 34% by SHS. Effects of elevated [CO2] on the response of C4 cycle enzyme
activities in both maize inbred lines during SHS were negligible and inconsistent. When comparing
averaged reduction of all three enzyme activities across combined treatments, it is obvious that
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Fig 2. Relative injury (RI) in response to sudden heat shock of two maize cultivars grown under ambient and
elevated [CO2]. Within each cultivar, values of different CO2 and heat treatments with same letter are not significantly
different (P<0.05) based on one-way ANOVA analysis. Vertical bars represent S.E. for n = 3.
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Fig 3. Photosynthetic rates (PN) and stomatal conductance (gs) in response to a 2 hour sudden heat shock
treatment in B76 and B106 grown under ambient or elevated [CO2]. Within each cultivar, values of different CO2 and
heat treatments with same letter are not significantly different (P<0.05) based on one-way ANOVA analysis.
SHS effects were greatest for the B76 genotype and were least for the B106 genotype when
comparing experiments performed in ambient [CO2] treatments (Fig 4).
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Fig 4. In vitro activities of C4 assimilation pathway enzymes in response to sudden heat shock treatments in B76
and B106 grown under ambient or elevated [CO2]. Within each cultivar, values of different CO2 and heat treatments with
same letter are not significantly different (P<0.05) based on one-way ANOVA analysis. Vertical bars represent S.E. for n = 3.
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Transcript expression of C4 enzymes in response to heat stress and CO2
Changes of transcript expression in response to SHS and CO2 treatments were also determined
for the three C4 enzymes as described above (Fig 5). Overall, the expression of PEPC gene
decreased from 14 to 31% in response to SHS compared with nonheat stress treatments in
either ambient or elevated [CO2] across two maize genotypes. In contrast, the expression of
NADP-ME gene decreased over 80% in both maize inbred lines and in either CO2 treatment.
The expression of NADP-ME in B76 grown under ambient [CO2] was reduced 88% for SHS
treated leaves relative to nonheat treated leaves. The inhibition effects of SHS on the expression
levels of NADP-MDH also were in excess of 80% in B76, while the expression levels decreased
around 50% in B106 across CO2 treatments. The gene expression of PEPC in either genotype
and in both temperature treatments was decreased 33% on average by elevated [CO2] relative
to ambient [CO2]. Conversely, the effects of SHS on the expression of NADP-ME were greater
in ambient [CO2] compared to elevated [CO2] treatment. Consistent effects of CO2
enrichment were not observed for changes of expression of NADP-MDH due to SHS.
Effects of heat stress and CO2 enrichment on soluble metabolite
concentrations in maize leaves
Pronounced changes of maize leaf metabolites occurred in response to SHS treatments in both
maize genotypes used in this study (Fig 6; Table 2). The effects of either maize inbred lines,
grown CO2 and SHS treatments, or their interactions on starch were significant. (Table 2).
Leaf starch levels decreased by 60 to almost 100% in both maize inbred lines following 2 h
SHS. In contrast to starch, glucose levels across CO2 treatments increased 3 ~ 8 fold and almost
2 fold due to SHS in B76 and B106, respectively. Both maltose and sucrose decreased in
response to SHS in B76 and in particular, maltose was reduced over 70%. Similar results for
maltose and sucrose were observed for the B106 except that sucrose slightly increased in
response to SHS in the ambient CO2 treatment. Other than starch there was no evidence that
elevated CO2 treatments influenced soluble carbohydrate concentrations in this study (Fig 6;
The SHS treatments also exerted dramatic effects on organic acids associated with either
the TCA cycle or with the C4 carbon assimilation metabolism in maize leaves. Citrate,
aconitate, malate, fumarate and succinate were decreased by SHS in B76. Both citrate and malate
decreased 70% or more in B76 in response to SHS. These organic acids in B106 were also
decreased by SHS, but the values of reductions by SHS were less than that observed in B76.
Interestingly, SHS treatments did not affect citrate levels in B106 across CO2 treatments.
Malate levels were 5 to 10 fold higher in B106 compared to B76 when measurements were
made following SHS across CO2 treatments. There were no consistent effects of CO2
enrichment on organic acids levels in this study (Fig 6; Table 2).
