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.