O3 effects on k and RGR
The meta-analysis allowed a quantitative re-evaluation of studies that may have suggested biologically meaningful effects of O3 on k or RGR, but lacked the statistical power to document significant differences (Bailar 1997). In the data available for the present analysis, only about 25% of observations and 50% of individual plant species exhibited a significant effect of O3 on k in the original studies. Of these, 74% showed that k declined. When all available data were pooled, 55% of observations reflected a reduction in k.
The meta-analysis demonstrated that mean k declined significantly by 5.6% (P < 0.05) following O3 exposure. The herbaceous dicots, mostly juvenile, that dominated the available data (74% of observations) exhibited a 2.6-fold smaller response than monocots, and a 7.7-fold smaller response than trees. The modest response of this over-represented plant group reduced the overall mean effect size determined by the meta-analysis.
The response in trees differed significantly from the response in dicots but, despite its large magnitude, was not itself significantly different from zero using the conservative, non-overlapping CI method (Waltz 2006). In the context of the meta-analysis, this may simply reflect the small number of observations for trees (a third of those available for monocots and less than a tenth of those for dicots). Yet, even among the original studies, there were few significant observations of O3 impacts on k. This must reflect a large variability relative to a small sample size in these studies, and may reflect the inherent difficulties of resolving the effects on growth and allocation in trees, perhaps because of longevity, woodiness or large plant size. In trembling aspen (Populus tremuloides), the sensitivity of gas exchange parameters to O3 varied with clone, leaf age and canopy position of same-aged leaves (Karnosky et al. 2005), and in sugar maple (Acer saccharum) with tree age, shading and (tree age × shading) (Chappelka & Samuelson 1998). In the latter species, shading enhanced O3 sensitivity in mature trees (Tjoelker et al. 1995), but not young trees (Laurence et al. 1996).
Despite the conservative estimate of O3 impact provided by the dominant herbaceous dicots, and the conservative test used to document this significant effect, the data do not support uncritical generalization nor prediction of O3 effects on allocation in specific instances. The diversity among plant groups was large and many changes in k were small, with 13% less than ± 0.03 in LRRk. This may reflect the relatively moderate O3 imposed in most available observations (Table 1), because a recent meta-analysis suggested that root growth was mostly inhibited when O3 exceeded 120 p.p.b. (Wittig et al. 2005).
The available data indicate a significant, but variable, reduction of k caused by O3. The present analysis did not isolate effects caused by plant age, growth habit or environmental conditions on O3 sensitivity of k. These, along with methods of scaling O3 impacts from seedlings to mature trees, remain important research questions.
Plant growth rate was also significantly reduced by O3, by 8.2% over all observations. This larger mean effect on RGR than on k was consistent with the detection of significant reductions in RGR in 50% of observations and 50% of species. The reductions were found in 89% of all observations. Observations that resolved O3-induced reductions were thus more common for RGR than for k.
The O3 impact on RGR, as for k, was largest in trees. The effects in trees and dicots were significantly greater than in monocots, but did not differ from each other. The large effects observed in trees suggest the potential for biologically meaningful O3 effects under ambient conditions. Even small O3 effects on growth or allocation in perennial species may impose large cumulative constraints on growth over several seasons (Retzlaff, Williams & DeJong 1997b). This is supported by simulation studies of long-lived trees (Retzlaff, Williams & DeJong 1992; Retzlaff et al. 2000). These O3 impacts on growth and allocation below ground may influence predictions of the biological effects of climatic change and the efficacy of proposed carbon sequestration schemes.
Although O3 impacts on RGR were larger and more consistent than on k, the significant observations were surprisingly much less uniformly distributed among the plant groups. These ranged from 0 to 63% of observations among the plant groups for RGR, compared with 22–38% for k. The herbaceous dicots exhibited a larger proportion of significant reductions and a larger mean effect than monocots, the reverse of the case for k. About 25% of all observations of changes in RGR were within ± 3% of the range, compared with only 13% in k. The meta-analysis confirmed the well-accepted impact of O3 on plant growth, but even in this case, generalizations may prove problematic because of variability among plant groups and experiments. Predictions of O3 impacts in specific cases may be more reliable for RGR than for k.
Carbohydrate allocation has some inherent limitations as a biological end point that may contribute to the difficulty of generalizing O3 impacts. The determination of root mass is confounded experimentally by unavoidable losses during separation from soil or growth medium. Relative losses may be large in the seedling experiments that constitute the bulk of available observations, because the young shoot dominates plant biomass and calculations of RGR. Furthermore, allometric relationships such as k are strongly conserved (Causton & Venus 1981; Hunt 1990; Farrar & Gunn 1998; Gunn et al. 1999) and may be more resistant than growth to perturbation by O3. Finally, whole-plant acclimation to O3 may obscure the effects on both RGR and k, through compensatory leaf initiation and enhanced gas exchange of young leaves (Pell, Eckardt & Enyedi 1992; Farage & Long 1995; McCrady & Anderson 2000). These limitations of allocation as an end point are not limited to documentation of O3 impacts. In studies of both root–shoot competition (Cahill 2002) and of shading (Hunt & Cornelissen 1997), it has proven more difficult to document the effects on allocation than on growth.
