SEARCH

SEARCH BY CITATION

Keywords:

  • Glycine max;
  • chlorophyll a fluorescence imaging;
  • leaf level;
  • photosynthesis;
  • spatial heterogeneity

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES
  9. Supporting Information

Experimental investigations of ozone (O3) effects on plants have commonly used short, acute [O3] exposure (>100 ppb, on the order of hours), while in field crops damage is more likely caused by chronic exposure (<100 ppb, on the order of weeks). How different are the O3 effects induced by these two fumigation regimes? The leaf-level photosynthetic response of soybean to acute [O3] (400 ppb, 6 h) and chronic [O3] (90 ppb, 8 h d−1, 28 d) was contrasted via simultaneous in vivo measurements of chlorophyll a fluorescence imaging (CFI) and gas exchange. Both exposure regimes lowered leaf photosynthetic CO2 uptake about 40% and photosystem II (PSII) efficiency (Fq′/Fm′) by 20% compared with controls, but this decrease was far more spatially heterogeneous in the acute treatment. Decline in Fq′/Fm′ in the acute treatment resulted equally from decreases in the maximum efficiency of PSII (Fv′/Fm′) and the proportion of open PSII centres (Fq′/Fv′), but in the chronic treatment decline in Fq′/Fm′ resulted only from decrease in Fq′/Fv′. Findings suggest that acute and chronic [O3] exposures do not induce identical mechanisms of O3 damage within the leaf, and using one fumigation method alone is not sufficient for understanding the full range of mechanisms of O3 damage to photosynthetic production in the field.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES
  9. Supporting Information

Tropospheric ozone (O3) is a phytotoxic air pollutant whose global background concentration has been rising since the Industrial Revolution, and is widely recognized as a problem affecting both natural plant communities and crops (Ashmore & Marshall 1999). Limited measurements from the nineteenth century suggest that pre-industrial ground-level O3 concentration ([O3]) was less than 10 ppb, and has risen 35–40 ppm in the Northern Hemisphere to a current average of 40 ppb (Volz & Kley 1988; Wittig, Ainsworth & Long 2007; Fowler 2008). Ozone enters the plant through the stomata and quickly reacts with the apoplast of the mesophyll cells lining the stomatal cavity, producing active oxygen species (AOS) and triggering a series of signalling cascades and plant defence responses which ultimately result in a range of effects, including visible foliar damage, decreased photosynthetic capacity and/or accelerated senescence (Omasa et al. 2000; Leipner, Oxborough & Baker 2001; Long & Naidu 2002; Kangasjarvi, Jaspers & Kollist 2005). Meta-analyses of field studies in which plants have been exposed to ambient air, and air from which the O3 has been removed, suggest that current [O3] is decreasing photosynthesis in trees in the Northern Hemisphere by between 9 and 13% (Wittig et al. 2007). Field studies suggest an even larger decrease in soybean, the second most important crop in the USA, in terms of total grain production (Morgan, Ainsworth & Long 2003).

Much of the rural temperate Northern Hemisphere is now subject to a significant elevation of background [O3], such that plants are subject to a small but persistent elevation of [O3] throughout the growing season (Emberson, Ashmore & Murray 2003). This type of long-term exposure to O3 (at concentrations <100 ppb) has generally been termed ‘chronic’ in the literature (Kangasjarvi et al. 2005; Fowler 2008). Acute [O3] exposure, in contrast, is generally defined as exposure to a high level of ozone ([O3] > 100 ppb) for a short period of time, typically on the order of hours (Long & Naidu 2002; Kangasjarvi et al. 2005). Such events are rarer and more likely to occur in locations close to urban centres, where air pollution is often produced at high levels (Emberson et al. 2003). To investigate elevated [O3] effects in a laboratory setting, it has been common practice to induce O3 damage by dosing plants with a short acute exposure, because such exposures are faster and easier to control than a long-term chronic fumigation. For these reasons, such short-term exposures have dominated investigations of the molecular basis of plant responses to O3, with the aim of understanding how to adapt plants to rising [O3] pollution (reviewed: Kangasjarvi et al. 2005). But, is acute [O3] exposure alone sufficient to induce the range of physiological effects observed under actual field conditions, which are dominated by conditions more similar to chronic [O3] exposure? And is it sufficient to gain an understanding of how crops may ultimately be bred or engineered to better tolerate O3? When studied separately, both types of O3 exposures have been found to produce losses in photosynthetic capacity and visible damage on the surface of leaves, but does the mechanism of damage or the pattern of O3-induced damage across the leaf differ between fumigation methods?

