Ozone tolerant maize hybrids maintain Rubisco content and activity during long‐term exposure in the field

Abstract Ozone pollution is a damaging air pollutant that reduces maize yields equivalently to nutrient deficiency, heat, and aridity stress. Therefore, understanding the physiological and biochemical responses of maize to ozone pollution and identifying traits predictive of ozone tolerance is important. In this study, we examined the physiological, biochemical and yield responses of six maize hybrids to elevated ozone in the field using Free Air Ozone Enrichment. Elevated ozone stress reduced photosynthetic capacity, in vivo and in vitro, decreasing Rubisco content, but not activation state. Contrary to our hypotheses, variation in maize hybrid responses to ozone was not associated with stomatal limitation or antioxidant pools in maize. Rather, tolerance to ozone stress in the hybrid B73 × Mo17 was correlated with maintenance of leaf N content. Sensitive lines showed greater ozone‐induced senescence and loss of photosynthetic capacity compared to the tolerant line.


| INTRODUCTION
Ozone (O 3 ) pollution formed in the troposphere compromises yields of many crop species and is estimated to reduce maize yields by as much as 10-15% (Avnery, Mauzerall, Liu, & Horowitz, 2011;McGrath et al., 2015;Mills et al., 2018c;Van Dingenen et al., 2009). Ozone is a short-lived pollutant and concentrations are dynamic and variable across the globe. The highest ozone concentrations are measured in the midlatitudes of the northern hemisphere, with lower concentrations measured in Australia, New Zealand and southern parts of South America (Mills et al., 2018). High concentrations of ozone are measured in important crop-growing regions in the United States, the Mediterranean region, India and China (Mills, Pleijel, et al., 2018). Physiological responses of crops to O 3 pollution include visible injury, reduced carbon assimilation and premature leaf senescence (Ainsworth, 2017). These responses are likely interlinked and scale from the cell to the leaf to the crop canopy, negatively impacting economic yields (Emberson et al., 2018). Many studies have investigated the mechanisms of O 3 stress on a variety of C 3 crops, which have been widely reviewed (Ainsworth, Yendrek, Sitch, Collins, & Emberson, 2012;Ashmore, 2005;Feng, Kobayashi, & Ainsworth, 2008;Fiscus, Brooker, & Burkey, 2005;Morgan, Ainsworth, & Long, 2003). However, fewer studies investigated the physiological impacts of O 3 stress on C 4 plants, including maize, the most widely produced grain crop in the world (FAO, 2018), in part because early studies showed that C 4 crops were more O 3 tolerant than C 3 crops (Heagle et al., 1988;Miller, 1988). Despite this, more recent modeling studies have predicted significant impacts of O 3 on C 4 crops (Avnery et al., 2011;McGrath et al., 2015;Mills et al., 2018b;Mills, Sharps, et al., 2018c;Van Dingenen et al., 2009), and experimental studies have shown that C 4 plants are also sensitive to O 3 pollution (Grantz & Vu, 2009;Grantz, Vu, Tew, & Veremis, 2012;Leisner & Ainsworth, 2012;Leitao, Bethenod, & Biolley, 2007;Leitao, Maoret, & Biolley, 2007;Li, Courbet, Ourry, & Ainsworth, 2019;. Ozone damage primarily occurs once O 3 diffuses into the leaf through the stomata and into the apoplast. There, O 3 reacts with the aqueous layers to form other reactive oxygen species (ROS), such as hydrogen peroxide, superoxide and hydroxyl radical (Heath, 2008). Apoplastic antioxidants, including ascorbate, glutathione and phenolic compounds, quench ROS, but if the ROS exceed the antioxidant-quenching capacity of the apoplast, reactions in the plasma membrane can occur, along with signalling cascades that cause metabolic changes within the cell (Luwe, Takahama, & Heber, 1993). In many species, underlying differences in the content of antioxidant compounds correlated with O 3 sensitivity (Betzelberger et al., 2010;Wellburn & Wellburn, 1996;Li, Calatayud, Gao, Uddling, & Feng, 2016), and in tropical maize, phenolic compounds, flavonoids and anthocyanin pigments increased with O 3 stress (Singh, Agrawal, Shahi, & Agrawal, 2014). However, it has proven difficult to generalize antioxidant responses to elevated O 3 across species and even genotypes within a species in part because the requirement for detoxification depends upon the amount of O 3 entering leaves, which can change with stomatal responses to elevated O 3 (Wellburn & Wellburn, 1996). Antioxidant compounds are also constantly changing and present in different cellular compartments at different concentrations, which complicate generalizations. Yet, development of accurate flux-based models of O 3 effects on crops requires fundamental knowledge of both stomatal behavior and detoxification capacity (Emberson et al., 2018). Accelerated senescence and reduced photosynthetic carbon assimilation are two major determinants of crop yield loss to O 3 pollution.