Shading and O3 exposure are analogous environmental perturbations. Both interact directly with plants only at the shoot and affect assimilation and source strength as well as carbohydrate allocation to sink tissues. The effects of O3 documented here are contrasted with the effects of shading reported for 28 species of seedlings by Hunt & Cornelissen (1997). No attempt is made to review the literature on shading. Shading [photosynthetic photon flux density (PPFD) of 125–135 versus 250 µmol m−2 s−1] significantly reduced k in 83% of observations with herbaceous dicots and 63% in herbaceous monocots (Hunt & Cornelissen 1997). The mean effects of shading in dicots were 8.4% for k and 20.3% for RGR, larger than the effects in monocots of 7.4% for k and 14.5% for RGR (calculated from data presented by Hunt & Cornelissen 1997). In addition to the larger mean effect on RGR, a smaller proportion of observations of RGR than of k yielded no detectable response (there were none for RGR). The mean effects of shading, as for O3, were larger, more frequent and more easily documented for RGR than for k.
Other impediments to documenting O3 effects on allocation are more specific to studies of O3 impacts. Some variability is related to experimental variability in O3 exposures. For example, exposures that track stochastic ambient concentrations will vary between experiments. Uncertainties in the rates of O3 transport to the leaf interior and in the rates of biochemical detoxification of O3 make it difficult to quantify the effective biological dose of O3, even when reproducible O3 exposures can be imposed (Lefohn 1992; Massman 2004). Furthermore, these physiological factors are modified by environmental conditions in poorly characterized ways.
In a survey of non-managed vegetation in Ohio (Showman 1991), less O3 injury was observed in a year with high ambient O3 than in a similar but rainier year with lower O3. This probably reflected a reduced biological dose, as stomatal uptake of O3 would be expected to decline with lower soil moisture and humidity in the drier year. Temperature increases of 1–3 °C, commonly associated with OTCs and climatic change, altered CO2 effects on the yield of wheat (Triticum aestivum) (Van Oijen et al. 1999) and may be shown in the future to interact with O3. Increases in root : shoot ratio of radish (Raphanus sativus), induced by root chilling from 18 to 13 °C, were reduced by exposure to O3 (Kleier, Farnsworth & Winner 2001). Factors such as soil type and pot size have not been found to influence O3 effects (Taylor et al. 1986; Whitfield, Davison & Ashenden 1996; Booker et al. 2005), but merit further scrutiny.
The uncertainty in resolving O3 impacts on biomass allocation has been associated with experimental and environmental variabilities, and with real and reproducible biological differences in the sensitivity of k to O3. The nearly eightfold range of sensitivities observed among the plant groups in the present analysis probably reflects all of these. A more robust generalization of O3 impacts on biomass allocation below the ground will require experiments over a broad range of plant types and environmental conditions, specifically designed to characterize this heterogeneity.
Towards the mechanism of O3 action
The mechanism by which biomass allocation in plants is regulated remains unknown, in general, and in response to environmental perturbations such as shading, soil water deficit and mineral deficiency, as well as exposure to O3. The diversion of biomass to shoot growth demonstrated here is believed to facilitate plant defense against reactive oxygen species generated from aqueous O3 (Lee & Bennett 1982; Mehlhorn et al. 1986; Sandermann 1996) and ultimately to enable repair of O3-induced foliar wounding (Barnes 1972; McLaughlin & Shriner 1980; Amthor & Cumming 1988; Sandermann 1996; Grulke & Balduman 1999). These responses of allocation are also consistent with a relaxed interpretation of the ‘theory of functional equilibrium’ (Brouwer 1983; van Noordwijk et al. 1998; Poorter & Nagel 2000). O3 induces compensatory shoot growth in response to reduced CO2 availability to the shoot, caused by a reduced ability to harvest CO2, even though absolute resource availability remains unchanged. Whatever the mechanism, O3 induces shoot growth at the expense of root growth, despite an unchanged resource availability in the rhizosphere.
The often distinct responses of k and RGR to O3 provide some insights into the possible mechanisms of O3 action in the whole plant. For example, the sensitivities of k and of RGR to O3 were not correlated. In 27% of all observations, O3 altered k without significantly affecting RGR. This implies that O3 altered allocation to roots without suppressing the productivity or the photosynthetic substrate availability. Such a separation of effects appears to be consistent with the inhibition of translocation of photosynthetic products rather than with substrate limitation caused by the inhibition of photosynthesis. This could reflect a direct inhibition of translocation, as previously suggested (Grantz & Farrar 2000), or a programmed restoration of functional equilibrium mediated by yet unknown controls.