Several factors suggest that the effects of elevated [O3] are heterogeneous at the leaf level. The clearest evidence that effects of elevated [O3] are non-uniform across the leaf surface is the characteristic symptom of visible damage. Stipples form on the leaf when local areas of cells lyse, releasing phenols into the intercellular space (Findley et al. 1996). Necrotic or chlorotic spots form when the plant initiates programmed cell death (PCD) in response to the O3 (Long & Naidu 2002; Kangasjarvi et al. 2005). Ozone enters the plant through the stomata and quickly reacts with the aqueous environment of the apoplast exposed to the intercellular air space to produce a range of oxidizing radicals which may react with the plasma membrane (Omasa et al. 2000; Leipner et al. 2001; Long & Naidu 2002). Therefore, it might be expected that the mesophyll cells which are closer to the stomata could incur greater damage under O3 exposure. The effects of O3 are strongly dependent on uptake or flux into the plant, so any variation in the aperture between stomata (i.e. stomatal heterogeneity) would also result in a non-uniform effect of O3 damage within the mesophyll (Fuhrer 2002). Therefore, there is a strong possibility that the pattern of O3 uptake and impacts within the leaf would differ between acute and chronic [O3] exposures, because short-term patterns of stomatal conductance (on the scale of hours) could differ from the long-term patterns resulting from lower exposures on the order of several days.

Modulated chlorophyll a fluorescence imaging (CFI) allows quantitative visualization of the in vivo efficiency of electron transport through photosystem II (PSII) (Fq′/Fm′), and heterogeneity at the leaf scale (Baker & Oxborough 2004; Oxborough 2004b). CFI has also been used to examine physiological processes or environmental stresses, such as mapping carbon assimilation rates (Genty & Meyer 1994), mapping intercellular [CO2] across leaf surfaces (Meyer & Genty 1998), tracking circadian rhythms (Rascher et al. 2001), assessing indirect effects of herbivory (Aldea et al. 2005), observing stomatal patchiness (West et al. 2005) and examining plant–pathogen interactions (Chaerle et al. 2004). The success of these previous applications suggests that CFI is well suited to probing the effects of O3 across the leaf.

In addition, chlorophyll fluorescence analysis can distinguish the factors underlying differences in the operating efficiency of PSII (Fq′/Fm′) across the leaf by partitioning the fluorescence ratio into its component ratios (Baker & Oxborough 2004; Oxborough 2004a). Fv′/Fm′ represents the maximum quantum efficiency of PSII photochemistry in the light; it can be interpreted as the relative integrity of the PSII reaction centres, because decreases in Fv′/Fm′ are associated with down-regulation of PSII or damage to the reaction centres (Baker & Oxborough 2004). The Fq′/Fv′ is non-linearly related to the oxidation state of the QA pool; changes in Fq′/Fv′ can be attributed to changes downstream of PSII, including ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and the Calvin cycle (Baker & Oxborough 2004).

Spatial analysis of the imaging data (e.g. quantification of spatial heterogeneity and/or gradients) can provide additional information about O3 effects. If acute and chronic [O3] exposure both decrease photosynthesis via the same basic mechanism, possibly by a form of PCD, then for a given reduction in photosynthesis, a similar increase in spatial heterogeneity of photosynthesis should be observed. In addition, the patterns of damage observed within leaves should also be similar, as well as the O3-induced changes in the fluorescence images, because this would also point towards similar mechanisms at work in the leaf.

The objective of this study was to test the hypothesis that the character and spatial pattern of O3-induced damage to leaf-level photosynthesis is similar under acute and chronic exposures to elevated [O3]. This hypothesis was tested by assessing the photosynthetic response of soybean (Glycine max cv. NE3399) via simultaneous CFI and gas exchange, and quantifying the leaf-level patterns of photosynthesis using spatial analytic techniques. Soybean was selected for this study because it is a major food crop known to be sensitive to elevated [O3] (Ashmore & Marshall 1999). As an inbreeding species, it also provided uniform genetic material for testing the hypothesis; furthermore, its internal leaf structure is heterobaric, which increases the chance of significant spatial heterogeneity of ozone uptake and effects within the leaf.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES
  9. Supporting Information

Growth chamber conditions

Soybean plants (Glycine max cv. NE3399) were grown in controlled environments (CEC38-15HLE-G, Rheem Scientific, Montgomery, AL, USA) under a photon flux of 500 µmol m−2 s−1 for 14 h d−1 at a temperature of 26 °C during the day and 20 °C during the night. Seeds were planted in 1 L pots using a commercial peat-based soil mix (Sunshine Mix LC1, Sun Gro Horticulture, Bellevue, WA, USA). Plants were fertilized with a 5 mm CaNO3 and 5 mm NH4NO3 nutrient solution once per week.

