Heat stress prevented the biomass and yield stimulation caused by elevated CO2 in two well-watered wheat cultivars

To investigate the interactive effects of elevated CO2 and heat stress (HS), we grew two contrasting wheat cultivars, early-maturing Scout and high-tillering Yitpi, under non-limiting water and nutrients at ambient (aCO2, 450 ppm) or elevated (eCO2, 650 ppm) CO2 and 22°C in the glasshouse. Plants were exposed to two 3-day HS cycles at the vegetative (38.1°C) and/or flowering (33.5°C) stage. At aCO2, both wheat cultivars showed similar responses of photosynthesis and mesophyll conductance to temperature and produced similar grain yield. Relative to aCO2, eCO2 enhanced photosynthesis rate and reduced stomatal conductance and maximal carboxylation rate (Vcmax). During HS, high temperature stimulated photosynthesis at eCO2 in both cultivars, while eCO2 stimulated photosynthesis in Scout. Electron transport rate (Jmax) was unaffected by any treatment. eCO2 equally enhanced biomass and grain yield of both cultivars in control, but not HS, plants. HS reduced biomass and yield of Scout at eCO2. Yitpi, the cultivar with higher grain nitrogen, underwent a trade-off between grain yield and nitrogen. In conclusion, eCO2 improved photosynthesis of control and HS wheat, and improved biomass and grain yield of control plants only. Under well-watered conditions, HS was not detrimental to photosynthesis or growth but precluded a yield response to eCO2. Key message High temperatures increased photosynthetic rates only at eCO2 and photosynthesis was upregulated after recovery from heat stress at eCO2 in Scout suggesting that eCO2 increased optimum temperature of photosynthesis.


Introduction 40
Ongoing climate change is threatening the production of agricultural crops including 41 wheat (Triticum aestivum) (Asseng et al., 2015;Mishra et al., 2021). By the end of 42 this century, atmospheric carbon dioxide concentration ([CO 2 ]) is expected to reach 43 700 ppm, increasing surface temperatures by 1.1°C to 2.6°C (IPCC, 2014). For 44 every degree of the temperature increase, global wheat production is predicted to 45 decrease by 6-10% (Asseng et al., 2015;García et al., 2015). Crop models are 46 important tools for assessing the impact of climate change (Asseng et al., 2013). 47 However, they largely lack the ability to consider genotype-specific responses to 48 elevated [CO 2 ] (eCO 2 ) and their interaction with other environmental conditions. 49 Hence, it is important to better understand how plants respond to eCO 2 interactions 50 with the environment. Photosynthesis, a fundamental process driving crop growth 51 and yield, can partially explain the interactive effects of eCO 2 with environmental 52 stresses and provide a mechanistic basis for crop models (Yin and Struik, 2009). 53 During photosynthesis, ribulose-1, 5-bisphosphate carboxylase (Rubisco) catalyzes 54 the carboxylation and oxygenation of ribulose-1, 5-bisphosphate (RuBP). eCO 2 55 increases photosynthetic rates (A sat ) and reduces photorespiration and stomatal 56 conductance (g s ). Generally, higher photosynthetic rates enhance the growth and 57 productivity of plants leading to increased leaf area, plant size and crop yield 58 (Krenzer and Moss, 1975;Sionit et al., 1981;Hocking and Meyer, 1991;Mitchell et al., 59 1993;Kimball et al., 1995;Mulholland et al., 1998;Cardoso Vilhena and Barnes, 2001;60 Högy et al., 2009;Kimball, 2016;Fitzgerald et al., 2016;Kimball, 1983). Following long 61 term CO 2 enrichment, photosynthetic capacity may diminish due to lower amount of 62 Rubisco (Nie et al., 1995;Rogers and Humphries, 2000;Ainsworth et al., 2003) or 63 reduced activation of Rubisco (Delgado et al., 1994). 64 Optimum temperature range for wheat growth is 17-23°C, with a minimum of 0°C 65 and maximum of 37°C (Porter and Gawith, 1999). Global warming involves a gradual 66 increase in mean temperature as well as increased frequency and intensity of heat 67 waves. Heat can adversely affect crop growth and disrupt reproduction depending on 68 the timing, intensity and duration (Sadras and Dreccer, 2015). Higher daytime 69 temperatures (below damaging level) increase photosynthesis up to an optimum 70 temperature, above which photosynthesis decreases mainly due to higher 71 Many studies have investigated the response of wheat to eCO 2 in enclosures and in 103 the field (Wang & Liu, 2021). However, only a few studies have considered eCO 2 104 interaction with temperature increases in wheat (Rawson, 1992;Delgado et al., 105 1994;Morison and Lawlor, 1999;Jauregui et al., 2015;Cai et al., 2016) and rarely 106 with HS (Coleman et al., 1991;Wang et al., 2008). Studies considering HS have 107 addressed mainly the biomass or yield aspects and not the physiological processes 108 such as photosynthesis (Stone and Nicolas, 1994, 1996, 1998. Interactive effects of 109 eCO 2 and HS on photosynthesis have been reported in a limited number of studies 110 (Wang et al., 2008(Wang et al., , 2011Macabuhay, 2016;Macabuhay et al., 2018;Chavan et al., 111 2019). Macabuhay et al., (2018) studied interactive effects of eCO 2 and 112 (experimentally imposed) heatwaves on wheat (cv Scout and Yitpi) grown in a 113 dryland cropping system and concluded that eCO 2 may moderate some effects of 114 HS on grain yield but such effects strongly depend on seasonal conditions and 115 timing of HS. In another glasshouse experiment on the interactive effects of severe 116 HS and eCO 2 in wheat (cv Scout), we found that eCO 2 mitigated the negative 117 impacts of HS at anthesis on photosynthesis and biomass, but grain yield was 118 reduced by HS in both CO 2 treatments (Chavan et al., 2019). However, HS can 119 occur throughout plant growth, including during vegetative, flowering or grain filling 120 stages. In addition, different crop genotypes may respond variably to the interaction 121 of eCO 2 with HS. 122 Here, we build on our previous work by comparing the interactive effects of eCO 2 123 and HS in two commercial wheat cultivars. Scout and Yitpi have similar genetic 124 background but distinct agronomic features. Scout is a mid-season maturity cultivar 125 with very good early vigor that can produce leaf area early in the season. Scout has 126 a putative water-use efficiency (WUE) gene, which has been identified using carbon 127 isotope discrimination (Condon et al., 2004). Yitpi is a good early vigor, freely 128 tillering, late flowering and long maturity cultivar (Bahrami et al., 2017;Pacificseeds, 129 photosynthetic acclimation (Delgado et al., 1994) compared to slow growing 136 counterparts with low sink capacity. Consequently, we hypothesized that Yitpi will 137 show greater photosynthetic, growth and yield response to eCO 2 due to its greater 138 vegetative sink capacity (tillering) relative to Scout with restricted tillering (Hypothesis 139 2). The greater growth stimulation at eCO 2 may buffer Yitpi against HS damage 140 compared to Scout. Thus, HS may decrease yield in Scout more than Yitpi and aCO 2 141 more than eCO 2 (Hypothesis 3). HS is more damaging at the reproductive relative to 142 the vegetative developmental stage (Farooq et al., 2011). Hence, we expect less 143 damage in plants exposed to HS at the vegetative stage relative to the flowering 144 stage (Hypothesis 4). 145 To test these hypotheses, Scout and Yitpi were grown at ambient or elevated CO 2 146 conditions and subjected to one or two heat stresses at the vegetative (HS1) and/or 147 flowering (HS2) stage. Growth, biomass and photosynthetic parameters were 148 measured at different time points across the life cycle of the plants. At the plant level, 149 we report that eCO 2 equally stimulated grain yield of Scout and Yitpi. While 150 moderate HS under well-watered conditions was not detrimental to photosynthesis 151 and growth in the long term due to the transpirational cooling, HS prevented the 152 wheat plant from reaping the benefits of eCO 2 on biomass and yield. 153

