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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., Ozone pollution significantly reduces maize yield, and understanding the responses underpinning ozone tolerance is critical to developing varieties to mitigate losses. In field‐grown maize, tolerance to elevated ozone is associated with increased leaf N and Rubisco content, maintaining carboxylation capacity.

We examine the impact of sustained elevated ozone concentration on the leaf transcriptome of 5 diverse maize inbred genotypes, which vary in physiological sensitivity to ozone (B73, Mo17, Hp301, C123, and NC338), using long reads to assemble transcripts and short reads to quantify expression of these transcripts. More than 99% of the long reads, 99% of the assembled transcripts, and 97% of the short reads map to both B73 and Mo17 reference genomes. Approximately 95% of the genes with assembled transcripts belong to known B73–Mo17 syntenic loci and 94% of genes with assembled transcripts are present in all temperate lines in the nested association mapping pan-genome. While there is limited evidence for alternative splicing in response to ozone stress, there is a difference in the magnitude of differential expression among the 5 genotypes. The transcriptional response to sustained ozone stress in the ozone resistant B73 genotype (151 genes) was modest, while more than 3,300 genes were significantly differentially expressed in the more sensitive NC338 genotype. There is the potential for tandem duplication in 30% of genes with assembled transcripts, but there is no obvious association between potential tandem duplication and differential expression. Genes with a common response across the 5 genotypes (83 genes) were associated with photosynthesis, in particular photosystem I. The functional annotation of genes not differentially expressed in B73 but responsive in the other 4 genotypes (789) identifies reactive oxygen species. This suggests that B73 has a different response to long-term ozone exposure than the other 4 genotypes. The relative magnitude of the genotypic response to ozone, and the enrichment analyses are consistent regardless of whether aligning short reads to: long read assembled transcripts; the B73 reference; the Mo17 reference. We find that prolonged ozone exposure directly impacts the photosynthetic machinery of the leaf., National Science Foundation Plant Genome Research Program (PGR‐1238030; ADBL, LMM, and EAA) MCA-PGR: genetic and genomic approaches to understand and improve maize responses to ozone, National Institute of General Medical Sciences R01GM128193 (LMM), National Cancer Institute R03CA222444 (AC, LMM), United States Department of Agriculture SoyFACE Global Change Research Project Number 5012-21000-030-17-S.

The maize pangenome has demonstrate large amounts of presence/absence variation and it has been hypothesized that presence/absence variation contributes to stress response. To uncover whether the observed genetic variation in physiological response to elevated ozone (a secondary air pollutant that causes significant crop yield losses) concentration is due to variation in genic content, and/or variation in gene expression, we examine the impact of sustained elevated ozone concentration on the leaf tissue from 5 diverse maize inbred genotypes (B73, Mo17, Hp301, C123, NC338). Analysis of long reads from the transcriptomes of the 10 conditions found expressed genes in the leaf are part of the shared genome, with 94.5% of expressed genes from syntenic loci. Quantitative analysis of short reads from 120 plants (twelve from each condition) found limited transcriptional response to sustained ozone stress in the ozone resistant B73 genotype (151 genes), while more than 3,300 genes were significantly differentially expressed in the more sensitive NC338 genotype. The genes underpinning the divergence of B73 from the other 4 genotypes implicates ethylene signaling consistent with some findings in Arabidopsis. For the 82 of the 83 genes differentially expressed among all 5 genotypes and the 788 of 789 genes differentially expressed in 4 genotypes (excluding B73) in sensitivity to ozone is associated with oxidative stress tolerance being associated with a weaker response to a reactive oxygen species (ROS) signal and suggests that genetic variation in downstream processes is key to ozone tolerance.