Chromosome Location Contributing to Ozone Tolerance in Wheat

Breeding wheat for higher grain yield can contribute to global food security and sustainable production on less land. Tropospheric ozone can injure wheat plants and subsequently reduce grain yield. Identification of ozone tolerance in the wheat genome can assist plant breeders in developing new sources of tolerant germplasm. Our objective was to use the ‘Chinese Spring’ monosomic lines to screen for ozone response and identify the chromosomic locations contributing to ozone tolerance based on foliar injury. Two methodologies, Continuous Stirred Tank Reactors and Outdoor Plant Environment Chambers, were used to expose wheat monosomic lines to varying concentrations and durations of ozone. Each wheat monosomic line in ‘Chinese Spring’ has a missing chromosome in each of the wheat subgenomes (A, B, and D). In both methodologies, we found significant and repeatable data to identify chromosome 7A as a major contributor to tolerance to ozone injury in ‘Chinese Spring’. In every experiment, the absence of chromosome 7A resulted in significant injury to wheat due to ozone. This was not the case when any other chromosome was missing.


Introduction
Tropospheric ozone (O 3 ) is a secondary air pollutant and a global warming gas in ambient air, the concentrations of which have been increasing since the industrial era and are expected to continue to increase in the future [1]. Once formed, the O 3 molecules can be inhaled by humans and animals, causing respiratory problems, and can diffuse through stomata into plant leaves, where ozone is considered the most phytotoxic air pollutant [2]. Inside the leaf, O 3 immediately dissolves, generating primary reactive oxygen species (ROS) in the apoplast [3]. If not detoxified, these non-biogenic ROS react directly in the apoplastic fluid with the cell wall and/or plasma membrane, inducing a self-propagating oxidative burst of biogenic ROS that spreads to the surrounding tissue [4]. The resulting oxidative stress causes visible injury and biomass and yield reduction in sensitive plants including major crops, such as wheat, soybean, corn, and rice, thereby threatening global food security.
Common bread wheat (Triticum aestivum L.) is one of the most sensitive crops to elevated O 3 at all growth stages [5]. Modeling based on O 3 concentrations in the atmosphere estimate current wheat yield reduction due to elevated O 3 to be 7-12% of the yield worldwide, and this number is expected to reach 9-18% by the year 2030 [6,7]. Recent modeling work based on the more biologically relevant flux-based approach predicts similar yield reductions, but proposes a different geographical distribution of the impacts on wheat production as the result of regional differences in the environmental conditions that control O 3 uptake through stomata [8,9]. Breeding wheat for O 3 tolerance is essential for increasing yield per area under O 3 stress, and is a critical adaptation needed to sustainably produce 70% more food by 2050 [10]. This is especially true in developing countries, where more food production is needed, and where O 3 precursors emission and population are expected to continue increasing. For example, both the highest stomatal O 3 fluxes and O 3 concentrations are observed in the most populated region of the world, in South and East Asia [8,9]. In particular, resource-poor farmers in developing countries rely for food on new abiotic and biotic stress-tolerant varieties that are freely available.
Currently, O 3 stress is not prioritized by wheat breeders [9,11,12]. This lack of breeding effort may be attributed to several factors. Wheat breeders are less familiar with O 3 as a stress factor and the magnitude of O 3 impacts on wheat. Research is lacking on the genetic control of O 3 tolerance in wheat, including information on genetic markers associated with the trait and the best approaches for phenotyping key varieties and breeding lines. Breeding for O 3 tolerance is further complicated by the temporal and spatial inconsistency of O 3 stress, so that testing requires specialized research facilities.
The development of O 3 -tolerant wheat varieties can be accelerated if better information was available on the genetic control of the trait, and molecular markers are identified for easier and quicker identification of tolerant germplasm. The complex, allohexaploid (2n = 6x = 42 chromosomes, AABBDD) genome of common wheat became better understood with the availability of aneuploids, in particular monosomics, which allowed genes to be assigned to chromosomes [13]. In this work, we investigated the O 3 response of 'Chinese Spring' and its monosomic series in order to identify the chromosome location of O 3 response using visible foliar symptoms.

Ozone Injury to 'Chinese Spring' Monosomics Using Continuous Stirred Tank Reactors
Previous work [14] showed that 'Chinese Spring' would be a suitable indicator for O 3 response because this variety exhibits a reduced O 3 -damage phenotype compared to other potential standard varieties. These results, coupled with the wealth of genotypic and phenotypic data on 'Chinese Spring', along with it being the reference variety for the sequencing of the wheat genome [15], make it ideal for O 3 research. For our Continuous Stirred Tank Reactor (CSTR) study, 'Chinese Spring' and its 21 monosomic lines were first grown in charcoal-filtered (CF) air from planting until early tillering, Zadoks growth stage 21-23 [16]. Our CSTRs were located within an environmentally controlled greenhouse. At that time, the plants were moved to the five O 3 treatments (CF, 50, 70, 90, and 110 ppb) for five days, and then two days after treatment they were visually assessed for O 3 damage. Visual assessments were based on the relative percent of a leaf exhibiting chlorosis and necrosis. At the time of our assessments, the fourth leaf provided the most uniform growth stage. Averaged over O 3 concentrations, 'Chinese Spring' averaged a mean O 3 injury score of 11% ( Figure 1).
The only monosomic line significantly different from 'Chinese Spring' was the line missing chromosome 7A, at an O 3 injury level of about 30%. When the responses of the monosomic lines were investigated within each O 3 treatment, the monosomic line missing chromosome 7A always showed the greatest injury. However, the effect was significantly different from 'Chinese Spring' at only 110 ppb (Supplementary Figure S1). Thus, chromosome 7A was associated with increased tolerance to O 3 . Because other monosomic lines differed (though not significantly at the 95% confidence level) from 'Chinese Spring', it could be inferred that multiple minor genes/alleles may be associated with the O 3 response in wheat, in addition to those major-effect gene(s) on chromosome 7A.