Ozone treatment

Ozone was generated and controlled within the growth environment using a custom-built system which employed a high-voltage O3 generator to generate O3 from oxygen (HTU-500AC, Azco Industries Ltd., Surrey, British Columbia, Canada), O3 analyser (1008-RS, Dasibi Environmental Corp., Glendale, CA, USA) and a programmable datalogger (CR10, Campbell Scientific, Logan, UT, USA). Ozone concentration in the growth chamber was controlled to within ± 10% of the target concentration during the course of all fumigations.

For both treatments, plants were grown for 14 d under the conditions described earlier to allow completion of expansion of the third trifoliate leaf. For the acute treatments, the plants were then exposed to a single O3 dose of 400 ppb for 6 h, in the middle of the photoperiod under the conditions described earlier. Gas exchange and fluorescence measurements were taken on the terminal leaflet of the third trifoliate on the morning before and following the acute treatment (n = 4). For the chronic treatment, the [O3] was elevated to 90 ppb for 8 h during the middle of the light period for 28 d following full expansion of the third trifoliate leaf. Control plants were grown in ambient air ([O3] < 20 ppb) for the same period. Measurements were taken on the terminal leaflet of the third trifoliate on the 13th, 20th and 28th day of O3 exposure (n = 4). Plants and treatments were rotated between chambers twice a week to minimize potential chamber effects.

Chlorophyll fluorescence terminology

The chlorophyll fluorescence terminology used here follows the convention of Baker & Oxborough 2004. The fluorescence ratio Fv/Fm, measured in the dark, represents the maximum potential or dark-adapted quantum efficiency of PSII photochemistry. Under actinic light, Fq′/Fm′ corresponds to the operating quantum efficiency of PSII photochemistry. The cause of change in Fq′/Fm′ can be partitioned between two component fluorescence ratios as follows:

  • image(1)

Fv′/Fm′ gives an estimate of the potential PSII operating efficiency under the current photon flux density, while Fq′/Fv′ is a non-linear estimate of the fraction of open PSII centres (i.e. the oxidation state of QA). The product of these two fluorescence ratios Fq′/Fm′ is the realized operating efficiency of PSII photochemistry in the light. False-colour maps (hereafter referred to as ‘images’) of Fv/Fm, Fv′/Fm′, Fq′/Fv′ and Fq′/Fm′ were constructed using fluorescence imaging software (Fluorimager; Technologica Ltd, Essex, UK), following Baker & Oxborough (2004).

Image capture and gas exchange measurements

Attached third trifoliates were enclosed in a custom-built temperature, humidity and ambient CO2-controlled gas exchange cuvette with a clear IR transmitting window (Tedlar polyvinyl fluoride film, Dupont, Wilmington, DE, USA) measuring 5 × 7.5 cm. The chamber was attached to an open gas exchange system incorporating infrared CO2 and water analysers to allow measurement of photosynthetic CO2 uptake (A) and stomatal conductance to water vapour (gs) (LI-6400, Li-Cor Inc., Lincoln, NE, USA). The ambient CO2 concentration (Ca) was held at 400 ppm and leaf temperature at 25 °C for all measurements. Steady-state A and gs were recorded while fluorescence images were being captured. The leaf chamber was placed within the imaging area of a pulse-modulated CFI system (CF Imager, Technologica Ltd.) to enable simultaneous measurements of gas exchange and fluorescence. The imaging system used 1600 blue LEDs (peak wavelength = 470 nm) to provide actinic illumination, measuring pulses and saturating pulses. The system employed a CCD camera which captured 8-bit images at a spatial resolution of 150 × 150 µm per pixel and a maximum count of 704 × 520 pixels per image. Plants were dark-adapted for 30 min, after which a single ‘saturating pulse’ was applied to capture Fo and Fm images. Plants were then exposed to an actinic photon flux of 500 µmol m−2 s−1. Light-adapted fluorescence images were taken by applying a ‘saturating pulse’ (3500 µmol m−2 s−1) every 2.5 min until the mean Fq′/Fm′ reached steady state. Typically, this required 30 min and roughly coincided with attainment of steady-state A.

Image analysis

Spatial heterogeneity of the fluorescence images was quantified statistically by calculating the local neighbourhood standard deviation (NbrSD) of each image. NbrSD at a given location is assessed by calculating the standard deviation of the population of data values which fall within a pre-defined neighbourhood centred on that location. The mean NbrSD of an image serves as a quantitative measure of the spatial heterogeneity; higher values of NbrSD indicate greater spatial heterogeneity. A detailed example of NbrSD calculation is given in the Supporting Information Appendix S1.

