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Ozone occurs at low concentrations in the troposphere where the natural sources are lightning strikes, the incursion of small amounts of stratospheric ozone and the interaction of hydrocarbons released by plants with nitrogen oxides and sunlight. But ozone is mostly generated anthropogenically in the troposphere and there is evidence of a global increase in background concentrations of this gas (Bytnerowicz et al., 2007). The phytotoxic effect of ozone was first discovered in the 1950s in the Los Angeles basin (Haagen-Smit et al., 1952). The harmful impact of mild chronic ozone exposure on higher plants has been highlighted in recent reviews (Ashmore, 2005; Karnosky et al., 2007; Renaut et al., 2009; Wittig et al., 2009). Plants exposed to realistic concentrations of ozone over long periods of time (chronic ozone exposure) show a range of successive modifications, leading to a reduction in growth rate, yield and biomass, possibly combined with hastened senescence (Matyssek & Sandermann, 2003). Cellular metabolism is affected early with a reduction of photosynthesis (Reich, 1983; Dizengremel, 2001; Heath, 2008). Gaseous ozone enters the leaf through the stomata and generates rapidly reactive oxygen species (ROS) such as , H2O2 and OH˙ (Langebartels et al., 2002). Plant detoxification systems, present in the apoplasm, the cytosol and the organelles, contribute to the scavenging of ROS, but a negative impact on photosynthesis can still occur (Pell et al., 1992; Dizengremel et al., 1994; Dizengremel, 2001; Le Thiec & Manninen, 2003; Bagard et al., 2008; Heath, 2008). An increase in catabolic processes occurs simultaneously, which allows detoxification processes to remain active through the supply of reducing power (Dizengremel, 2001; Dizengremel et al., 2008; Gillepsie et al., 2011). In response to ozone, a large part of carbon skeletons is also diverted towards pathways leading to the synthesis of secondary compounds (flavonoids, phenolic compounds, lignins and so on) putatively used for defence (Kangasjärvi et al., 1994; Booker & Miller, 1998; Dizengremel, 2001; Cabanéet al., 2004; Kontunen-Soppela et al., 2007; Castagna & Ranieri, 2009). In plants emitting volatile organic compounds, such as isoprenoids, part of the carbon could be redirected towards their production for protection against the ozone-induced damage (Loreto & Velikova, 2001; Vickers et al., 2009). Focusing on phosphoenolpyruvate (PEP), which is in decreased supply and increased demand under ozone exposure, could provide an insight into a metabolic node that influences a number of downstream processes important in plant responses to ozone. PEP is at the origin of four major metabolic routes (Fig. 1): (1) mitochondrial respiration (associated with pyruvate kinase), a process that carries out the breakdown of organic acids (mostly pyruvate) and the production of ATP by using NADH-producing processes and the respiratory chain; (2) isoprenoid formation in chloroplasts through the methylerythritol 4-phosphate (MEP) pathway, starting with the condensation of pyruvate and glyceraldehyde 3-phosphate; (3) the shikimate–phenylpropanoid pathway, starting in chloroplasts through the condensation of PEP with erythrose 4-phosphate to give aromatic amino acids, precursors of phenolic compounds and monolignols; and (4) the anaplerotic pathway starting with PEP carboxylase (PEPc) activity. This last pathway, which could be favoured in the light (Tcherkez et al., 2011), would serve to replenish the TCA cycle through the input of OAA and/or malate. A noncyclic functioning of the TCA cycle would ensue (Sweetlove et al., 2010), allowing the subsequent use of tricarboxylic acids (citrate and/or oxoglutarate) for further amino acid synthesis (O’Leary et al., 2011; Werner et al., 2011). These four routes are not always present together in plants since many, especially herbaceous plants, do not produce isoprenoids (Sharkey et al., 2008). Several reviews on the effects of ozone on plant metabolism have been published recently, including results stemming from ‘omics’ tools (Bohler et al., 2007; Cho et al., 2008, 2011; Heath, 2008; Renaut et al., 2009; Galant et al., 2012), but a comprehensive review of the physiological integration of the changes in carbon allocation is still lacking, especially in isoprene-emitting plants.
In this letter, we want to draw attention to the problems emerging from the source and the fate of PEP in oxidative stress conditions. In particular, owing to the decrease in recently assimilated carbon, the question arises as to how PEP can fulfil the increased demand in carbon skeletons. An imbalance between PEP production and use could lead to a shift from homeostasis towards death. In this context, the relative importance of the different routes employing PEP will be explored, as well as the sequence of their utilization under ozone exposure.
Ozone-mediated metabolic changes
A survey of published papers shows that ozone generally causes an increase in each of the four pathways (1–4) described in the previous section (Fig. 1) with which PEP is connected:
2 The MEP pathway Following ozone exposure, the use of PEP is amplified for the synthesis of isoprenoids in isoprene-emitting plants (Valkama et al., 2007). Endogenous isoprene biosynthesis is known to play an antioxidant role (Loreto & Velikova, 2001; Vickers et al., 2009) and/or to prevent photosynthetic electron transport damage (Magel et al., 2006). Isoprene synthesis has been shown to be inversely correlated to PEPc activity and positively correlated to the rate of mitochondrial respiration (Loreto et al., 2007). These results, however, were obtained mainly by varying the CO2 concentration, and the effects of ozone exposure have not been directly assessed.