Materials and methods 154
Plant culture and treatments 155 The experiment was conducted in the glasshouse facility located at the Hawkesbury 156 campus of Western Sydney University (WSU). Seeds of commercial winter wheat 157 cultivars Scout and Yitpi were procured from Agriculture Victoria (Horsham). 158 Cultivars were selected based on their use in the Australian Grains Free Air CO 2 159 Enrichment (AGFACE) project investigating climate change impacts on wheat growth 160 and yield (Houshmandfar et al., 2017). For germination, 300 seeds of each cultivar 161 were sterilized using 1.5 % NaOCl 2 for 1 min followed by incubation in the dark at 162 28°C for 48 hours in petri plates. Sprouted seeds were planted in germination trays 163 using seed raising and cutting mix (Scotts, Osmocote ® ) at ambient CO 2 (aCO 2 , 400 164 μ l L -1 ), temperature (22/14 °C day/night), relative humidity (RH, 50 to 70%) and 165 natural light (midday average 500 μ mol m -2 s -1 ) ( Figure S1). The growth stages are 166 denoted by decimal code (DC) according to (Zadoks et al., 1974) along with the time 167 points here after. Two-week-old seedlings (DC12) were transplanted to individual 168 cylindrical pots (15 cm diameter and 35 cm height) using sieved soil collected from 169 local site. At transplanting stage (T0) pots were distributed into two aCO 2 (400 μ l L -1 ) 170 and two eCO 2 (650 μ l L -1 ) chambers ( Figure S1B). Some plants were exposed to 171 one or two HS cycles at the vegetative (HS1, 10 weeks after planting, WAP, DC 32) 172 and/or the flowering (HS2, 15 WAP, DC 63) stages for 3 days with temperature ramp 173 up from 14°C night temperature (8 pm to 6 am) to 38°C during mid-day (10 pm to 4 174 pm) at 60% daytime RH (Figures S1 and S2). The two HS cycles created four sets of 175 heat treatments at each CO 2 concentration as follows: (1) Control -plants were not 176 exposed to HS at any stage, (2) HS1 -plants were exposed to HS at vegetative 177 (DC32) stage only, (3) HS2 -plants were exposed to HS at reproductive (DC63) 178 stage only and (4) HS1+2 -plants were exposed to both the heat stresses HS1 and 179 HS2 ( Figure S2). 180 Thrive all-purpose fertilizer (Yates) was applied monthly throughout the experiment 181 to maintain similar nutrient supply in all treatment combinations. Pots were regularly 182 swapped between left and right benches as well as between front and back for 183 randomization within chamber. Pots and treatments were also swapped between the 184 two ambient and two elevated CO 2 chambers for randomization among chambers. 185

Leaf gas exchange measurements 198
The youngest fully developed leaf (which was the flag leaf at T3) was used to 199 measure gas exchange parameters. Instantaneous steady state leaf gas exchange 200 measurements were performed at time points T1, T2 and T3 using a portable open 201 gas exchange system (LI-6400XT, LI-COR, Lincoln, USA) to measure light-saturated 202 (photosynthetic photon flux density (PPFD) =1500 µmol m -2 s -1 ) photosynthetic rate 203 (A sat ), stomatal conductance (g s ), ratio of intercellular to ambient CO 2 (C i /C a ), leaf 204 transpiration rate (E), dark respiration (R d ) and dark-and light-adapted chlorophyll 205 fluorescence (Fv/Fm and Fv`/Fm`, respectively). Dark adapted leaf measurements 206 were conducted by switching off light for 15 minutes. Steady state leaf gas exchange 207 measurements were also performed during and after heat shock along with recovery 208 stage. Plants were moved to a neighboring chamber with ambient CO 2 levels for 209 short time (20-30 min for each plant) where air temperature was separately 210 manipulated to achieve the desired leaf temperature. The LI-COR 6400-40 leaf 211 chamber fluorometer (LCF) was used to measure gas exchange at a PPFD of 1500 212 μ mol m -2 s -1 at two CO 2 concentrations (400 and 650 μ l L -1 ) and two leaf 213 temperatures (25 and 35 °C). Photosynthetic down regulation or acclimation was 214 examined by comparing the measurements at common CO 2 (ambient and elevated 215 CO 2 grown plants measured at 400 μ l L -1 CO 2 partial pressure) and growth CO 2 216 (aCO 2 grown plants measured at 400 μ l L -1 CO 2 partial pressure and eCO 2 grown 217 plants measured at 650 μ l L -1 CO 2 partial pressure). 218 Dark respiration (R d ) was measured after a dark adaptation period of 15 minutes. 219 Photosynthetic water use efficiency (PWUE) was calculated as A sat (μmol m -2 s -1 )/ g s 220 (mol m -2 s -1 ). The response of A sat to variations in sub-stomatal CO 2 mole fraction 221 (Ci) (A-Ci response curve) was measured at T3 in 8 steps of CO 2 concentrations (50, 222 100, 230, 330, 420, 650, 1200 and 1800 μ l L -1 ) at leaf temperature of 25°C. 223 Measurements were taken around mid-day (from 10 am to 3 pm) on attached last 224 fully expanded or flag leaves of the main stems. Before each measurement, the leaf 225 was allowed to stabilize for 10-20 minutes until it reached a steady state of CO 2 226 uptake and stomatal conductance. Ten replicate plants per treatment were 227