After initial testing of the sensitivity of NbrSD at different neighbourhood sizes (3 × 3, 5 × 5 or 7 × 7 pixels) and consideration of the physical size of the mesophyll air spaces between minor veins in the heterobaric soybean leaf, a 7 × 7 pixel neighbourhood was chosen for all calculations of NbrSD because it exhibited the most sensitivity to spatial heterogeneity induced by O3 (data not shown). This represented an area of 1.1 mm2 and approximately 1500 palisade mesophyll cells. NbrSD was calculated for the images of Fv′/Fm′, Fq′/Fv′ and Fq′/Fm′ derived from all leaves under all treatments with the aid of a software that has been specifically designed for analogous analyses of digital raster images in the field of remote sensing (ERDAS IMAGINE 8.6; Leica Geosystems, Heerbrugg, Switzerland).

Base-to-tip gradients within Fv/Fm images were quantified by dividing each leaf into 10 latitudinal sections of equal width and calculating the mean Fv/Fm within each section. The base-to-tip gradient was quantified in all leaves in the acute [O3] treatment and on all leaves at the last time-point of the chronic [O3] treatment. To further investigate the nature of O3-induced physiological changes in the photosynthetic apparatus, the mean and variance of Fv′/Fm′, Fq′/Fv′ and Fq′/Fm′, as well as the correlation between each pair of ratios, were calculated for the fluorescence images of all leaves in the acute treatment and on the last day of the chronic treatment.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES
  9. Supporting Information

Overall photosynthetic response

In both acute and chronic [O3] treatments, A and Fq′/Fm′ declined and spatial heterogeneity (NbrSD) of Fq′/Fm′ increased after O3 exposure (Figs 1 & 2). The acute [O3] treatment caused about a 20% decrease in the efficiency of electron transport (Fq′/Fm′) relative to the untreated control and a decrease in CO2 assimilation rate (A) by 43%, while the NbrSD of Fq′/Fm′ increased by 46% (Fig. 1). Over the course of the 28 d chronic [O3] exposure, A and Fq′/Fm′ declined, and NbrSD of Fq′/Fm′ increased in both the control and O3-exposed plants, but the changes were significantly greater in the O3-exposed plants (Fig. 2). After 28 d of the chronic [O3] treatment, Fq′/Fm′ and A had decreased by 20 and 40%, respectively, relative to the control (Fig. 2a,b). The extent of these chronic [O3]-induced changes in photosynthetic capacity was therefore very similar in magnitude to those induced by the acute treatment. In contrast, spatial heterogeneity of Fq′/Fm′ in the chronic [O3] treatment was only increased slightly, 10% relative to the control, compared to about 50% in the acute treatment (Figs 1c & 2c).

image

Figure 1. Average photosynthesis and spatial heterogeneity resulting from the acute [O3] exposure of 400 ppb for 6 h. Closed bars (▪) are O3 treated; open bars (□) are untreated controls (n = 4). (a) Efficiency of electron transport (Fq′/Fm′) calculated from chlorophyll fluorescence measurements. (b) Leaf net CO2 assimilation rate (A) was calculated from gas exchange measurements. (c) Spatial heterogeneity of Fq′/Fm′ (NbrSD) was calculated from chlorophyll fluorescence images as described in the Methods and is in units of Fq′/Fm′. Each bar is the mean of four independent plants (+1 SE), and asterisks indicate P < 0.05.

Download figure to PowerPoint

image

Figure 2. Mean photosynthesis and spatial heterogeneity resulting from chronic exposure to 90 ppb [O3] for 8 h d−1 for 28 d; data are shown for the last 16 d. Closed symbols (●) are O3 treated; open symbols (○) are untreated control plants. (a) Efficiency of electron transport (Fq′/Fm′) (●). (b) Leaf net CO2 assimilation rate (A) (▪). (c) Spatial heterogeneity of Fq′/Fm′ (NbrSD) (▴). Each point is the mean of four independent plants (± 1 SE) and P values are given in the table below.

Download figure to PowerPoint

Some chlorosis was visible in both the acute and chronic [O3]-treated leaves (Fig. 3b,c). The images of maximum dark-adapted photosynthetic efficiency of PSII (Fv/Fm) were relatively uniform across the surface of control leaflets (Fig. 3a). However, after O3 exposure, discrete patches of lower Fv/Fm were apparent (Fig. 3b,c). Frequency histograms of the Fv/Fm images showed that control leaves possessed a normal distribution of Fv/Fm across the leaf, while O3-treated leaves had a wider, right-skewed distribution.

image

Figure 3. Digital colour and fluorescence images of soybean leaflets representative of the different treatments. The top row are digital-reflected colour images, middle row are false-colour fluorescence images of Fv/Fm and the bottom row are the corresponding frequency histograms and colour palettes of the fluorescence images. (a) Control leaflet (fully expanded terminal leaflet of the third trifoliate), (b) following a 6 h 400 ppb acute [O3] dose and (c) following chronic exposure to 90 ppb [O3] for 8 h per day over 28 d. Leaflets exposed to chronic [O3] were marked to ensure that the same area was sampled across each time-point.

