Discussion
The major aim of the present study was to assess the effect of elevated [O3] on the metabolites in different crop species which is little known so far. Wheat, rice and soybean feed more than half of the world population, yet they are the most sensitive crops to O3 pollution. Previous model studies predicted their huge yield loss under elevated [O3] in the past 20 years (Sitch et al ., 2007; Avnery et al ., 2011b). Although many papers demonstrated the damage of O3 to crops, few researches were performed to compare the adverse effecs of O3 on different species, especially between monocots and dicots. Therefore, understanding how elevated [O3] affects crops via metabolic pathway is crucial for efforts to imporve their performance. Here, we found that some metabolic changes were common to the three species, whereas others were specific in monocots (wheat and rice) or dicots (soybean).
Photosynthesis is the primary source of chemical energy and biomass accumulation, it is therefore essential for plant growth and development (Kruse et al ., 2005; Melis, 2013). Previous findings reported that chronic exposure to O3impaired stomata conductance and photosynthesis in plant leaves through change of calcium influxes and increased photorespiration (Ainsworth, 2008; Avneryet al ., 2011a). Consistent with this, CO2assimilation rates (A sat), stomatal conductivity (g s) and transpiration rate (E ) were reduced in wheat, rice and soybean under O3 stress in our study. It has been observed that the reduction of photosynthesis coupled to alteration of cellular metabolism (Dizengremel, 2001; Heath, 2008; Reich, 1983). We found the pentose phosphate pathway was disturbed by O3 in three crop plants with highly increased concentration of the purines, which are converted from Ribulose-1,5-bisphosphate carboxylase/oxygenase (Ribulose D-Ribulose-5-P). The glycerol phosphate pathway was impaired only in wheat and rice by O3, leading to the increased concentration of phospholipids. This metabolic trait seems to be associated with different species in response to O3. Subsequently, the shikimate-phenylpropanoid pathway starting in chloroplasts was affected in three crop plants by O3.In this pathway salicylate-derived methyl salicylate (SA) was generously generated in soybean, while the concentration of salicylate was reduced in rice and wheat. SA and methylated salicylic acid (Me-SA) have been reported to be required for adaptive responses to certain biotic and abiotic stresses (Rekhter et al ., 2019). Furthermore, low concentrations of SA generally enhance the antioxidant capacity in plants, but high concentrations of SA may cause cell death or susceptibility to abiotic stresses (Vlot et al ., 2009). These results implied that SA or Me-SA is involved in the regulation of O3 response but it functions in different manner in monocots as wheat and rice, and dicots as soybean. These two types of plants may evolved different strategies to minimize the adverse effects of O3 stress. Similarly, the synthesis of flavonoids, lignin and phenylpropanoids also altered in this pathway. These metabolites are induced by various adverse environmental stresses (Tzin & Galili, 2010). The study presented here pointed to increased concentration of the lignin and phenylpropanoids induced by O3 in soybean, which was consistent with that in poplar (Cabané et al ., 2004). On the contrary, the level of lignin and phenylpropanoids in wheat was decreased. It suggests that stimulation of enzyme activities by O3 involved in the more specific lignin pathway is different according to the different species. Previous studies showed that the change of lignin biosynthesis could alter the synthesis of other secondary metabolites (Baxter & Stewart, 2013). Comparable reduction in flavonoids occured across wheat, rice and soybean, suggesting that the three species share same metabolic pathway leading to the reduced synthesis of flavonoid in response to O3. This was strongly associated with the decrease of cinnamic acid. Flavonoids, as an effective abiotic elicitor, not only showed significant positive correlations with seed yield but also act as photoprotective compounds and antioxidants (Mao et al ., 2017; Middleton & Teramura, 1993). To this end, flavonoids may play an important regulatory role in the seed yield rather than protecting plants against elevated [O3].