measured. 228
Mesophyll conductance and temperature response 229 Mesophyll conductance (g m ) was determined by concurrent gas exchange and stable 230 carbon isotope measurements using portable gas exchange system (LI-6400-XT, LI-231 COR, Lincoln, NE, USA) connected to a tunable diode laser (TDL) (TGA100, 232 Campbell Scientific, Utah, USA) for the two wheat cultivars grown at ambient 233 atmospheric CO 2 partial pressures. A sat and 13 CO 2 / 12 CO 2 carbon isotope 234 discrimination were measured after T1 at five leaf temperatures (15, 20, 25, 30 and 235 35°C) and saturating light (1500 µmol quanta m -2 s -1 ). Leaf temperature sequence 236 started at 25°C decreasing to 15°C and then increased up to 35°C. Response of A sat 237 to variations in Ci was measured at each leaf temperature. Dark respiration was 238 measured by switching light off for 20 minutes at the end of each temperature curve. 239 Measurements were made inside a growth cabinet (Sanyo) to achieve desired leaf 240 temperature. The photosynthetic carbon isotope discrimination (Δ) to determine g m 241 was measured as follows (Evans et al., 1986) (2) 245 C ref and C sam are the CO 2 concentrations of dry air entering and exiting the leaf 246 chamber, respectively, measured by the TDl. g m was calculated using correction for 247 ternary and second-order effects (Farquhar and Cernusak, 2012;Evans and Von 248 Caemmerer, 2013) following the next expression: 249 Where, fractionation of 13 C due to respiration and photorespiration respectively (Evans and 253 Von Caemmerer, 2013). 254 The constants used in the model were as follows: E denotes transpiration rate; g t ac is 259 total conductance to diffusion in the boundary layer (ab = 2.9‰) and in air (a = 260 4.4‰); a′ is the combined fractionation of CO 2 across boundary layer and stomata; 261 net fractionation caused by RuBP and PEP carboxylation (b = 27.3‰) (Evans et al., 262 1986); fractionation with respect to the average CO 2 composition associated with 263 photorespiration (f = 11.6‰) (Lanigan et al., 2008) and we assumed null fractionation 264 associated with mitochondrial respiration in light (e = 0). 265

Leaf nitrogen and carbon estimation 266
Leaf discs were cut from the flag leaves used for gas exchange measurements at 267 time points T2 and T3 then oven dried. Leaf discs were processed for nitrogen (N) 268 and carbon (C) content using elemental analyzer (Dumas method). N and C were 269 also estimated from other plant components including leaf, stem, root and grain 270 harvested at T1, T3 and T4. Ground samples were processed for C & N with a CHN 271 analyzer (LECO TruMac CN-analyser, Leco corporation, USA) using an automated 272 dry combustion method (Dumas method). Leaf N per unit area (N area ) was calculated 273 as N (mmol g -1 ) × LMA (g m -2 ). Photosynthetic nitrogen use efficiency (PNUE) was 274 calculated as A sat (μmol m -2 s -1 )/leaf N area (mmol m -2 ). Protein content was 275 determined using N and multiplication factor of 5.7 (Mosse, 1990;Bahrami et al., 276 2017). 277

Statistical and temperature analysis 278
All data analyses and plotting were performed using R computer software (R Core 279 Team, 2020). The effect of treatments and their interactions was analyzed using 280 linear modeling with 'anova' in R. Significance tests were performed with anova and 281 post hoc Tukey test using the 'glht' function in the multcomp R package. Coefficient 282 means were ranked using post-hoc Tukey test. The Farquhar-von Caemmerer-Berry 283 (FvCB) photosynthesis model was fit to the A sat response curves to C i (A-Ci 284 response curve) or chloroplastic CO 2 mole fraction (Cc), which was estimated from 285 the g m measurements (A-Cc response curve). We used the plantecophys R package 286 (Duursma, 2015) to perform the fits, using measured g m and R d values, resulting in 287 estimates of maximal carboxylation rate (V cmax ) and maximal electron transport rate 288 (J max ) for D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) using 289 measured R d values. Temperature correction parameter (Tcorrect) was set to False 290 while fitting A-C i curves. Temperature response of V cmax and J max were calculated by 291 Arrhenius and peaked functions, respectively (Medlyn et al., 2002). Estimated V cmax 292 and J max values at five leaf temperatures were then fit using nonlinear least square 293 (nls) function in R to determine energy of activation for V cmax (EaV) and J max (EaJ) 294 and entropy (ΔSJ). Temperature responses of V cmax and R d were fit using Arrhenius 295 equation as follows, 296 Where, Ea is the activation energy (in J mol -1 ) and k 25 is the value of R d or V cmax at 298 25 °C. R is the universal gas constant (8.314 J mol -1 K -1 ) and Tk is the leaf 299 temperature in K. The activation energy term Ea describes the exponential rate of 300 rise of enzyme activity with the increase in temperature. The temperature coefficient 301 Q 10 , a measure of the rate of change of a biological or chemical system as a 302 consequence of increasing the temperature by 10 °C was also determined for Rd 303 using the following equation: 304 A peaked function (Harley et al., 1992) derived Arrhenius function was used to fit the 306 temperature dependence of J max , and is given by the following equation: 307 The temperature response of A sat was fit using a simple parabola equation (Crous et 315 al., 2013) to determine temperature optimum of photosynthesis: Where, T is the leaf temperature of leaf gas exchange measurement for A sat , T opt 318 represents the temperature optimum and A opt is the corresponding A sat at that 319 temperature optimum. Steady state gas exchange parameters g m , g s , C i and J max to 320 V cmax ratio were fit using nls function with polynomial equation: Two commercial wheat cultivars Scout and Yitpi were grown under aCO 2 or eCO 2 325 (daytime average of 450 or 650 μ l L -1 , respectively; 65% RH and 22°C), natural 326 sunlight and well-watered conditions ( Figure S1). Both aCO 2 and eCO 2 grown plants 327 were exposed to two 3-day HS cycles at the vegetative (HS1, 10 WAP, DC32, 328 daytime average of 38°C) and flowering stage (HS2, 15 WAP, DC63, daytime 329 average of 33.5°C), while daytime RH was maintained at 60%. HS2 was lower in 330 intensity relative to HS1 due to the cool winter conditions. Both HS cycles had similar 331 overall effects on growth and yield parameters, refuting our fourth hypothesis that HS 332 during the reproductive stage is more damaging. Hence, we mostly compare the 333 control plants to those exposed to both heat stresses. Grain filling started 17 WAP 334 (DC65) and final harvest occurred 25 WAP (DC90) ( Figure S2). 335