Download figure to PowerPoint

The base-to-tip gradient of O3 damage

The acute [O3] treatment resulted in significantly greater damage at the base of the leaf than at the tip, as shown by a significant interaction between O3-induced decline in Fv/Fm and position along the longitudinal axis of the leaflet (Fig. 4). Fv/Fm declined 0.048 at the base of the leaf relative to the control compared to 0.016 at the tip, a statistically significant difference (Fig. 4a). In contrast, in the chronic [O3] treatment, the decrease in Fv/Fm was least at the base (0.014) and greatest at the tip (0.031), although this was not statistically significant (Fig. 4b). Normalizing the mean Fv/Fm of each treatment against its respective control for each leaf position revealed a slight decline from base to tip in the chronic treatment and an obvious increase in the acute treatment (Fig. 4c).

image

Figure 4. Comparison of the average Fv/Fm of leaflets from the base (1) to the tip (10), where each section was of equal width across the leaf. (a) Acute [O3] treatment of 400 ppb for 6 h. Each point is the mean of four plants (± 1 SE) (b) Chronic [O3] exposure to 90 ppb [O3] for 8 h d−1 over 28 d. Each point is the mean of four plants (± 1 SE). (c) Ratio of O3 treatment to control in each exposure regime. (d) Table of P values showing the statistical significance level of the effects of O3 treatment, longitudinal position from base to tip and the interaction of O3 treatment with position along the leaf.

Download figure to PowerPoint

Partitioning change in Fq′/Fm′ between Fv′/Fm′ and Fq′/Fv

In the acute [O3] treatment, Fv′/Fm′ and Fq′/Fv′ both decreased by about 11%, suggesting that both factors contributed similarly to the resulting 20% decrease in Fq′/Fm′ (Fig. 5a). By contrast, in the chronic [O3] treatment, no significant difference in Fv′/Fm′ was found, but Fq′/Fv′ was significantly decreased by 16%, and was presumably the main contributor to the 19% decline in Fq′/Fm′ (Fig. 5b). This suggests that while both treatments induced similar losses in efficiency of electron transport, the underlying causes were quite different.

image

Figure 5. (a) & (b) Comparison of fluorescence ratios Fv′/Fm′, Fq′/Fv′ and Fq′/Fm′ in both acute (a) and chronic (b) [O3] treatments. (c) & (d) Comparison of spatial heterogeneity (NbrSD) of Fv′/Fm′, Fq′/Fv′ and Fq′/Fm′ in both acute (c) and chronic (d) [O3] treatments. Open bars are control; closed bars are O3 treated. Each bar is the mean (+1 SE) of four plants. *P < 0.10; **P < 0.05.

Download figure to PowerPoint

In the acute [O3] treatment, spatial heterogeneity of Fv′/Fm′ and Fq′/Fv′ increased by 75 and 37%, respectively, while it increased by 46% in the Fq′/Fm′ images (Fig. 5c). As in the case of fluorescence ratio itself, the NbrSD of Fv′/Fm′ was affected relatively little by the chronic [O3] treatment (17%), while the NbrSD of Fq′/Fv′ was increased to a similar amount as in the acute treatment (35%) (Fig. 5d). The NbrSD of Fq′/Fm′ images for the chronic [O3] treatment increased by only 10%, less than a quarter of the change observed in the acute treatment (Fig. 5c,d).

The relationship between Fv′/Fm′, Fq′/Fv′ and Fq′/Fm

The variance within and correlation between the light-adapted fluorescence ratio images Fv′/Fm′, Fq′/Fv′ and Fq′/Fm′ showed significant differences between control and O3 treatment in both the acute and chronic [O3] fumigations (Table 1). In the acute [O3] treatment, there were significant increases after O3 exposure in the variance of all three ratios and in the correlation between ratios, except for the correlation between Fq′/Fv′ and Fq′/Fm′, which was already very high in the control (r2 = 0.80, Table 1). In the chronic [O3] experiment, no differences were found between the control and O3 treatments at the α = 0.05 level. However, at the α = 0.10 level, the correlation between Fq′/Fv′ and Fq′/Fm′ was significantly higher under chronic [O3] than in the control (P = 0.06). This implies that decreases in Fq′/Fm′ observed under chronic [O3] were driven primarily by changes in Fq′/Fv′.