The sensitivity of k to O3 was significantly related to RGR in the absence of O3 exposure. Reductions in k caused by O3 were most pronounced in slow-growing plants, while rapidly growing plants exhibited less negative or even positive changes in k. This decreasing inhibition of k by O3 with increasing RGRcontrol suggests that the sensitivity of k to O3 was not mediated by the stomatal flux of O3, as stomatal conductance and carbon assimilation would likely be correlated with the rate of plant growth. There was a strong positive relationship between k and RGR, when both were determined in the presence of O3, but no significant relationship under control conditions. In the shading studies of Hunt & Cornelissen (1997), herbaceous monocots and trees (woody species) exhibited consistent positive relationships between k and RGR, but the sensitivity of k to shading increased with increasing RGRcontrol. The resource ratio model (Tilman 1988), predicting that faster-growing plants would invest resources in photosynthetic tissues at the expense of roots, was not supported by the analyses of O3 (here) or shading (Hunt & Cornelissen 1997). Maintenance of RGR in the presence of O3 is a clear manifestation of O3 resistance, and the strong correlation between kozone and RGRozone implies that such resistance is also reflected in the maintenance of allocation to roots.
The current meta-analysis indicated that the sensitivity of RGR to O3 was not significantly related to RGRcontrol. In contrast, the sensitivities of RGR to elevated CO2 and to shading were both positively related to RGRcontrol (measured at ambient CO2) (van Noordwijk et al. 1998) or at higher PPFD (Hunt & Cornelissen 1997). A similar positive relationship has been observed previously for O3 (e.g. in mixed pasture species) (Bungener et al. 1999) and in mixed forest species (Karnosky et al. 2005). In the latter case, rapidly growing pioneer species (trembling aspen; paper birch, Betula papyrifera) were more responsive than slower-growing species (sugar maple, A. saccharum).
On the other hand, in cotton, leaf soluble carbohydrates increased following O3 exposure (Grantz & Farrar 2000), and short exposures (0.75 h) to O3 inhibited phloem loading and carbohydrate export more severely than photosynthesis (Grantz & Farrar 1999). Longer exposures altered allocation differently than did source limitation induced by partial defoliation (Grantz & Yang 2000). These data are consistent with a primary effect on phloem loading and secondary feedback inhibition of assimilation, perhaps as one of multiple targets of O3 attack.
Exposure to O3 has been shown to reduce carbohydrate export from source leaves of Plantago major (Zheng et al. 2000), to reduce the amount of 14C transported to roots of clover (Trifolium repens) (Blum, Mrozek & Johnson 1983), and at higher concentrations to nearly abolish the export of recent photosynthate from source leaves of cotton (Grantz & Farrar 1999). Compartmental efflux analysis indicated that phloem loading from the cytoplasmic transport pool was inhibited, whereas exchange with the vacuolar storage pool was unaffected (Grantz & Farrar 2000). The velocity of the transport of sugars within the phloem, in contrast, was unaffected in both loblolly pine (Spence, Rykeil & Sharpe 1990) and wheat (Mortensen & Engvild 1995).
O3 impacts on k do not appear to be obligately linked to O3 effects on RGR or on photosynthesis at the leaf or whole-plant scale. Inhibition of photosynthetic processes may be inadequate to fully explain the range of responses to O3 observed in whole plants and communities.
The lower leaves act as preferential sources of assimilate for roots, and the upper leaves for shoots (Wardlaw 1968; Thorpe, Walsh & Minchin 1998). Both O3 and shading have greater effects on older, lower leaves than on younger, more exposed leaves (Nie, Tomasevic & Baker 1993; Soja & Soja 1995; Mulholland et al. 1997). Accelerated senescence and reduced phloem loading in the lower canopy could each contribute to reduced allocation to roots. A simple model of phloem transport (Minchin, Thorpe & Farrar 1993) could account for the effects of shading and O3 on allocation. The flux of carbohydrate along two paths, and thus, the allocation of carbohydrate between two non-equivalent sinks, is shown to be altered by changes occurring only in source strength. If root and shoot are non-equivalent sinks, changes in k following both shading and O3 exposure could be caused by a reduced flux of carbohydrate from the source leaves. However, the mechanisms of source strength reduction could differ.
Effect of exposure technology
The current meta-analysis found no significant effects of the various exposure technologies represented in the available data on the O3 sensitivity of either k or RGR. Both CECs and OTCs yielded significant reductions of both k and RGR caused by O3. The available data were not ideally distributed among the exposure categories. Most of the observations involved herbaceous dicotyledonous species exposed in CECs of various designs. Fewer observations were available under GH or OTC conditions and none under non-chamber field conditions.
While the relationship between effective dose and imposed O3 exposure depends in part on chamber characteristics, there is no indication from these somewhat limited data that O3 sensitivity is a function of exposure technology. Nevertheless, further experimentation may resolve subtle effects of exposure technology and improve current methods of scaling from exposure experiments to extensive field environments.