Photosynthetic temperature responses of the two wheat cultivars at aCO 2 336
A-C i curves together with g m were measured at five leaf temperatures to characterize 337 the thermal photosynthetic responses of the two wheat cultivars grown at aCO 2 338 ( Figure 1; Table 1) Table 1). V cmax and J max exponentially increased with leaf temperature, but J max 349 declined above T opt (30°C) in both cultivars ( Figures 1E-F). There was no significant 350 difference in V cmax , J max or their activation energies between the two wheat cultivars 351 ( Figure 1E-G, Table 1). The ratio of J max /V cmax was similar for the two cultivars and 352 linearly decreased with leaf temperature ( Figure 1G). 353

High temperature during HS enhanced photosynthesis under eCO 2 364
The two HS cycles did not reduce A growth measured at 25℃ during or after HS ( Figure  365 3A-D, Tables S1-S3). During both HS1 and HS2, eCO 2 stimulated A growth measured 366 at 25℃ in Scout but not Yitpi. Relative to 25°C, A growth increased at 35°C in Scout 367 (10-14%) and Yitpi (12-18%) grown at eCO 2 but not at aCO 2 . Immediately after the 368 recovery from HS, A growth was upregulated in eCO 2 -grown Scout (Figures 3A-D and 369 S3). During both HS cycles, dark-adapted Fv/Fm measured at 25℃ tended to be 370 lower in Yitpi grown at eCO 2 relative to aCO 2 . In both cultivars, Fv/Fm decreased at 371 35℃ relative to 25 o C, indicating transient damage to PSII due to HS at both CO 2 372 treatments ( Figure 3E-H, Tables S1-S3). 373 Following long-term recovery from HS1 and/or HS2, the eCO 2 stimulation of A growth 374 was still marginally apparent in all T3 plants, being the strongest in eCO 2 -grown Yitpi 375 ( Figure 4A-B, Tables S1-S3). The reduction of g s at eCO 2 was weak in all plants 376 ( Figure 4C-D, Tables S1-S3). Hence, PWUE was stimulated by eCO 2 in all 377 treatments, while PNUE was unaffected ( Figure 4E-H, Tables S1-S3). There was a 378 good correlation between A growth and g s (r 2 = 0.51, p <0.001) across all treatments 379 ( Figure 5A). 380 V cmax and J max were derived from A-C i response curves measured at 25°C during the 381 recovery stage after HS2. For control and HS plants, growth at eCO 2 marginally 382 reduced V cmax in Scout (-14%, p = 0.09) and Yitpi (-15%, p = 0.06) but had no effect 383 on J max . HS had no effect on V cmax or J max in either cultivar ( Figure 4I-L, Tables S1-384 S3). V cmax and J max correlated well (r 2 = 0.75, p <0.001) across treatments ( Figure  385 5B). 386

eCO 2 similarly stimulated wheat biomass and grain yield under non-HS 406 conditions 407
The increase in plant biomass at eCO 2 depended on the growth stage 408 Tables S4 and S5). However, the overall stimulation was not different between the 409 two cultivars as evident from the non-significant eCO 2 x cultivar interaction at all 410 harvests (Table S4). By T3 (anthesis), when both cultivars were still within the 411 exponential growth stage, eCO 2 stimulated plant biomass of Yitpi (+29%, p < 0.001) 412 and Scout (+9%, p < 0.001) under control conditions. The number of tillers, total leaf 413 area, mean leaf size or leaf mass area were not significantly affected by eCO 2 in 414 either cultivar ( Figure 6, Tables S4 and S5). eCO 2 increased allocation to stem 415 relative to leaf biomass, particularly in Yitpi. Accordingly, there was a strong 416 correlation across treatments between stem and leaf biomass (r 2 = 0.83, p < 0.001) 417 and between total biomass and leaf area (r 2 = 0.83, p < 0.001) in Scout but not in 418 Yitpi. However, the two cultivars followed common relationship for root versus shoot 419 biomass (r 2 = 0.41, p < 0.001) and leaf area versus leaf number (r 2 = 0.82, p < 0.001) 420 across all treatments suggesting no effect of cultivar, eCO 2 or HS on these common 421 allometric relationships ( Figure S5, Table S5). 422 At the final harvest T4 (seed maturity), eCO 2 enhanced biomass and equally 423 stimulated grain yield by increasing grain number in both cultivars (+64% in Scout 424 and +50% in Yitpi) under control conditions only ( Figure 7A-D, Tables S4-S6). 425 Harvest index was not directly affected by any treatments but showed a significant 426 interaction (p <0.05) between CO 2 and cultivar, such that HI was higher in Yitpi 427 under eCO 2 (Tables S5 and S6). 428

eCO 2 did not stimulate the grain yield of HS plants 429
At T3, moderate HS (34-38°C) applied under well-watered conditions and 60% RH 430 during the vegetative (HS1 applied after T1) and flowering (HS2 applied after T2) 431 stages had no significant impact on plant biomass of either wheat cultivar or CO 2 432 treatment. By T4, there were significant HS x CO 2 x cultivar interactions (p < 0.01) 433 for biomass and grain yield. HS1+2 reduced the biomass and grain yield of eCO 2 -434 grown Scout relative to aCO 2 -grown counterparts. Unlike control plants, the biomass 435 and yield of HS plants were not enhanced by eCO 2 (Figure 7, Tables S4 and S5). 436

eCO 2 reduced grain N in Yitpi but not in Scout 437
Neither eCO 2 or HS had a significant effect on flag leaf N content in either cultivar at 438 T2 or T3, but eCO 2 reduced aggregate leaf N content (-18%) at T3 in Yitpi only 439 (Cultivar x CO 2 p < 0.05) (Table S7). Yitpi had higher grain N content (+26%) than 440 Scout in control plants grown at aCO 2 ( Figure 7G-H, Table S6). In control plants, 441 eCO 2 significantly reduced grain protein content in Yitpi (-18%, p < 0.05) but not in 442 Scout due to significant cultivar X CO 2 interaction (p < 0.01), while HS had no effect 443 on protein content in either cultivar ( Figure 7G-H, Table S6). 444