Table 1.  Variances of pixel values within fluorescence ratio images Fv′/Fm′, Fq′/Fv′ and Fq′/Fm′, and between parameter correlations of the images, for both the acute and chronic [O3] treatments (n = 4) Thumbnail image of

In the acute [O3] treatment, there was a sharp and significant increase in the correlation of Fv′/Fm′ to Fq′/Fm′ from 0.27 to 0.74, in contrast to the decrease observed in the chronic treatment, from 0.47 to 0.37. This provides strong evidence that the decline in Fv′/Fm′ strongly influenced the decline in the operating efficiency of electron transport (Fq′/Fm′) in the acute [O3] treatment, but not in the chronic [O3] treatment.

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES
  9. Supporting Information

Because the spatial patterns and fluorescence signature between O3 exposure regimes were dissimilar, the hypothesis that acute and chronic [O3] induce the same effects in soybean leaves was rejected. Although both the acute and chronic [O3] treatments resulted in similar overall photosynthetic impairment compared to their respective controls, the fluorescence imaging data revealed that the physiological mechanisms underlying the decreases differed.

The greater increase in spatial heterogeneity in the acute [O3] treatment over the chronic [O3], in spite of the similar extent of photosynthetic depression observed in both treatments, could have several bases. Firstly, high concentrations of O3 typically induce oxidative shock and the hypersensitive response within a matter of hours, leading to PCD; very high concentrations of O3 can even directly damage leaf tissue (Long & Naidu 2002; Kangasjarvi et al. 2005). Secondly, by the end of the chronic [O3] treatment, control leaves were already exhibiting an increase in spatial heterogeneity of photosynthesis because of the process of natural leaf ageing. Literature suggests that chronic [O3] exposure accelerates the process of ageing and senescence (Pell, Schlagnhaufer & Arteca 1997; Long & Naidu 2002). Therefore, the results would be consistent with the hypothesis that chronic [O3] treatment simply caused accelerated ageing, resulting in smaller differences in spatial heterogeneity of photosynthesis between the O3 and control treatments at any given point in time compared to the acute [O3] treatment.

The two different O3 exposure regimes produced differing patterns of O3 damage at the leaf level, as seen in the chlorophyll fluorescence images (Fig. 3). Acute [O3] damage was characterized by small local areas of depressed photosynthetic capacity, most of which are found in areas near the major veins (Fig. 3b). This was consistent with studies that have found that AOS accumulate near the veins during O3 stress, possibly linked to formation of localized foci of PCD (Langebartels et al. 2002; Wohlgemuth et al. 2002). In contrast, in the chronic [O3]-exposed leaf the areas of lowest photosynthetic capacity were found in the interveinal regions and were of more variable size and shape (Fig. 3c).

The base-to-tip gradient of O3 damage also differed between the acute and chronic [O3] treatments (Fig. 4). The acute [O3] exposure produced a strong base-to-tip gradient of photosynthetic efficiency, whereas the chronic [O3] treatment did not produce any significant gradient, although a slight tip-to-base gradient was indicated. The reasons for the strong base-to-tip gradient of O3 damage in the acute [O3] treatment are not clear. During the acute [O3] fumigation, the soybean leaflets were observed to tilt downwards slightly. This caused the area nearest the petiole to remain exposed to the highest amount of light; high light can exacerbate O3 damage via photoinhibition and increased ‘excitation pressure’ (Guidi, Tonini & Soldatini 2000). Another possible explanation is that the tilting of the leaves caused a gradient of stomatal opening across the leaf. The amount of O3 assimilated by different parts of the leaf is directly proportional to stomatal opening, given a uniform [O3] in the air around the leaf, as in these experiments.

The integrated fluorescence data suggested that under acute [O3] exposure, the reduction in Fq′/Fm′ was caused by both direct damage to PSII and down-regulation of downstream processes (Fig. 5a). However, in the chronic [O3] treatment, the majority of the decrease in Fq′/Fm′ was caused by a decrease in downstream processes and not direct damage to PSII, even though both acute and chronic [O3] treatments produced approximately the same reduction in Fq′/Fm′ (∼20%). This is consistent with the hypothesis that acute [O3] can damage PSII reaction centres through oxidative stress as well as affect Rubisco downstream, but chronic [O3] largely affects Rubisco (McKee, Farage & Long 1995; Farage & Long 1999). The spatial heterogeneity of these component ratio images showed a similar trend (Fig. 5c,d). Under acute [O3] exposure, the spatial heterogeneity of both Fv′/Fm′ and Fq′/Fv′ images increased significantly, while under chronic [O3] exposure there was a much greater increase in spatial heterogeneity of Fq′/Fv′ compared to Fv′/Fm′.