3 The shikimate and phenylpropanoid pathways Ozone elicits increases in gene transcription and activity of a large number of enzymes of the chloroplast shikimate pathway and of the phenylpropanoid pathway, leading to the production of flavonoids, phenolic compounds and monolignols (Toumainen et al., 1996; Booker & Miller, 1998; Cabanéet al., 2004; Betz et al., 2009). The activities of shikimate dehydrogenase, phenylalanine ammonia-lyase (PAL) and cinnamyl alcohol dehydrogenase are enhanced by ozone, leading to a slight increase in the amount of lignins and to changes in their monomeric composition (Cabanéet al., 2004; Betz et al., 2009).
4 The anaplerotic pathway This pathway is increased following ozone stress linked to a large increase in the activity of PEPc. In C3 plants, RuBisCO is the carboxylating enzyme for carbon assimilation, whereas the cytosolic PEPc is thought to participate in anaplerotic CO2 fixation and the recapture of respiratory CO2 (Melzer & O’Leary, 1987). In the presence of ozone, a decrease in RuBisCO activity is commonly observed, while a strong increase in the activity of PEPc also occurs (Landolt et al., 1997; Fontaine et al., 1999; Dizengremel, 2001; Dalstein et al., 2002; Renaut et al., 2009). Gene expression studies have shown that photosynthesis-related genes were repressed, whereas genes related to catabolic processes were enhanced (Dizengremel, 2001; Cho et al., 2008; Heath, 2008; Renaut et al., 2009). OAA produced from PEPc can be transported into the mitochondrion or produce malate in the cytosol. Malate can thus produce pyruvate through the cytosolic NADP-malic enzyme, providing additional NADPH for detoxification purposes (Fig. 2). In addition, the transfer of OAA and/or malate into the mitochondrion could feed the so-called anaplerotic pathway and could lead subsequently to different pathways for amino acid synthesis (Fig. 2). In illuminated leaves, citrate exported from the mitochondrion is thereafter transformed to oxoglutarate in the cytosol via isocitrate dehydrogenase (IDH)) with the production of NADPH as a result (Fig. 2). Oxoglutarate can also result from the mitochondrial NAD-dependent IDH activity and can be exported towards the cytosol. Two different nonexclusive pathways could occur thereafter (Fig. 2). Oxoglutarate may enter the chloroplast to be integrated into the glutamine synthetase 2 (GS2)/glutamate oxoglutarate aminotransferase (GOGAT) cycle to give glutamate. Conversely, oxoglutarate may give glutamate in the cytosol by aspartate aminotransferase, and glutamate could then be aminated to glutamine by glutamine synthetase 1 (GS1) (Fig. 2). To cope with a decreased availability as a result of the ozone-decreased photorespiration, the required ammonium may come from phenylalanine (shikimate pathway) or from protein degradation. Within the mitochondrion, oxoglutarate can also be converted into glutamate by glutamate dehydrogenase (GDH). Glutamate, exported from the mitochondrion, can be converted by glutamate decarboxylase (GAD) to gamma-aminobutyric acid (GABA) (Fig. 2). An increase in both GDH and GAD has been demonstrated in ozone-treated rice leaves (Cho et al., 2008). Increased concentrations of GABA are known to occur in response to stress and could represent an adaptive mechanism, through the GABA shunt, to maintain the rate of respiration (Araujo et al., 2012). Recent results (Bagard, 2008; Galant et al., 2012) have shown an increased activity of the cytosolic glutamine synthetase isoform GS1 in leaves of poplars and soybeans fumigated with ozone. This activity of GS1 is often linked to senescence (Masclaux et al., 2001) and is also concomitant with an increased PAL activity (Bernard & Habash, 2009). In the case of ozone exposure, the increased activity of GS1 could possibly be devoted to the reassimilation of ammonia released from increased PAL activity and also from proteins denatured by the hastened senescence process. Mobile glutamine could be further transported from the senescent leaves to the whole plant. Ammonia is also well known to increase both the transcripts and the activity of PEPc, the enzyme at the origin of this anaplerotic pathway (Scheible et al., 1997). It is well known that changes in the activities of PEPc and GS are positively correlated (Britto & Kronzucker, 2005). Under ozone stress, the increased ammonium reassimilation by GS could concur with an increase in the anaplerotic role of PEPc to support the demand in organic acids, substrates for N assimilation.
How to reconcile PEP needs and supplies?
We propose an analysis of the two crucial problems raised under ozone exposure: on the one hand the increased requirement in PEP for feeding the different pathways allocating carbon, and on the other hand, the capacity to replenish this metabolite (Fig. 1). The processes described earlier increase in the four pathways and raise the question of how they are regulated. Surprisingly, the particular role of PEP is scarcely studied, whereas the regulation of glycolysis, which gives a prominent role to pyruvate kinase, and that of the Krebs cycle (Araujo et al., 2012) have been deciphered.