Two wheat cultivars with contrasting morphology and phenology, but similar 446 photosynthesis and grain yield 447
The effects of future climate conditions, including eCO 2 , will depend on the 448 environmental conditions (e.g., water and heat stress) and the crop's agronomic 449 features. Here, we compared the interactive effects of eCO 2 and HS on two 450 commercial wheat cultivars, Scout and Yitpi, with contrasting phenology and growth 451 habit. Plants were grown under well-watered and fertilized conditions to remove any 452 confounding effects of water or nutrient limitations on the eCO 2 or HS responses. RH 453 was kept constant to minimize the negative impact of dry air during HS. Finally, we 454 compared the effects of applying HS at the vegetative and flowering stages. 455 Free tillering Yitpi produced substantially more tillers, leaf area and biomass relative 456 to the faster developing Scout. Accordingly, our first hypothesis predicted that Yitpi 457 will have higher grain yield. The results only partially supported this hypothesis, 458 because relative to Yitpi, Scout had higher harvest index (HI) due to its early 459 maturing and senescing habit. While Yitpi initiated more tillers, a lower proportion of 460 these tillers produced ears and filled grains. In contrast, Scout produced less tillers 461 but flowered earlier which allowed enough time for all its tillers to produce ears and 462 fill bigger grains by the final harvest. Hence, both cultivars had relatively similar 463 yields due to bigger grain size in Scout and higher grain number in Yitpi. It is worth 464 noting that some field trials have reported slightly higher grain yields in Scout than 465 Yitpi (National variety trial report, GRDC, 2014). Our results are consistent with a 466 previous study using different wheat cultivars with contrasting source-sink 467 relationships which reported that the freely tillering cultivar "Silverstar" translated into 468 more spikes while restricted tillering cultivar "H45" had more and heavier kernels per 469 spike than "Silverstar" (Tausz-Posch et al., 2015). Thus, early vigor and maturity 470 compared to high tillering capacity seem to be equally beneficial traits for high grain 471 yield in the Australian environment. 472 The two wheat cultivars showed similar photosynthetic traits and response to 473 temperature and eCO 2 . In contrast to our expectations that Scout would have higher 474 WUE due to its selection based on a carbon isotope discrimination gene (Condon et 475 al., 2004), both wheat cultivars showed similar PWUE under most measurement and 476 growth conditions in this study ( Figure 4E-F, Table 2). 477

Elevated CO 2 stimulated photosynthesis but reduced photosynthetic capacity 478 in both cultivars 479
Long term exposure to eCO 2 may reduce photosynthetic capacity due to lower 480 amount of Rubisco in a process referred as 'acclimation' (Nie et al., 1995;Rogers 481 and Humphries, 2000;Ainsworth et al., 2003). Alternatively, eCO 2 may 'down-482 regulate' photosynthetic capacity by reducing Rubisco activation or other regulatory 483 mechanisms without affecting Rubisco content (Delgado et al., 1994). In the current 484 study, eCO 2 similarly increased A growth (+21%) measured at growth CO 2 and reduced 485 both A sat (-12%) measured at common CO 2 ( (Table S4) led to a relatively small observed photosynthetic downregulation in response to 497 growth at eCO 2 . This allowed a sustained photosynthetic stimulation, which in turn 498 led to a significant biomass and yield enhancement by CO 2 enrichment in both wheat 499 cultivars (Figures 6-7). Photosynthetic responses of wheat in current study are in 500 agreement with earlier enclosure studies which generally have higher response to 501 eCO 2 than the FACE studies (Kimball et al., 1995;Hunsaker et al., 1996;Osborne et 502 al., 1998;Kimball et al., 1999;Long et al., 2006;Cai et al., 2016). 503

Elevated CO 2 stimulated grain yield similarly in both wheat cultivars 504
In disagreement with our second hypothesis, eCO 2 similarly stimulated plant 505 biomass and grain yield in early-maturing Scout and high tillering Yitpi (Figures 6-7, 506 Table S4-S5). In Scout, the biomass stimulation was associated with increased 507 tillering (one extra tiller per plant). In contrast, Yitpi produced many tillers at aCO 2 508 and the additional fixed carbon at eCO 2 was allocated to the existing tillers. At seed 509 maturity, eCO 2 stimulated grain yield similarly in both cultivars as a result of the 510 trade-off between grain yield components (Dias de Oliveira et al., 2015). In 511 particular, eCO 2 stimulated grain number in both cultivars, while grain size increased 512 in Scout only (Figure 7, Table S6). Generally, eCO 2 stimulates grain yield by 513 increasing the number of tillers and consequently, ears per plant (Zhang et al., 2010;514 Bennett et al., 2012), which has also been reported in FACE studies (Högy et al., 515 2009;Tausz-Posch et al., 2015;Fitzgerald et al., 2016). However, in our study, the 516 increase in grain yield at eCO 2 was mainly due to the increase in the number of 517 grains per ear. In line with our results, Tausz-Posch et al., (2015) reported 518 comparable grain yield stimulation by eCO 2 in two different wheat cultivars with 519 contrasting source-sink relationships. Moreover, grain yield of twenty wheat cultivars 520 that differed in tillering propensity, water soluble carbohydrate accumulation, early 521 vigor and transpiration efficiency responded similarly to eCO 2 in glasshouse settings 522 (Ziska et al., 2004;Bourgault et al., 2013). 523

Elevated CO 2 reduced grain N in Yitpi only 524
Overall, there is a negative relationship between grain yield and quality (Taub et al., 525 2008;Pleijel and Uddling, 2012). Hence, increased grain yield at eCO 2 results in 526 lower grain N and hence protein content (Seneweera and Conroy, 1997;Bahrami et 527 al., 2017). In our study, eCO 2 reduced grain N in Yitpi under control conditions. 528 Scout was characterized by having larger grains which accumulated less N than 529 Yitpi. Moreover, eCO 2 reduced total leaf N (-18%) at T3 and grain N (-17 %) at T4 in 530 Yitpi but not in Scout. This is consistent with the results from FACE study with same 531 cultivars which reported -14% reduction in N content by eCO 2 in above ground dry 532 mass in Yitpi but not in Scout under well-watered conditions (Bahrami et al., 2017). 533 The higher biomass accumulation in free tillering Yitpi may have exhausted the 534 nutrient supply, such that further biomass stimulation by eCO 2 lead to a significant 535 dilution in N content (Taub and Wang, 2008). 536 Wheat cultivars with early vigour such as Scout have greater root biomass 537 accumulation as well as greater early N uptake which may have avoided a negative 538 effect of eCO 2 on leaf and grain N (Liao et al., 2004;Bahrami et al., 2017). 539 Accordingly, Scout maintained a higher N utilization efficiency (grain yield per total 540 plant N) relative to Yitpi under all treatments (Table S6). Increased grain yield is 541 strongly associated with higher grain number per unit area (Zhang et al., 2010;542 Bennett et al., 2012) which dilutes the amount of N translocated per grain. Quality 543 deterioration due to lower protein via reduced N is of critical concern in future high 544 CO 2 climate considering that even additional supply of N does not prevent N dilution 545 in grain under eCO 2 (Tausz et al., 2017). In addition, eCO 2 has strong detrimental 546 effect on other nutrient availability and remobilization from leaves to grains (Tcherkez 547 et al., 2020). 548