Field experiments with maize, soybean and wheat have provided evidence that loss of photosynthetic capacity is a repercussion of accelerated leaf senescence in elevated O 3 (Morgan, Bernacchi, Ort, & Long, 2004;Feng, Pang, Kobayashi, Zhu, & Ort, 2011;Yendrek, Erice, et al., 2017). Degradation of Rubisco protein and reduced Rubisco activity measured in vitro and in vivo in response to O 3 stress has been observed in C 3 crops (Enyedi, Eckardt, & Pell, 1992;Goumenaki, Taybi, Borland, & Barnes, 2010;Junqua et al., 2000). In C 4 crops, Rubisco is located in the bundle sheath cells, and, therefore, may be more isolated from O 3 -induced ROS. However, previous work indicated that bundle sheath proteins are more susceptible to oxidative damage than mesophyll cell proteins (Kingston-Smith & Foyer, 2000), and Rubisco activity and transcript levels were significantly reduced in sugarcane (Grantz et al., 2012), switchgrass (Li et al., 2019) and juvenile maize (Leitao et al., 2007, b) exposed to elevated O 3 . Our previous research revealed significant genetic variation in the photosynthetic response of maize hybrids and indicated that the mechanisms of response to O 3 may also vary among diverse hybrids (Choquette et al., 2019).
This study further investigates physiological and biochemical responses of six maize hybrids containing parents Hp301 and NC338, which previously exhibited greater photosynthetic sensitivity to elevated O 3 (Choquette et al., 2019). These hybrids were grown in elevated O 3 using Free Air Concentration Enrichment (FACE), which enables crops to be grown under field conditions, but with an altered atmospheric composition. Specifically, we test for genetic variation in O 3 response by examining the effects of elevated O 3 on the photosynthetic capacity in vivo and in vitro, stomatal limitation to photosynthesis, antioxidant pools and nitrogen (N) content. Based on previous experiments of midday gas exchange (Choquette et al., 2019), we predict that variation in sensitivity to elevated O 3 will be correlated to differential stomatal responses to O 3 stress, as well as to differences in antioxidant stores. . Within experimental and control plots, each genotype was planted in two 3.5 m rows spaced 0.76 m apart. Plant density was 8 plants m −1 . The six maize genotypes occupied one half of each 20 m diameter octagonal plot and were exposed to either ambient or elevated O 3 . The layout of the experiment was a randomized complete block design with n = 4. The O 3 target set-point was 100 nl L −1 and was applied from 10:00 to 18:00 throughout the growing season, as described in . In 2018, the 1 min. average O 3 concentration within the elevated plots was within 20% of the target concentration for 81.6% of the time. When it was raining, leaves were wet, or the wind speed was lower than 0.5 m s −1 , the O 3 treatment was not applied. Average temperature and precipitation from the growing season were recorded in an on-site weather station, and average developmental stages were estimated from growing degree days ( Figure 1).

| Gas exchange measurements
Gas exchange was measured from June 18-21, 2018 to July 2-5, 2018 on the eighth leaf, which was the youngest fully expanded leaf in the June measurements. The eighth leaf was tagged for tissue sampling and gas exchange measurements for both time points. Measuring the same leaf number in two time points provided information about the cumulative effects of chronic O 3 exposure on photosynthetic and biochemical mechanisms over time. Leaves from one block of the experiment (i.e., one ambient and one elevated O 3 plot) were excised before dawn for measurement in a field laboratory. Leaves were recut under water and placed in 50 ml tubes filled with water.