In the acute [O3] experiment, control leaves showed a low correlation between Fv′/Fm′ and Fq′/Fm′, and a good correlation between Fq′/Fv′ and Fq′/Fm′ (Table 1). Control leaves in the chronic [O3] experiment showed a greater variance in both Fv′/Fm′ and Fq′/Fm′, and a higher correlation between the two parameters, compared to the control of the acute [O3] experiment. This is not an unexpected result, because the control leaves in the chronic [O3] experiment were ageing, and thus, it was reasonable to assume that there would be greater natural variation across the leaf in PSII integrity as it aged. These results indicated a major difference between acute and chronic [O3] effects; spatially heterogeneous decreases in Fq′/Fm′ under acute [O3] were driven by changes in both Fv′/Fm′ and Fq′/Fv′, whereas the decreases in Fq′/Fm′ under chronic [O3] were mainly driven by changes in Fq′/Fv′. These conclusions are consistent with existing models of the mechanism of ozone damage (Pell et al. 1997).

In the chronic [O3] experiment, significant differences between control and treatment were more difficult to detect as a whole because photosynthetic capacity naturally decreased and spatial heterogeneity naturally increased as both the control and O3-treated leaves aged. However, the results in this study follow the prevailing view in the literature regarding long-term O3 effects, namely, that chronic [O3] exposure accelerates ageing and senescence in leaves (Pell et al. 1997), and that it is not driven by effects at the reaction centre (Farage & Long 1999).

In summary, this study found that, while the acute and chronic [O3] treatments produced very similar reductions in whole-leaf CO2 uptake rate, the within-leaf spatial patterns of factors underlying this change differed markedly. Acute [O3] caused significantly more spatial heterogeneity, and photosynthetic depression was greatest at the base of the leaf. Decreases in Fq′/Fm′ under acute [O3] exposure resulted from decreased Fv′/Fm′ and Fq′/Fv′, but similar decreases in Fq′/Fm′ under chronic [O3] exposure were the result of changes in only Fq′/Fv′.

Although plants in the field are typically exposed to long-term elevations in background [O3], they can also experience short-term, mildly acute levels of [O3] (∼100 to 200 ppb) sporadically throughout their growing season, on days when the ambient [O3] spikes because of a confluence of high temperature, air movements bringing in high levels of NOx and clear skies (Wang et al. 2001). The results of this study indicate that acute and chronic [O3] exposures induce some different effects via different mechanisms of damage, and suggest that both are necessary for understanding the full range of effects of rising tropospheric [O3] on plants. The most all-encompassing strategy, then, is to use an O3 exposure regime which tracks ambient O3 concentrations throughout the day, since it would provide consistent low-level [O3] elevation with occasional spikes of acute [O3] during the midday hours.

ACKNOWLEDGMENT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES
  9. Supporting Information