HS had little effects on wheat photosynthesis or yield at aCO 2 549
A key finding of this study was that the application of HS events (HS1, HS2 or 550 HS1+2) was not detrimental to aCO 2 -grown wheat plants ( Figures 5 and 7, Tables 551 S4-S6). Thus, our hypothesis that HS will reduce photosynthesis, biomass and yield 552 at aCO 2 was rejected. This finding is in contrast to previously reported studies where 553 HS reduced the grain yield and negatively affected the growth and development in 554 wheat (Stone andNicolas, 1996, 1998;Farooq et al., 2011;Coleman et al., 1991). relative humidity, there is sufficient water vapour gradient to sustain high 563 transpiration rates when soil water is available, as was the case in our experiment. In 564 most cases, g s was not significantly affected (Tables S1 and S2), and even slightly 565 higher at T3 in HS-pants relative to the control (Figure 4c,d) Well-watered crops can 566 maintain grain-filling rate, duration and size under HS (Dupont et al., 2006), and high 567 temperatures can increase crop yields if not exceeding critical optimum growth 568 temperature (Welch et al., 2010). Also, in the current study, the night temperatures 569 were not increased during HS which favors plant growth by reducing respiratory 570 losses (Prasad et al., 2008). 571 In particular, HS did not elicit a direct negative impact on photosynthesis or 572 chlorophyll fluorescence in either cultivar or CO 2 treatment. During HS, high 573 temperature transiently reduced maximum efficiency of PSII (Fv/Fm) in both cultivars 574 and CO 2 treatments ( Figure 3E-H). However, unchanged Fv/Fm measured at 25 o C 575 confirmed that photosynthesis did not suffer long-term damage during or after HS. 576 Moreover, HS was not severe enough to negatively affect A growth measured at 25°C. 577 complex from the photosystems (Wahid et al., 2007;Poudel, 2020). We were unable 584 to measure leaf temperatures in the current study, but we speculate that, in well-585 watered wheat plants growing at moderate RH, leaf temperatures might not have 586 increased beyond damaging levels to the membranes during the HS events. fourth hypothesis, and this may additionally be due to the short term duration of the 592 two HS cycles (3 days each). Hence, our study demonstrated the benign effect that 593 HS has on crop yield when separated from water stress and plants are able to 594 transpire. 595

HS precluded an eCO 2 response in biomass and grain yield 596
In our study, the impact of HS depended on the wheat cultivar and growth CO 2 597 (Tables S1 and S4). Elevated CO 2 and temperature interactions can be complex, 598 dynamic and difficult to generalize as they can go in any direction depending on 599 plant traits and other environmental conditions (Rawson, 1992). Plant development 600 is generally accelerated by increased temperature; eCO 2 can accelerate it further in 601 some instances or may have neutral or even retarding effects in other cases 602 (Rawson, 1992). 603 While eCO 2 stimulated wheat biomass and grain yield under control (non-HS) 604 conditions, HS precluded a yield response to eCO 2 in Yitpi and reduced biomass and 605 yield in eCO 2 -grown Scout relative to aCO 2 -grown counterparts (Figure 7). These 606 results are in contrast with previous studies that reported similar wheat yield 607 reduction at ambient or elevated CO 2 in response to severe (Chavan et al., 2019) or 608 moderate HS (Zhang et al., 2018). The results also partially refuted our third 609 hypothesis that HS may decrease yield more at aCO 2 than eCO 2 , while partially 610 agreeing that HS will have a more negative impact on Scout relative to Yitpi, albeit 611 for different reasons than what we originally suggested. The negative effect of HS on 612 Scout biomass and grain yield at eCO 2 occurred despite the eCO 2 stimulation of 613 A growth under HS (T3, Figure 4). However, over the long term, A growth was stimulated 614 in eCO 2 -grown Yitpi and not Scout (Figure 4). 615 Lack of a biomass stimulation despite high photosynthetic rates during HS under 616 eCO 2 could be due to the short duration of HS (3 days), which may not have been 617 long enough to stimulate biomass gain. In addition, nutrient limitation at eCO 2 may 618 have restricted the eCO 2 growth response. Typically, eCO 2 studies show reduced N 619 content in wheat and other crops (Taub and Wang, 2008;Leakey et al., 2009;620 Bahrami et al., 2017). Hence, the wheat plants may have exhausted available 621 nutrients due to increased demand by growing sinks at eCO 2 , which may limited 622 further stimulation by high temperature. HS may be more damaging at eCO 2 due to 623 reduce transpirational cooling as a result of reduced g s at eCO 2 , leading to higher 624 leaf temperatures. However, A growth increased in response to high temperature 625 (35°C) under eCO 2 but not under aCO 2 during HS ( Figure 3A-D), which refutes the 626 suggestion of HS-damage to photosynthesis. 627 Higher g s during HS at moderate RH in well-watered conditions may increase A growth 628 by increasing C i in both aCO 2 and eCO 2 grown plants. Furthermore, lower 629 photorespiration under eCO 2 allows additional increase in A growth with temperature 630 when measured at 35°C relative to 25°C (Long, 1991). Under aCO 2 , photorespiration 631 increases with temperature reducing A growth measured at 35°C relative to 25°C. Our 632 results also point to a shift in T opt of photosynthesis (~ 24°C at aCO 2 ) to higher 633 temperatures for plants grown at eCO 2 (Sage and Kubien, 2007). This would come 634 about as a result of lower photorespiration at eCO 2 as well as the slight upregulation 635 of photosynthetic rates observed in eCO 2 -grown Scout at the recovery stage of HS 636 ( Figure 3A, C). However, at T3, A growth was similar between aCO 2 and eCO 2 grown 637 plants ( Figure 4A) indicating the short-term nature of this photosynthetic 638 upregulation. 639

Conclusions 640
The two wheat cultivars, Scout and Yitpi differed in growth and development but 641 produced similar grain yield. Under control conditions, eCO 2 stimulated biomass and 642 yield similarly in both cultivars. HS was not damaging to photosynthesis, growth, 643 biomass or grain yield under well-watered and moderate RH conditions. However, 644 HS interacted with eCO 2 , leading to similar or lower biomass and grain yield at eCO 2 645 relative to both aCO 2 in plants exposed to HS. This interactive effect precluded the