Before starting gas exchange measurements, leaves were placed under grow lights (YG 600 W Grow Light, YGROW) with light spectrum of 380-740 nm for 20 min to acclimate to high light before starting gas exchange measurements. Leaves were then placed in the leaf cuvette of a portable gas exchange system (LI-6800, LICOR, Lincoln, NE) to measure the response of net carbon assimilation (A) to intercellular CO 2 (c i ). Once steady-state values of A and g s (at 400 μmol mol −1 CO 2 , 1800 μmol m −2 s −1 PPFD, 30.0 C leaf temperature and 1.5 kPa vapour pressure deficit) were reached, measurements were taken at 400, 300, 200, 100, 10, 400, 400, 600, 800, 1,000, 1,200, and 400 μmol mol −1 CO 2 . After the A/c i response curves finished, leaves were left to reach steady state at 400 μmol mol −1 . Once A/c i curves reached steady state at 400 μmol mol −1 , three leaf disks of 1.46 cm 2 from the portion of the leaf that was enclosed in the cuvette were cut, snap-frozen in liquid N for later quantification of Rubisco content and activation status.
From the A/c i response curves, the maximum apparent rate of phosphoenolpyruvate (PEP) carboxylase activity (V pmax ) and CO2-saturated photosynthetic rate (V max ) were estimated. Two leaves per genotype per plot per time point were measured for a total of 192 measurements. The initial slope of the A/c i curve was used to calculate V pmax according to von Caemmerer (2000). A four-parameter nonrectangular hyperbolic function was used to estimate V max as the horizontal asymptote of the A/c i curve. Stomatal limitation was estimated at a CO 2 concentration of 400 μmol mol −1 from fitted C 4 curves using the equation: where A 0 is the rate of photosynthesis that would occur at infinite stomatal conductance (Farquhar & Sharkey, 1982).

| Quantifying Rubisco content, activation state and activity
Rubisco activation state was determined from measurements of initial and total (or fully activated) Rubisco catalytic sites (Butz & Sharkey, 1989;Galmés et al., 2011;Ruuska et al., 1998;von Caemmerer et al., 2005). Catalytic sites were measured by the binding of carboxypentitol-1,5-bisphosphate ( 14 C-CPBP) using size exclusion chromatography following Kubien, Brown, and Kane (2011), with modifications to quantify initial sites as described in Butz and Sharkey (1989). Purified C 3 Rubisco was inactivated and measured to ensure the protocol did not return artificially high estimates of activation states ( Figure S1). Leaf samples were ground in an ice-cooled  (Butz & Sharkey, 1989;Pierce, Tolbert, & Barker, 1980). After isotope exchange, samples underwent size exclusion chromatography, as described below. To quantify total Rubisco content in the sample, 100 μl of supernatant was aliquoted into a tube containing activation buffer and was incubated at room temperature for 20 min. This activated sample was then incubated with 3 mM 14 C-CPBP at room temperature for 30 min.
Rubisco-bound 14 C in initial and total samples was separated from unbound 14 C via size exclusion chromatography using 0.7 × 30 cm columns packed with Sephadex G-50 (Sigma-Aldrich G5050), equilibrated with 20 mM EPPS, 75 mM NaCl (pH 8.0). Aliquots were measured by liquid scintillation counting (Packard Tri-Carb 1900 TR, Canberra Packard Instruments Co., Downers Grove, IL). Activation state was calculated as the ratio of the number of initial Rubisco active catalytic sites to fully activated Rubisco catalytic sites (Butz & Sharkey, 1989). A Bradford assay (BioRad 5,000,001) was used to determine total soluble protein in the supernatant.
RuBP for these assays was synthesized and purified as described by Kane, Wilkin, Portis, and Andrews (1998).

| Quantification of ROS scavenging metabolites
At midday on June 23, 2018 and July 6, 2018 leaf samples for measuring phenolic content, ascorbate content, glutathione content, sugar content and chlorophyll content were taken on the marked eighth leaf. These samples were taken with a cork borer and immediately frozen in liquid N. The antioxidants pools were measured to gain insight in the antioxidant capacity of the plant. Chlorophyll and foliar glucose, fructose and sucrose were measured as described in Yendrek, Leisner, and Ainsworth (2013). A leaf sample of 1.34 cm 2 was processed for total foliar phenolic content as described in Ainsworth and Gillespie (2007). In short, samples were extracted in 95% methanol and incubated in the dark at room temperature for 48 hr. The samples were then incubated with 10% Folin-Ciocalteu solution and 700 mM Na 2 CO 3 at room temperature for 2 hr. Finally, absorbance was measured at 765 nm and compared to a standard curve of gallic acid. A GSH/GSSG-Glo Assay kit (Promega Corporation, Madison, WI) was used to quantify glutathione content using a luminescence reaction following the manufacturer's protocol. Ten milligram of leaf tissue was mixed with 1× phosphate-buffered saline with 2 mM EDTA (pH 8.0).