Discussion
The goal of this study was to determine the individual chromosome contribution towards the known O 3 tolerance in 'Chinese Spring' wheat. This objective was achieved by screening the cultivar with complete genome and its 21 monosomic lines under different O 3 treatments, in two different gas exposure systems. Ozone injury data showed that chromosome 7A is critical for O 3 tolerance in 'Chinese Spring'. Our finding supports a previous report [17] attributing O 3 tolerance in hexaploid wheat to subgenomes AABB. Biswas et al. [17] found that durum wheat (Triticum durum; AABB) was the most O 3 -tolerant, followed by T. monococcum, a successive sister of T. urartu, the more likely origin of genome AA. In this study, we provided direct evidence, using common bread wheat, that chromosome 7A has a major effect on the tolerance to O 3 in 'Chinese Spring'. The O 3 -induced injury reported here had a direct effect on reducing plant biomass [14], but further research is needed to relate the biomass reduction to grain yield.
Davydov [18] showed that the absence of chromosome 7A was associated with the least stomatal length amongst all monosomic lines, which indicates that the increased sensitivity observed in the absence of 7A reported in the present study is likely derived by other mechanisms that are not affected by the size of stomata. These mechanisms could include lower O 3 detoxification or injury containment abilities. Interestingly, wheat chromosome 7A also carries other important stress-and disease-related traits, such as resistance to powdery mildew [19], salinity tolerance [20], and resistance to Fusarium Head Blight [21]. This research serves as an initial step in our identification of O 3 tolerance in wheat. We are currently conducting a genome-wide association analysis of O 3 injury data on a double-haploid population made between O 3 -sensitive and O 3 -tolerant wheat germplasms, with the goal of better pinpointing those genetic loci associated with O 3 tolerance.
Evidence suggests that several mechanisms contribute to O 3 tolerance in crops. For both rice [12] and soybean [22], multiple quantitative trait loci have been associated with O 3 tolerance. It is reasonable to expect the same for wheat. Mills et al. [9] described a wheat ideotype with specific characteristics relevant to O 3 stress responses. Characteristics include leaf traits that limit O 3 uptake, metabolic traits that regulate antioxidant metabolism and programmed cell death, and root traits that enhance water and nutrient uptake under stress [9]. Each characteristic is potentially associated with different sets of markers. It is clear that yield superiority under O 3 stress is the ultimate predictor for O 3 tolerance. However, for screening large numbers of entries, biomass and visible symptoms are key indicators. Visible symptoms determine the active photosynthetic area, as well as the healthy host tissue interacting with O 3 at later growth stages.

Materials and Methods
The 'Chinese Spring' monosomics were obtained from the USDA-ARS National Small Grains Collection in Aberdeen, ID. For both exposure systems, dry seeds were planted in 170 ml plastic containers (20.7 cm length, 4 and 2.5 cm diameter at the top and the bottom, respectively) filled with Fafard #2 Pro Mix (Fafard, Anderson, SC, USA). Plants were irrigated from the bottom in a water basin. Water soluble fertilizer (20-10-20 NPK) was applied to the water basin once a week. Seedlings were grown in CF air (O 3 concentration = 5 ppb) in the greenhouse for 30 days after planting. Plants at an early tillering growth stage of 21-23 in Zadok's scale were selected for growth stage and canopy status. The fully expanded fourth leaf on the top of the main stem was tagged and then plants moved to the Continuous Stirred Tank Reactors (CSTRs) [23,24] or the Outdoor Plant Environment Chambers (OPECs) [25].
In both systems, plants were acclimated for two days in CF before treatment with O 3 . In CSTRs, the five target O 3 treatments (CF, 50, 70, 90, and 110 ppb) were planned for five days (7 h/day, at 25 • C and 60% relative humidity (RH)). The actual achieved treatments were 5, 49, 70, 92, and 107 ppb, respectively. The experiment design involved 15 chambers in three blocks. Each treatment was randomly assigned to one chamber per block. One plant per genotype per chamber was used. In OPECs, plants were exposed for 14 days to one of four O 3 treatments using a predefined diurnal O 3 profile (CF, 50, 70, 90 ppb, 12 h average, at 25/16 • C day/night and 50% RH). The actual achieved treatments were 5, 46, 65, and 84 ppb, respectively. Eight OPEC chambers divided into two blocks were used. Each treatment was randomly assigned to one chamber per block. Two plants per genotype were randomized to each chamber.
Foliar injury was assessed visually following a 0-100% scale (Figure 3)  However, only the third to fifth leaves were considered for statistical analysis, as the first and second leaves showed some signs of senescence, and not all plants had fully expanded leaves above the fifth at the time of assessment. Data were subjected to analysis of variance using Glimmix procedure in SAS 9.4 (SAS Inc., Cary, NC, USA) as a split-plot design (O 3 treatments in the main plot, and genotype in the subplot). Random effects of block and sampling were accounted for. Significant effects were determined at a significance level of 5% (p ≤ 0.05) and Dunnett's test was used to compare each monosomic line to 'Chinese Spring'.  Figure S1: Visible injury responses of spring wheat variety "Chinese Spring" and it's monosomic lines exposed to different ozone levels in CSTRs. Figure S2: Visible injury responses of spring wheat variety "Chinese Spring" and it's monosomic lines exposed to different ozone levels in OPECs.