Differential heat-response of electrolyte leakage in two maize genotypes
Chen et al. [
] observed substantial differences in leaf damage due to high temperatures stress
under both field and controlled environment conditions when various maize inbred lines were
screened for abiotic stress tolerance. In particular, visible differences in leaf damage were
observed between two inbred lines B76 and B106, after intact plants were exposed to 37 to 39
oC maximum air temperatures for 1 to 2 d in the field. These findings were confirmed when
38/30 oC growth treatments were applied to the same maize inbred lines in a greenhouse study
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Fig 5. Relative transcript abundance of C4 assimilation pathway genes in response to sudden heat shock
treatments in B76 and B106 grown under ambient or elevated [CO2]. Within each cultivar, values of different CO2 and
heat treatments with same letter are not significantly different (P<0.05) based on one-way ANOVA analysis. Vertical bars
represent S.E. for n = 3.
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Fig 6. Effects of sudden heat shock treatments on maize leaf metabolites in B76 and B106 grown under ambient and elevated [CO2]. Values for
some metabolites were multiplied by 10 for clarity, as indicated by the axis label. Within each metabolite, values of different CO2 and heat treatments with
same letter are not significantly different (P<0.05) based on one-way ANOVA analysis. Vertical bars represent S.E. for n = 3.
]. In the current field study, open-top chambers (OTCs) were applied to evaluate
physiological and metabolic response of the two maize lines to a 2 h sudden heat shock (SHS) treatments
under different grown CO2 conditions. Compared with FACE, OTCs remain a workable
alternative with relatively stable CO2 control, simple technical requirements and economize
]. We observed that relative injury as assayed by electrolyte leakage was greater in B106
than in B76 following the SHS treatments (Fig 2). Similar results were observed when these
experiments were repeated in controlled environment chambers (not shown). The above
findings suggested that B76 exhibited greater tolerance of heat stress than B106 and were in
agreement with results published by Chen et al. [
]. Our previous study [
] on a different maize
line demonstrated that more ion leakage was observed at elevated than at ambient CO2 in
response to 45 oC heat stress. This was also found in the present study for maize inbred line
B106, but not for B76.
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Photosynthetic response to heat stress under elevated CO2
In a prior study [
], we hypothesized that elevated [CO2] could protect photosynthesis (PN)
from high temperature treatments by decreasing stomatal conductance (gs) and
transpiration, and improve the leaf water use efficiency. However, in that study, the 45 oC high
temperature treatments eliminated CO2 concentration effects on gs in one maize inbred line.
In current study, using two maize cultivars, B76 and B106 with contrasting heat tolerance,
we found that the reduction of gs by elevated [CO2] during SHS treaments in the field did
protect PN of inbred line B76 (Fig 3). In contrast, there was much less reduction in gs in
B106 at elevated CO2 and thus no protection of PN by elevated CO2 in response to SHS
Metabolite responses to heat shock
In this study, gas chromatography±mass spectrometry (GC/MS)-based metabolomics profiling
method was used, since GC-MS has very high sensitivity and can therefore be used for the
analysis of less commonly encountered types of samples [
]. Metabolite changes in response
to SHS were similar to prior findings for soybean plants that were grown at 8ÊC above ambient
]. First, transitory starch in maize leaves was diminished by exposure to SHS.