Total glutathione content was measured by detecting a luciferase signal, which was proportional to glutathione content. Total and reduced ascorbate were measured using the methods of Gillespie and Ainsworth (2007) using a leaf sample of 1.9 cm 2 .
Samples for specific leaf area (SLA) were taken with a cork borer (1.9 cm 2 ) and placed into a coin envelope. SLA samples were dried at 60 C for 10 days until they reached a constant mass. They were weighed and then ground to a fine powder. A small amount of each sample was weighed into a tin capsule for C and N analysis. An elemental analyzer (Costech 4010CHNSO Analyzer, Costech Analytical Technologies Inc. Valencia, CA) was used to measure C and N content.
Acetanilide and apple leaves (National Institute of Science and Technology, Gaithersburg, MD) were used as standards.

| Statistical analysis of physiological and biochemical traits
The field design was a random complete block design (n = 4).  Reduced photosynthetic capacity resulted in lower net photosynthetic rates (A) in elevated O 3 , with rates decreased by 16% in June and by 34% in July (Table 1). Although no significant genotypetreatment interaction was detected in the statistical model (Table S2), the magnitude of the response of A to elevated O 3 ranged from a 7% decrease in elevated O 3 in B73 × Mo17 to a 40% decrease in NC338 × Hp301 in June. Stomatal conductance (g s ) was also significantly lower in elevated O 3 in June, but not in July (Tables 1 and S2). Thus, prolonged exposure to elevated O 3 in hybrid maize altered the linear relationship between A and g s (Figure 4). Intercellular [CO 2 ] (c i ) was also significantly greater in elevated O 3 , especially in July (Table 1).
NC338 × Hp301 showed the greatest change in c i in both June and July, whereas B73 × Mo17 showed no change in g s or c i under elevated O 3 compared to ambient O 3 in July (Table 1). Stomatal limitation to photosynthesis (l) did not consistently respond to elevated O 3 , and tended to be slightly higher in elevated O 3 in June and lower in elevated O 3 in July (Tables 1 and S2). Pairwise comparisons showed that only genotype B73 × Mo17 showed a significant reduction in stomatal limitation in elevated O 3 in July (Table 1).

| Biochemical responses of hybrid maize to elevated O 3
Overall, percent nitrogen (%N) decreased in elevated O 3 in both June and July (Figure 5a,b; Table S3) with a much greater reduction in July.  in June and were too low to be detected in July (Table S4).
Phenolic, ascorbate and glutathione contents were measured in all six genotypes in June and July to determine if there were genotypic differences in antioxidant responses to elevated O 3 (Table 3). Across all hybrids, there was a significant O 3 effect on phenolic content with increased levels in elevated O 3 in June (p < .05), but there was no effect of O 3 on phenolic compounds in July (Table S5) (Table 4). There was a consistent increase in foliar fructose under elevated O 3 in June, but the response was inconsistent in July (- Table S6). Similarly, glucose showed no consistent pattern in June, but decreased in elevated O 3 across all hybrids in July (Table S6). Sucrose remained unchanged between ambient and elevated O 3 in both June and July.