Previous studies reported that O3 entered leaves of plants primarily via stomata, and then generated reactive oxygen species (ROS), such as superoxide O2.-, single oxygen, hydroxyl radicals and hydrogen peroxide in plant cells, which can destroy the structure of DNA, proteins, lipids and carbohydrates due to oxidation capacity (Vaultier & Jolivet, 2015). In the study, O3 induced significant increase of H2O2 in rice and soybean. In order to resist O3-induced oxidative stress, plants have elvolved various mechanism to avoid detrimental reactions, such as motivate activities of antioxidant enzymes (Vendruscolo et al ., 2007). The activities of SOD and POD have been considered as indicators for eliminating oxidative stress and scavenging ROS (Vendruscolo et al ., 2007). Our data demonstrated O3 up-regulated activities of SOD, POD and CAT in rice, suggesting rice utilized high activities of these antioxidant enzymes as a strategy to detoxify O3-induced stress. However, activities of POD and CAT reduced in soybean but H2O2 was increased with great extant after O3 exposure. This suggests that the O3-induced oxidative stress is beyond the ability of this antioxidant enzyme to detoxify ROS (Biswas et al ., 2008). The elevated SOD activity was accompanied with decreased activities of POD and CAT in wheat, indicating these antioxidants can compensate for each other in removing ROS toxicity.
Damage of mitochondrial respiration generated ROS when plants were fumigated by  O3 (Dizengremel et al ., 2012). NADH, which is produced by the photorespiration-associated glycine decarboxylase (GDC) in the mitochondrion, induces retroinhibition on the decarboxylating enzymes of the TCA cycle (Igamberdiev & Gardeström, 2003). The TCA-cycle is one of the important metabolic pathways (Dizengremel et al ., 2012), which aims to produce energy to sustain plant growth. In this study, TCA cycle was impaired in both rice and soybean under O3 stress. However, the intermediate product changes were not consistent in rice (isocitrate) and soybean (citrate and 2-oxoglutarate). Moreover, the expression of genes coding citrate-synthase and isocitrate dehydrogenase was down-regulated in soybean treated with elevated O3, providing a reasonable explanation for the reduced accumulation of citrate and 2-oxoglutarate. Meanwhile, the expression of genes coding citrate (Si)-synthase in rice was significantly enhanced by O3. This increased citrate may be largely converted to isocitrate in rice. The change in TCA-cycle intermediates in the two species may serve as an energy conservation fashion to cope with O3 stress. Moreover, it also indicates that the soybean as dicot and rice as monocots may evolved different strategies to minimize the adverse effects of O3 stress. Subsequently, these changes lead to the high level of aspartate-derived asparagine or aspartate in wheat, rice and soybean. However, the elevated expression of genes coding aspartate transaminase might create a negative feedback loop in this metabolic pathway. Previous studies proved that asparate is the central regulator between carbon and nitrogen metabolism (Less & Galili, 2008). Carbon and nitrogen are the two most essential elements for plant growth and development, especially for crop productivity and quality. Therefore, coordinted carbon and nitrogen metabolism regulates plant development and metabolic in responding to varied environmental conditions (Kang & Turano, 2003). In this study, the significant O3-induced accumulation of aspartate implies that maintaining cellular carbon/nitrogen balance was an important metabolic mechanism and these amino acids are important for the adaptation of the plants to O3 conditions. Aspartate-semialdehyde dehydrogenase is a control point in isoleucine, methionine, lysine, and threonine synthesis (Schroeder et al ., 2010). In the present study, the production of methionine in rice was significantly enhanced. Pyrimidines converted by glutamine were elevated in rice and wheat rather than in soybean, which coincied with the SOD, POD and CAT facilitating the regeneration of redox ascorbate and glutathione metabolites (Foryer & Noctor, 2000). Arginine acts as a storage form of organic nitrogen due to its highest nitrogen/carbon ratio. It has been reported that accumulation of arginine attenuated oxidative stress in higher plants under salinity stress (Qados, 2010; Winter et al ., 2015). In this study, arginine in soybean and derived-arginine agmatine in wheat were strongly enhanced under elevated [O3]. These findings indicate a high demand for nitrogen release during O3 stress.
Collectively, the metabolic response of plants to O3stress is currently receiving more attention. This is driven by global crop supply facing challenge from continuous growth of population, agricultural land lost and climate change, especially O3pollution (Stocker et al ., 2013). Our current data suggests that elevated [O3] induces a wide range of changed metabolites in three species which contribute to avoiding O3 stress. It is also found that oxidative damage induced by O3 leads to the change of activity in key metabolic enzymes of different species. Together, these results have direct implications for different crop improvement strategies. Ultimately, understanding how environmental O3 affects metablolic pathways in different plants provides theoretical foundation for improving agronomic traits of crop plants under the environment of global climate change, specially O3 pollution.