The effect of OzT8 on photosynthesis in rice under ozone stress
A previous ozone tolerance screening of various rice cultivars and subsequent QTL mapping study identified several QTLs associated with higher or lower ozone tolerance (Frei et al. 2008). A subsequent work has confirmed and characterized two of these QTLs (OzT9 and OzT3), which are associated with higher and lower leaf bronzing under ozone stress (Frei et al. 2010). However, this study is the first to confirm the dry weight effect of the QTL OzT8 through the SL SL46, and found that this effect is accompanied by a higher relative midday photosynthetic carbon assimilation rate under ozone stress (Fig. 1; Table 1). Although the photosynthetic rate in ozone was higher in SL46 than in NB, stomatal conductance showed no significant difference between the two genotypes (Table 1). In fact, in some instances, it appeared that stomatal conductance under ozone stress was higher in SL46 than in NB (Fig. 4b). Therefore, we can confidently reject the hypothesis that OzT8 confers ozone tolerance via stomatal closure and avoidance of ozone.
However, the hypothesis that OzT8 is associated with higher biochemical photosynthetic capacity under ozone stress was confirmed (Fig. 2). Therefore, the results here show that the underlying mechanism of ozone tolerance associated with the OzT8 locus is relative maintenance of leaf photosynthetic capacity and daily carbon assimilation rate while under ozone stress.
The comparison of midday photosynthetic rate, stomatal conductance and chlorophyll fluorescence at days 5 and 23 of the ozone fumigation (Tables 1–3) showed a notable difference between leaves that had developed prior to onset of fumigation, and leaves that had developed during the treatment. Although there was a significant decrease in photosynthetic rate and stomatal conductance under ozone in both genotypes at day 5, there was no treatment effect for ΦPSII or its component fluorescence ratios (Table 2). However, for leaves that developed and grew under constant ozone stress, the ΦPSII was significantly decreased in NB, but not in SL46. This was a direct result of a significant decrease in qP in NB, which is non-proportionally related to the oxidation state of QA, that is, processes downstream of the PSII reaction centre. This result points towards the presence of ozone effects on Rubisco carboxylation capacity and the Calvin cycle, and is confirmed by a large observed decrease in Vc,max in NB (Fig. 2). This is consistent with the hypothesis in the literature that one of the major impacts of chronic [O3] is its negative effect on Rubisco (Farage & Long 1999; Goumenaki et al. 2010).
Leaves that developed under control conditions and were exposed to ozone responded differently than leaves that had developed under ozone-stressed conditions. This suggests that care should be taken when conducting experiments using only short-term ozone fumigations (e.g. several hours for a single day), because some effects may not appear within such a short treatment period. Other studies have also found that different physiological mechanisms occur in response to ozone at different timescales (Chen, Frank & Long 2009).
The analysis of the theoretical CO2 response curves indicated that stomatal limitation for both genotypes was similar under ozone stress (Table 4). This further makes the case that the higher midday photosynthetic rates in SL46 over NB under ozone stress could be attributed primarily to its higher photosynthetic capacity (Vc,max and Jmax) under stress conditions, and not caused by any differences in stomatal response.
If resistance to ozone stress in SL46 cannot be attributed to differences in ozone uptake into the plant, what are other possible reasons? Previous research has suggested that differences in antioxidant and radical-scavenging capability, especially in the apoplastic space, may confer resistance to ozone effects (Conklin & Barth 2004). Another possibility is that the defence response and signalling pathways that typically accompany ozone stress are altered, resulting in lower levels of key stress hormones such as ethylene, which would then limit the defence response-induced oxidative stress and foliar injury, as well as acceleration of senescence and down-regulation of Rubisco (Vahala et al. 2003; Kangasjarvi et al. 2005).
Under control conditions, the Vc,max and Jmax of SL46 were slightly lower than those of NB (Fig. 2); stomatal conductance was also lower in SL46 at some time-points (Fig. 4). This could potentially be attributed to a trade-off between stress tolerance and maximum achievable productivity, but could also be caused by other Kasalath chromosome inserts in SL46 that are not associated with ozone stress tolerance. However, a comparison of the grain yield between NB and SL46 under non-stressed conditions showed no significant difference at the end of the growing season (Chen et al. in press). Future fine-mapping of the Kasalath inserts in SL46 via additional SL46/NB crosses will be able to clarify this issue. Regardless, the results of this current study indicate a strong potential for identification of novel genes associated with increased ozone tolerance in rice within the OzT8 locus.
The sequence of ozone effects on photosynthesis in rice
Some previous literature has suggested that stomatal response to ozone is mediated via changes in the mesophyll, that is, the decrease of stomatal conductance under ozone stress follows the decrease in photosynthetic biochemical capacity. This hypothesis was confirmed in oak under acute ozone stress conditions, and in wheat and pea plants exposed to either acute or chronic ozone conditions (Farage & Long 1995, 1999). In particular, it was found that decreases in Rubisco carboxylation capacity preceded any changes in stomatal conductance or other photosynthetic components.
However, the results of this study indicate that the short-term response to ozone in the case of rice is fundamentally different. Stomatal conductance in rice plants responded within 3 h of exposure to a moderate concentration of ozone and precluded the decrease in steady-state photosynthetic rate (Fig. 4). This result implies that the plant was able to sense the presence of ozone and respond by closing its stomata even before the photosynthetic capacity in the mesophyll had been affected. Similar results have been found in the rice cultivar Koshihikari, whose stomatal conductance showed a significant decrease within even the first hour of exposure to ozone (Imai & Kobori 2008). This rapid closure of stomata upon exposure to even moderate levels of ozone may prevent a significant amount of ozone from entering into the leaf intercellular space in the field, and may provide a partial explanation for why rice has been found to be relatively tolerant to ozone compared to other agricultural crop species. However, this tolerance mechanism is only viable in the short term. If the plants are exposed to high ozone concentrations on a daily basis and react by consistently decreasing their stomatal conductance, the photosynthetic rate and long-term growth or biomass accumulation will suffer. Therefore, they must develop some mechanism of acclimation or biochemical tolerance to allow the stomata to remain open enough to conduct the gas exchange necessary for photosynthesis without accumulating additional damage from ozone. The exact mechanism for this long-term tolerance is not yet clear; however, the results of this study suggest that the QTL OzT8 may confer this type of ozone tolerance.