This likely represented the mobilization of stored starch rather than decreases of PN. CO2
stimulates starch accumulation for both heat stress and non-heat stress leaves, and the interactions
of CO2 and SHS on starch were significant (Table 2). Glucose accumulated 2 to 8 fold in
response to SHS in the leaves of both maize inbred lines and this result differed from soybean
leaflets in which hexose levels were unaffected by elevated growth temperatures [
accumulation of glucose in maize leaves in response to drought was attributed previously to the
induction of a specific vacuolar acid invertase [
] and it is possible that a similar mechanism
functions during SHS in the current study. The transformation of starch into glucose would be
expected to boost the osmotic potential of leaves exposed to abiotic stress and this would be
favorable for stress tolerance [
]. Fructose and sucrose are major end products of
photosynthesis in plants. Sucrose decreased in three of four instances in this study (Fig 6), and this
compound also decreased 20 to 30% in heat treated soybean leaves [
]. Also, results for fructose
varied in this study. Foliar concentrations of this reducing sugar increased in B76 and
decreased in B106 in response to SHS under both CO2 treatments (Fig 6). Overall, all of the
major carbohydrates in this study decreased in genotype B76 after SHS treatments, except
glucose. In comparison, fructose, glucose and in one instance sucrose increased in genotype
All of the organic acids in this study decreased in B76 leaves, and in particular, some
organic acids, such as citrate and malate, decreased over 70% due to SHS (Fig 6). The organic
acids measured in this study were all associated with the TCA cycle and our findings suggested
that TCA cycle activity was suppressed by SHS treatments in B76. The results were consistent
with the evidence observed in soybean leaflets grown at high temperatures [
] and with prior
observations involving abiotic stress effects on respiration [
]. However, large genotypic
differences were observed in current study in regard to SHS effects on leaf metabolites. Citrate
was unaffected by SHS in B106 and aconitate increased almost 2 fold in response to SHS in
this genotype. Also the effects of SHS on malate, fumarate and succinate levels in B106 were
less than that was observed in B76 (Fig 6). Taken together, down regulation of the TCA cycle
due to SHS treatments was less severe in B106 compared to B76 (Fig 6), and interactive effects
of CO2 and SHS on chosen compounds in TCA cycle were not significant except for citrate
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Disruption of C4 carbon assimilation cycle by heat stress
Law and Crafts-Brandner [
] suggested that the primary site of high-temperature inhibition
of PN in maize leaves was decreased ribulose-1,5-bisphophate carboxylase/oxygenase
(Rubisco) activity and activities of Rubisco can be inhibited by temperatures above 40ÊC.
However, studies on C4 carbon assimilation metabolism in response to heat stress were less
reported. Previous study suggested that the C4 carbon assimilation cycle can be inhibited by
water stress [
], but the question that if heat stress is able to inactivate this pathway as well
remains unclear. In the present study, our findings demonstrated that relevant components of
the C4 photosynthetic pathway were inactivated by heat stress (Figs 4 and 5). The three C4
enzyme activities measured in this study were inhibited a minimum of 34% by SHS treatments
in both inbred lines (Fig 4). Both transcript abundance and enzyme activities of NADP-ME
in B76 decreased over 85% by SHS in ambient [CO2] treatment (Figs 4 and 5). This enzyme is
important in the conversion of malate to phospho-(enol) pyruvate, which is the substrate
for PEPCase and is vital for CO2 fixation. Overall, the decreases of gene expression for
NADPMDH and NADP-ME in response to SHS were greater than for PEPC in both maize inbred
lines. Malate is synthesized from oxalacetic acid in reactions catalyzed by NADP-MDH. The
dramatic reductions of malate in response to SHS discussed above confirmed that the C4
photosynthetic pathway was inhibited by the SHS treatments imposed in this study (Figs 4 and 5).
The magnitude of the reduction in C4 enzyme activities closely matched the reductions in PN
for both lines and CO2 treatments, suggesting that the inhibition of the C4 cycle was more
important than disruption of the C3 photosynthetic pathway for this SHS. In contrast, for
water stress treatments, PN yielded relatively more inhibition by SHS than were C4 enzyme
activities in these same maize lines [
The relative injury measurements taken immediately after sudden heat shock (SHS)
treatments were performed in this field study confirmed that maize inbred line B76 was more
thermo-tolerant than B106. However, various other measurements in this study including PN,
gs, C4 enzyme activities, transcript abundance, and metabolite analysis, consistently showed
that inbred line B106 was more tolerant to SHS than B76, particularly under ambient CO2.
These findings indicated that heat stress tolerance is a complex trait. Therefore, it may be
difficult to identify biochemical, physiological or molecular markers that accurately and
consistently predict heat stress tolerance.
This work is supported by National Natural Science Foundation of China (31700201) and
Sailing Project, Shanghai Municipal Science and Technology Commission, China (17YF1421800).
Data curation: James A. Bunce, Xiaocen Zhu.
Formal analysis: James A. Bunce, Xiaocen Zhu.
Funding acquisition: James A. Bunce, Bo Gao.
Investigation: Genyun Chen.
Methodology: Mingnan Qu, Richard C. Sicher, Xiaocen Zhu.
Project administration: James A. Bunce, Genyun Chen.
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Resources: Richard C. Sicher, Bo Gao.
Software: Mingnan Qu.
Supervision: Richard C. Sicher.
Validation: Mingnan Qu, Richard C. Sicher.
Writing ± original draft: Mingnan Qu.
Writing ± review & editing: Xiaocen Zhu.
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