Beyond unravelling the control of the portioning of PEP between all of these pathways, the problem of supplying sufficient PEP must be raised. In the presence of light, photosynthetically generated triose phosphate is the source of PEP through glycolysis. Upstream of PEP, the removal of carbon skeletons for the pentose phosphate pathway is increased under ozone fumigation, as demonstrated by an increased glucose-6-phosphate dehydrogenase activity (Dizengremel et al., 1994, 2009; Gaucher et al., 2006). The NADPH provided by the pentose phosphate pathway is very useful for detoxification, and significant carbon is thus recycled but at the expense of carbon available for producing PEP through glycolysis. Knowing that the total amount of available carbohydrates will progressively decline, as a result of the decrease in photosynthesis (Calvin cycle) following ozone exposure, the enhanced sink strength, linked to the strong demand in the four defence/repair processes described earlier, could ultimately lead to an unavoidable disequilibrium and to cell death. The main question is thus to know how it is possible to provide sufficient PEP to allow the further PEP-consuming pathways to function as efficiently as possible for both saving carbon and ensuring the correct functioning of the defence pathways.
Aside from photosynthetically derived carbon skeletons, another source of PEP, as demonstrated for isoprene biosynthesis, could be carbon compounds transported from xylem and/or derived from starch and other carbon-containing compounds present in the leaves (Schnitzler et al., 2004). It is well known that starch and sucrose concentrations in leaves are lowered under ozone exposure along with a stimulation of carbohydrate catabolism (Einig et al., 1997). Finally, the possibility of producing PEP directly from pyruvate by using pyruvate phosphate dikinase must also be evoked since this activity has been shown to occur in illuminated leaves (Tcherkez et al., 2011) and could be enhanced under stressful conditions (Doubnerova & Ryslava, 2011), even though it has never been demonstrated under ozone fumigation.
Roughly speaking, in terms of carbon balance at the leaf level, mitochondrial respiration in the light, linked to PK and the TCA cycle, reaches no greater than 15% of CO2 fixation (Bagard et al., 2008). Based on the activity of PEPc, the carbon flux through PEPc and the anaplerotic pathway could represent nearly 5% of CO2 fixed by photosynthesis (Tcherkez et al., 2012). The MEP pathway in isoprene-emitting plants is only c. 2% of photosynthetic CO2 fixation (Sharkey et al., 2008). The main flux of carbon is incorporated into the shikimate pathway, accounting for up to 25% of CO2 fixation (Amthor, 2003). Under ozone exposure, the MEP pathway would remain minor (Loreto et al., 2007) whereas, based on the degree of stimulation of enzyme activities (Cabanéet al., 2004; Dizengremel et al., 2009), we can estimate that there is a general increase in the carbon flux through the three other pathways. The factor of stimulation would be 1.5 for the shikimate–phenylpropanoid pathway (Cabanéet al., 2004), 2.5 for the light respiration and 3.5 for the anaplerotic pathway (Dizengremel et al., 2009). The respective percentages of carbon flux could thus be estimated at c. 35% in the shikimate pathway and between 20 and 25% in the two other pathways. As the fraction of the photosynthates devoted to the pentose phosphate pathway is also enhanced under ozone stress (× 1.5; Dizengremel et al., 2009), the available pool of PEP could be rapidly depleted, leading to insufficient energy and carbon compounds needed for cell life. Clearly, these estimations need to be refined in future research.
It is nevertheless possible to mitigate a disastrous scenario concerning the enhanced consumption of PEP, considering that the metabolic events caused by chronic ozone exposure may not occur simultaneously according to the cumulative ozone dose (Cho et al., 2008; Heath, 2008). Attempts to define the succession of metabolic events in illuminated leaves could give the following order:
• The decrease in the biochemical step of photosynthesis (RuBisCO) and the increase in respiration (glycolysis coupled to the TCA cycle) could occur rapidly after ozone exposure and be concomitant with isoprene biosynthesis.
• The phenylpropanoid pathway increases rather rapidly, with synthesis of aromatic acids, various flavonoids and phenolic compounds. The concomitant increase in PEPc could be linked to the production of NADPH for detoxification and to the anaplerotic pathway associated with the GS2/GOGAT system.
• Until the onset of senescence, the maintenance of high PEPc activity could serve to provide organic acids for amino acid synthesis, in parallel with the increased GS1 activity. The NH4 would come from phenylalanine and damaged proteins.
In illuminated leaves, the central role of PEP in allocating carbon to pathways implicated in cellular respiration and anabolic processes is enhanced in response to ozone. It is of crucial interest to determine how PEP partitioning is regulated during ozone exposure, as photosynthetic carbon assimilation declines and other carbon sources could become available. The proposed cascades of the different steps would allow sufficient PEP to meet the various demands set against the increasingly damaging problems linked to continuous ozone uptake. Inhibition of photosynthesis leads to a decreased carbon provision and the reduction in the different pathways downstream of PEP leads to a decrease in the scavenging power against ROS. The balance between the supply and the use of PEP is thus of upmost importance for estimating the life span of leaf cells exposed to ozone.
This work was supported by the French Agence Nationale de la Recherche project Vulnoz ANR-08-VULN-012.