Conflicts of interest 668
The authors declare that they have no conflict of interest. 669

Availability of data 670
All data supporting the findings of this study are available within the paper and within 671 its supplementary materials published online. Reuse of the data is permitted after 672 obtaining permission from the corresponding author. 673

Code availability 674
Software application and codes used are all publicly available. 675

Authors contributions 676
All authors conceived the project. SGC maintained the plants and collected the data. 677 SGC and RAD analysed the data. SGC and OG prepared the manuscript with input 678 from other co-authors. 679 A  i  n  s  w  o  r  t  h  E  A  ,  D  a  v  e  y  P  A  ,  H  y  m  u  s  G  J  ,  O  s  b  o  r  n  e  C  P  ,  R  o  g  e  r  s  A  ,  B  l  u  m  H  ,  N  ö  s  b  e  r  g  e  r  J  ,  L  o  n  g  S  P   .  2  0  0  3  .  I  s   s  t  i  m  u  l  a  t  i  o  n  o  f  l  e  a  f  p  h  o  t  o  s  y  n  t  h  e  s  i  s  b  y  e  l  e  v  a  t  e  d  c  a  r  b  o  n  d  i  o  x  i  d  e  c  o  n  c  e  n  t  r  a  t  i  o  n  m  a  i  n  t  a  i  n  e  d  i  n  t  h  e  l  o  n L  a  n  i  g  a  n  G  J  ,  B  e  t  s  o  n  N  ,  G  r  i  f  f  i  t  h  s  H  ,  S  e  i  b  t  U   .  2  0  0  8  .  C  a  r  b  o  n  I  s  o  t  o  p  e  F  r  a  c  t  i  o  n  a  t  i  o  n  d  u  r  i  n  g   P  h  o  t  o  r  e  s  p  i  r  a  t  i  o  n  a  n  d  C  a  r  b  o  x  y  l  a  t  i  o  n  i  n  S  e  n  e  c  i  o  .  P  l  a  n  t  P  h  y  s  i  o  l  o  g  y   1  4  8   ,  2  0  1  3  -2  0  2  0  .   L  e  a  k  e  y  A  D  B  ,  A  i  n  s  w  o  r  t  h  E  A  ,  B  e  r  n  a  c  c  h  i  C  J  ,  R  o  g  e  r  s  A  ,  L  o  n  g  S  P  ,  O  r  t  D  R   .  2  0  0  9  .  E  l  e  v  a  t  e  d  C  O  2  e  f  f  e  c  t  s  o  n   p  l  a  n  t  c  a  r  b  o  n  ,  n  i  t  r  o  g  e  n  ,  a  n  d  w  a  t  e  r  r  e  l  a  t  i  o  n  s  :  s  i  x  i  m  p  o  r  t  a  n  t  l  e  s  s  o  n  s  f  r  o  m  F  A  C  E  .  J  o  u  r  n  a  l  o Z  h  a  n  g  X  ,  H  ö  g  y  P  ,  W  u  X  ,  S  c  h  m  i  d  I  ,  W  a  n  g  X  ,  S  c  h  u  l  z  e  W  X  ,  J  i  a  n  g  D  ,  F  a  n  g  m  e  i  e  r  A   .  2  0  1  8  .  P  h  y  s  i  o  l  o  g  i  c  a  l   a  n  d  P  r  o  t  e  o  m  i  c  E  v  i  d  e  n  c  e  f  o  r  t  h  e  I  n  t  e  r  a  c  t  i  v  e  E  f  f  e  c  t  s  o  f  P  o  s  t  -A  n  t  h  e  s  i  s  H  e  a  t  S  t  r  e  s  s  a  n  d  E  l  e  v  a  t  e  d  C  O  2  o  n   W  h  e  a  t  .  P  R  O  T  E  O  M  I  C  S   1  8   ,  1  8  0  0  2  6  2  .   Z  h  a  n  g  H  ,  T  u  r  n  e  r  N  C  ,  S  i  m  p  s  o  n  N  ,  P  o  o  l  e  M  L   .  2  0  1  0  .  G  r  o  w  i  n  g  -s  e  a  s  o  n  r  a  i  n  f  a  l  l  ,  e  a  r  n  u  m  b  e  r  a  n  d  t   Values are means with standard errors. Derived parameters include temperature optima (T opt ) of photosynthesis (A opt ); activation energy for carboxylation (EaV); activation energy (EaJ)¸ entropy term (∆SJ) and T opt and corresponding value for J max with deactivation energy (Hd) assumed constant; and activation energy (EaR) and temperature coefficient (Q 10 ) for dark respiration. Letters indicate significance of variation in means.

Figure 2
Response of leaf gas exchange parameters to eCO 2 under non-HS conditions. Measurements were made at 25°C before each harvest (T1, T2 and T3) for CO 2 assimilation rates (a, b) and stomatal conductance (c, d) in Scout (Circles) and Yitpi (Triangles). Plants were grown and measured at aCO 2 (blue solid cultivars), grown and measured at eCO 2 (red solid cultivars), and grown at eCO 2 and measured at 400 μ L CO 2 L -1 (red dashed cultivars). Statistical significance levels (ttest) for the growth condition within each cultivar are shown and they are: * = p < 0.05; ** = p < 0.01: *** = p < 0.001.

Figure 3 Response of photosynthesis and chlorophyll fluorescence to HS in
Scout and Yitpi grown at aCO 2 or eCO 2 . CO 2 assimilation rates (a, b, c, d) and dark-adapted chlorophyll fluorescence, Fv/Fm (e, f, g, h) were measured at growth CO 2 and 25 °C in Scout (Circles) and Yitpi (Triangles). Open and closed symbols represent control and HS plants, respectively. In addition, plants were measured at 35°C (*) during both HS cycles.

Figure 4
Response of photosynthetic parameters to eCO 2 and HS at anthesis (T3) in Scout and Yitpi. CO 2 assimilation rate (a, b), stomatal conductance (c, d), photosynthetic water use efficiency (e, f) and photosynthetic nitrogen use efficiency (g, h) were measured at growth CO 2 . V cmax (i, j) and J max (k, l) were derived from ACi curves measured at 25 o C. Cultivars indicate means and shaded region is 95% confidence interval. Data shown for control (not exposed to any heat stress) and plants exposed to both heat stress cycles (HS1+2). Statistical significance levels (ttest) for the growth condition within each cultivar are shown and they are: * = p < 0.05; ** = p < 0.01: *** = p < 0.001.