| Correlation between A, N, Rubisco content and yield
Exposure to elevated O 3 significantly decreased yield and individual seed weight (Table S7). Foliar N was strongly correlated with A ( Figure 6b) and Rubisco content (Figure 6a), and weakly correlated with seed yield (Figure 6c). Total Rubisco content was positively and significantly correlated with A and seed yield across O 3 treatments (Figure 6d,e) and A was weakly correlated with seed yield, largely because the hybrids showed a range of yield values in ambient O 3 , but little variation in A (Figure 6e). B73 × Mo17, which maintained high %N and photosynthetic capacity, was more tolerant to O 3 stress than the other hybrids (indicated by stars in Figure 6).  (Mills, Sharps, et al., 2018c), which translates to losses up to $9 billion in the US (McGrath et al., 2015). Note: Asterisks (*) and bold font represent significant pairwise comparison within each genotype for each time point (p < .05). Antioxidant capacity in the apoplast provides the first line of defence against O 3 and other ROS (Kangasjärvi, Jaspers, & Kollist, 2005) and is linked to tolerance to multiple environmental stresses (Scebba, Pucciarelli, Soldatini, & Ranieri, 2003). Phenolic molecules directly scavenge ROS (Grace & Logan, 2000), respond to stresses that impair photosynthesis (Koricheva, Larsson, Haukioja, & Keinanen, 1998) and increase under elevated O 3 in a variety of species (Gillespie, Rogers, & Ainsworth, 2011;Kangasjärvi, Talvinen, Utriainen, & Karjalainen, 1994;Peltonen, Vapaavuori, & Julkunen-Tiitto, 2005;Yendrek, Koester, & Ainsworth, 2015). Therefore, we hypothesized that antioxidant and phenolic compounds would vary among tolerant and sensitive maize lines. However, we did not find evidence for genotypic variation in antioxidant and phenolic responses to O 3 . While there was a significant effect of O 3 on phenolic content across all genotypes in June (Table 3), older maize leaves measured in July had lower phenolic content and no response to elevated O 3 . A study of wheat and maize also showed decreased phenolic content over time with exposure to O 3 (Li, Shi, & Chen, 2008).
Antioxidants, glutathione and ascorbate, are important to ROS scavenging (Foyer & Noctor, 2005), and reduced glutathione donates an electron to dehydroascorbate, which regenerates oxidized ascorbate into reduced ascorbate (Kangasjärvi et al., 1994). Although there were genotypic differences in the pool sizes of total glutathione across both time points, the redox state was the same between ambient and elevated O 3 . The redox state of ascorbate generally increased in elevated O 3 consistent with a study of juvenile maize grown under oxidative stress, which found no change in the redox state of glutathione and an increase in the redox state of ascorbate (Kingston-Smith, Harbinson, & Foyer, 1999).
Previous studies of these maize genotypes identified variation in leaf-level photosynthetic responses to O 3 , and it was hypothesized that variation in stomatal limitation may be associated (Choquette et al., 2019). However, contrary to our hypothesis, there was no evi- Rubisco activity in many different species under elevated O 3 (Brendley & Pell, 1998;Dann & Pell, 1989;Eckardt & Pell, 1994;Galant, Koester, Ainsworth, Hicks, & Jez, 2012;Pell & Pearson, 1983;Pelloux, Jolivet, Fontaine, Banvoy, & Dizengremel, 2001;Reid, Fiscus, & Burkey, 1998). In an experiment on juvenile maize, PEP carboxylase and Rubisco content and activity decreased with increasing O 3 exposure (Leitao, Bethenod, & Biolley, 2007), which is consistent with our results for sensitive hybrids. A meta-analysis of Rubisco content and activity also found that O 3 reduced Rubisco concentration (Galmés, Aranjuelo, Medrano, & Flexas, 2013), possibly because of reduced synthesis of Rubisco messenger RNA (Heath, 2008) or enhanced degradation of Rubisco (Eckardt & Pell, 1994). It has been shown that ROS can accelerate Rubisco degradation in chloroplasts (Feller, Anders, & Mae, 2008), which is consistent with decreased Rubisco, but not overall soluble protein content as found in this study ( Figure 3). Rubisco can also be regulated through redox potential (Huffaker, 1982) and has sulfhydryl groups that become oxidized and signal degradation under stress conditions and nutrient deficits (Garcia-Ferris & Moreno, 1993;Garcia-Ferris & Moreno, 1994;Pell & Pearson, 1983). It seems likely that chronic exposure to O 3 in maize overwhelmed the detoxification potential of the cells, resulting in signalling cascades that triggered degradation of Rubisco enzymes, as has been postulated for other C 3 species (Goumenaki et al., 2010).