We thank Dr Patrick Morgan for his assistance in development of the custom leaf chamber.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES
  9. Supporting Information
  • Aldea M., Hamilton J.G., Resti J.P., Zangerl A.R., Berenbaum M.R. & DeLucia E.H. (2005) Indirect effects of insect herbivory on leaf gas exchange in soybean. Plant, Cell & Environment 28, 402411.
  • Ashmore M.R. & Marshall F.M. (1999) Ozone impacts on agriculture: an issue of global concern. Advances in Botanical Research Incorporating Advances in Plant Pathology 29, 3152.
  • Baker N.R. & Oxborough K. (2004) Chlorophyll fluorescence as a probe of photosynthetic productivity. In Chlorophyll a Fluorescence: A Signature of Photosynthesis (eds G.C.Papageorgiou & Govindjee), pp. 6582. Kluwer Academic, London, UK.
  • Chaerle L., Hagenbeek D., De Bruyne E., Valcke R. & Van der Straeten D. (2004) Thermal and chlorophyll-fluorescence imaging distinguish plant–pathogen interactions at an early stage. Plant & Cell Physiology 45, 887896.
  • Emberson L.D., Ashmore M.R. & Murray F. (eds) (2003) Air Pollution Impacts on Crops and Forests: A Global Assessment. Imperial College Press, London, UK.
  • Farage P.K. & Long S.P. (1999) The effects of O-3 fumigation during leaf development on photosynthesis of wheat and pea: an in vivo analysis. Photosynthesis Research 59, 17.
  • Findley D.A., Keever G.J., Chappelka A.H., Eakes D.J. & Gilliam C.H. (1996) Sensitivity of red maple cultivars to acute and chronic exposures of ozone. Journal of Arboriculture 22, 264269.
  • Fowler D. (2008) Ground-level Ozone in the 21st Century: Future Trends, Impacts and Policy Implications. The Royal Society, London, UK.
  • Fuhrer J. (2002) Ozone impacts on vegetation. Ozone: Science & Engineering 24, 6974.
  • Genty B. & Meyer S. (1994) Quantitative mapping of leaf photosynthesis using chlorophyll fluorescence imaging. Australian Journal of Plant Physiology 22, 277284.
  • Guidi L., Tonini M. & Soldatini G.F. (2000) Effects of high light and ozone fumigation on photosynthesis in Phaseolus vulgaris. Plant Physiology and Biochemistry 38, 717725.
  • Kangasjarvi J., Jaspers P. & Kollist H. (2005) Signalling and cell death in ozone-exposed plants. Plant, Cell & Environment 28, 10211036.
  • Langebartels C., Wohlgemuth H., Kschieschan S., Grun S. & Sandermann H. (2002) Oxidative burst and cell death in ozone-exposed plants. Plant Physiology and Biochemistry 40, 567575.
  • Leipner J., Oxborough K. & Baker N.R. (2001) Primary sites of ozone-induced perturbations of photosynthesis in leaves: identification and characterization in Phaseolus vulgaris using high resolution chlorophyll fluorescence imaging. Journal of Experimental Botany 52, 16891696.
  • Long S.P. & Naidu S.L. (2002) Effects of oxidants at the biochemical, cell and physiological levels, with particular reference to ozone. In Air Pollution and Plant Life (eds J.N.B.Bell & M.Treshow), pp. 6988. John Wiley & Sons, West Sussex, UK.
  • McKee I.F., Farage P.K. & Long S.P. (1995) The interactive effects of elevated CO2 and O3 concentration on photosynthesis in spring wheat. Photosynthesis Research 45, 111119.
  • Meyer S. & Genty B. (1998) Mapping intercellular CO2 mole fraction (C-i) in Rosa rubiginosa leaves fed with abscisic acid by using chlorophyll fluorescence imaging – significance of C-i estimated from leaf gas exchange. Plant Physiology 116, 947957.
  • Morgan P.B., Ainsworth E.A. & Long S.P. (2003) How does elevated ozone impact soybean? A meta-analysis of photosynthesis, growth and yield. Plant, Cell & Environment 26, 13171328.
  • Omasa K., Tobe K., Hosomi M. & Kobayashi M. (2000) Absorption of ozone and seven organic pollutants by Populus nigra and Camellia sasanqua. Environmental Science & Technology 34, 24982500.
  • Oxborough K. (2004a) Imaging of chlorophyll a fluorescence: theoretical and practical aspects of an emerging technique for the monitoring of photosynthetic performance. Journal of Experimental Botany 55, 11951205.
  • Oxborough K. (2004b) Using chlorophyll a fluorescence imaging to monitor photosynthetic performance. In Chlorophyll a Fluorescence: A Signature of Photosynthesis (eds G.C.Papageorgiou & Govindjee) pp. 409428. Kluwer Academic, London, UK.
  • Pell E.J., Schlagnhaufer C.D. & Arteca R.N. (1997) Ozone-induced oxidative stress: mechanisms of action and reaction. Physiologia Plantarum 100, 264273.
  • Rascher U., Hutt M.T., Siebke K., Osmond B., Beck F. & Luttge U. (2001) Spatiotemporal variation of metabolism in a plant circadian rhythm: the biological clock as an assembly of coupled individual oscillators. Proceedings of the National Academy of Sciences of the United States of America 98, 1180111805.
  • Volz A. & Kley D. (1988) Evaluation of the Montsouris series of ozone measurements made in the 19th-century. Nature 332, 240242.
  • Wang T., Wu Y.Y., Cheung T.F. & Lam K.S. (2001) A study of surface ozone and the relation to complex wind flow in Hong Kong. Atmospheric Environment 35, 32033215.
  • West J.D., Peak D., Peterson J.Q. & Mott K.A. (2005) Dynamics of stomatal patches for a single surface of Xanthium strumarium L. leaves observed with fluorescence and thermal images. Plant, Cell & Environment 28, 633641.
  • Wittig V.E., Ainsworth E.A. & Long S.P. (2007) To what extent do current and projected increases in surface ozone affect photosynthesis and stomatal conductance of trees? A meta-analytic review of the last 3 decades of experiments. Plant, Cell & Environment 30, 11501162.
  • Wohlgemuth H., Mittelstrass K., Kschieschan S., Bender J., Weigel H.J., Overmyer K., Kangasjarvi J., Sandermann H. & Langebartels C. (2002) Activation of an oxidative burst is a general feature of sensitive plants exposed to the air pollutant ozone. Plant, Cell & Environment 25, 717726.

Supporting Information

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES
  9. Supporting Information

Appendix S1. How to calculate neighbourhood standard deviation.

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

FilenameFormatSizeDescription
PCE_1923_sm_Appendix_S1.doc26KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.