Figure 5
Relationships with leaf gas exchange and grain yield across treatments. CO 2 assimilation rate plotted as a function of stomatal conductance (a) (both aCO 2 and eCO 2 grown plants measured at 400 μ l L -1 ), J max plotted as a function of V cmax (b) and grain protein plotted as a function of yield (c) in Scout (Circles) and Yitpi (Triangles). Ambient and elevated CO 2 are depicted in blue and red, respectively. Control and heat stressed plants depicted using open and closed symbols. Panel a depicts data for control, HS1, HS2 and both heat stresses (HS1+2), while panels b and cinclude only control and HS1+2. Figure 6 Response of plant growth and morphological traits to elevated CO 2 and HS: Total dry mass (a, b), tillers or number of tillers (c, d), leaf area (e, f), leaf number (g, h) and height (i, j) were measured at different time points across the life cycle of wheat cultivars Scout (Circles) and Yitpi (Triangles). Ambient and elevated CO 2 are depicted in blue and red color, respectively. Open symbols connected with solid cultivars and closed symbols connected with dashed cultivars represent control and HS plants, respectively. HS1 and HS2 depict the timing of HS applied at 10 and 15 weeks after planting respectively.

Figure 7
Response of total plant dry mass and grain parameters to growth at eCO 2 and HS at maturity (T4): Total dry mass (a, b), grain dry mass (c, d), grain number (e, f) and grain nitrogen (g, h) were measured at the final harvest. Cultivars indicate means and shaded region is 95% confidence interval. Ambient and elevated CO 2 are depicted in blue and red color respectively. Heat stress levels include plants not exposed to any heat stress (control) and both heat stresses (HS1+2). Statistical significance levels (t-test) for the growth condition within each cultivar is shown and they are: * = p < 0.05; ** = p < 0.01: *** = p < 0.001. Table S1. Summary of statistics for gas exchange parameters. Table S2. Response of Scout gas exchange parameters to elevated CO 2 and heat stress. Table S3. Response of Yitpi gas exchange parameters to growth at elevated CO 2 and heat stress. Table S4. Summary of statistics for plant dry mass and morphological parameters. Table S5. Response of plant dry mass and morphological parameters to elevated CO 2 and HS.       Figure 1 Temperature response of photosynthetic parameters: CO 2 assimilation rate (a), mesophyll conductance (b), stomatal conductance (c) and intercellular CO 2 (d), V cmax (e), J max (f), J max / V cmax (g) and dark respiration (h) over leaf temperatures (15, 20, 25, 30 and 35 °C) in plants grown at aCO 2 . Scout and Yitpi are depicted using circles with solid cultivars and triangles with broken cultivars respectively. Data in panels (a), (b), (c), (d), (e), (f) and (h) are fit using nonlinear least square (nls) function in R.

Figure 2
Response of leaf gas exchange parameters to eCO 2 under non-HS conditions. Measurements were made at 25°C before each harvest (T1, T2 and T3) for CO 2 assimilation rates (a, b) and stomatal conductance (c, d) in Scout (Circles) and Yitpi (Triangles). Plants were grown and measured at aCO 2 (blue solid cultivars), grown and measured at eCO 2 (red solid cultivars), and grown at eCO 2 and measured at 400 μ L CO 2 L -1 (red dashed cultivars). Statistical significance levels (ttest) for the growth condition within each cultivar are shown and they are: * = p < 0.05; ** = p < 0.01: *** = p < 0.001.

Figure 3 Response of photosynthesis and chlorophyll fluorescence to HS in
Scout and Yitpi grown at aCO 2 or eCO 2 . CO 2 assimilation rates (a, b, c, d) and dark-adapted chlorophyll fluorescence, Fv/Fm (e, f, g, h) were measured at growth CO 2 and 25 °C in Scout (Circles) and Yitpi (Triangles). Open and closed symbols represent control and HS plants, respectively. In addition, plants were measured at 35°C (*) during both HS cycles.

Figure 4
Response of photosynthetic parameters to eCO 2 and HS at anthesis (T3) in Scout and Yitpi. CO 2 assimilation rate (a, b), stomatal conductance (c, d), photosynthetic water use efficiency (e, f) and photosynthetic nitrogen use efficiency (g, h) were measured at growth CO 2 . V cmax (i, j) and J max (k, l) were derived from ACi curves measured at 25 o C. Cultivars indicate means and shaded region is 95% confidence interval. Data shown for control (not exposed to any heat stress) and plants exposed to both heat stress cycles (HS1+2). Statistical significance levels (t-test) for the growth condition within each cultivar are shown and they are: * = p < 0.05; ** = p < 0.01: *** = p < 0.001.

Figure 5
Relationships with leaf gas exchange and grain yield across treatments. CO 2 assimilation rate plotted as a function of stomatal conductance (a) (both aCO 2 and eCO 2 grown plants measured at 400 μ l L -1 ), J max plotted as a function of V cmax (b) and grain protein plotted as a function of yield (c) in Scout (Circles) and Yitpi (Triangles). Ambient and elevated CO 2 are depicted in blue and red, respectively. Control and heat stressed plants depicted using open and closed symbols. Panel a depicts data for control, HS1, HS2 and both heat stresses (HS1+2), while panels b and cinclude only control and HS1+2.

Figure 6
Response of plant growth and morphological traits to elevated CO 2 and HS: Total dry mass (a, b), tillers or number of tillers (c, d), leaf area (e, f), leaf number (g, h) and height (i, j) were measured at different time points across the life cycle of wheat cultivars Scout (Circles) and Yitpi (Triangles). Ambient and elevated CO 2 are depicted in blue and red color, respectively. Open symbols connected with solid cultivars and closed symbols connected with dashed cultivars represent control and HS plants, respectively. HS1 and HS2 depict the timing of HS applied at 10 and 15 weeks after planting respectively.

Figure 7
Response of total plant dry mass and grain parameters to growth at eCO 2 and HS at maturity (T4): Total dry mass (a, b), grain dry mass (c, d), grain number (e, f) and grain nitrogen (g, h) were measured at the final harvest. Cultivars indicate means and shaded region is 95% confidence interval. Ambient and elevated CO 2 are depicted in blue and red color respectively. Heat stress levels include plants not exposed to any heat stress (control) and both heat stresses (HS1+2). Statistical significance levels (t-test) for the growth condition within each cultivar is shown and they are: * = p < 0.05; ** = p < 0.01: *** = p < 0.001.