Rubisco carboxylation can be inhibited by sugar phosphates and
Rubisco activase is important for removing the inhibitors from catalytic sites in an ATP-dependent manner. Rubisco activase restores Rubisco from an inactive to active conformation (Portis Jr., 2003) and is imperative to maintain photosynthesis in C 4 plants, even with carbon concentration mechanisms . In this study, activation state of Rubisco, reflecting Rubisco activase activity, was not affected by elevated O 3 and did not contribute to a loss in photosynthetic capacity in elevated O 3 (Figure 3). A previous study in maize showed that Rubisco activase transcript levels did not change with O 3 exposure in the fifth and 10th leaf (Leitao, Maoret, & Biolley, 2007), consistent with our findings. Although a study in Pinus halepensis M. demonstrated a small decrease in Rubisco activase concentration after exposure to O 3 , carbamylation of Rubisco remained unchanged (Pelloux et al., 2001). Our results support these previous experiments and extend the findings that activation of Rubisco by Rubisco activase was not affected by elevated O 3 in maize.
In this study, maize Rubisco activation state ranged from 65 to 82% in June and 60-75% in July, consistent with other estimates of activation state in maize (Carmo-Silva et al., 2010;Sharwood et al., 2016). In C 4 plants, total Rubisco content is lower than in C 3 species, yet activation state in C 4 maize was similar or lower than in C 3 species (Perdomo, Capo-Bauca, Carmo-Silva, & Galmes, 2017;Sharwood et al., 2016). In other studies, Rubisco activation state in C 4 species was reported as low as 45-55% (Carmo-Silva et al., 2010;von Caemmerer et al., 2005). It is generally believed that Rubisco carboxylation of CO 2 is the ultimate limitation in C 4 species (Edwards, Furbank, Hatch, & Osmond, 2001;von Caemmerer, Millgate, Farquhar, & Furbank, 1997), and over-expression of Rubisco content in maize resulted in increased plant height and biomass (Salesse-Smith et al., 2018). This begs the question, why would the activation state of Rubisco in C 4 species be the same or lower than C 3 species? It is possible that the carbon concentrating mechanism of Kranz leaf anatomy of C 4 species might play a role as the CO 2 concentration inside the bundle sheath is 10-fold higher than the atmosphere (Furbank & Hatch, 1987;von Caemmerer & Furbank, 2003). The carbon concentrating mechanism may make it feasible for C 4 species to over-invest in Rubisco by a negligible amount in terms of N storage and maintain low-levels of inactivated Rubisco. It is interesting that under different oxidative stress conditions and different leaf ages, the activation state of maize Rubisco remained relatively constant (Figure 3e,f).
Leaf N content was strongly correlated with Rubisco activity, Rubisco content and A in maize leaves exposed to elevated O 3 , which is predicted as Rubisco content and activity control net carbon assimilation and C 4 plants allocate 5-10% of leaf N to Rubisco (Figure 6; Ghannoum et al., 2005;Sharwood et al., 2016). We also found that Rubisco content measured in a mature leaf in July was correlated to yield in maize lines exposed to elevated O 3 ( Figure 6). Previous work has demonstrated that Rubisco is an important storage protein for N, sulfur and carbon skeletons (Liu, Ren, White, Cong, & Lu, 2018;Sage & Pearcy, 1987), and is crucial for remobilization of N to seeds (Feller et al., 2008;Millard & Grelet, 2010). The fact that the tolerant hybrid B73 × Mo17 did not show significant reductions in Rubisco content in July in contrast to other hybrids supports the notion that acceleration of senescence is a key determinant of productivity responses to O 3 .
O 3 is found to trigger the expression of genes involved in senescence in plants (Lim, Kim, & Nam, 2007;Miller, Arteca, & Pell, 1999), and when a leaf undergoes senescence, many nutrients, such as nitrogen, phosphorus and metals, are recycled in the plants and nutrient-rich molecules are degraded (Lim et al., 2007). Accelerated loss of photosynthetic capacity and leaf aging from elevated O 3 has been demonstrated in many crops of wheat, rice, soybean and maize (Betzelberger et al., 2010;Emberson et al., 2018;Feng et al., 2011;Pang, Kobayashi, & Zhu, 2009), and is a target for improving crop tolerance to O 3 (Yendrek, Erice, et al., 2017).

| CONCLUSIONS
Ozone pollution is an important stressor on plants that reduces crop yields around the world. Ozone damage to plants is considered a threat to food security and could be exacerbated in a changing climate (Tai, Martin, & Heald, 2014). For maize, global O 3 stress causes equivalent damage as nutrient deficiency, heat and drought stress (Mills, Sharps, et al., 2018c