Examining ozone effects on the tropical C₄ crop Sorghum bicolor

Introduction

Tropospheric ozone (O₃) is one of the most important air pollutants produced naturally by a series of photochemical reactions between the precursors: nitrogen oxides (NO_x), volatile organic compounds (VOCs), methane (CH₄) and carbon monoxide (CO) (Ainsworth et al., 2012; Schneider et al., 2017). Since the start of the industrial revolution, ground-level O₃ concentrations have significantly increased globally due to increasing abundance of O₃ precursor emissions (Monks et al., 2015; Young et al., 2013), and the influence of changing atmospheric chemistry under contemporary climate change (Brown et al., 2022). Ozone is well known to have a detrimental impact on plant growth, yield, and productivity (Ainsworth et al., 2012; Emberson et al., 2018). Indeed, it has been suggested that current O₃ exposure reduces global yields of wheat, soybean, maize, and rice by 227 Tg annually (Mills et al., 2018). Even accounting for the positive CO₂ fertilization effect, Tai et al. (2021) estimated global yield losses of 3.6 ± 1.1% for maize, 2.6 ± 0.8% for rice, 6.7 ± 4.1% for soybean, and 7.2 ± 7.3% for wheat. Such substantial crop losses due to O₃ are a threat to global food production and security.

Generally, O₃ from the atmosphere enters plant leaves through stomata (Ainsworth et al., 2012). When inside the leaf, O₃ reacts rapidly with molecules in the liquid surrounding leaf cells and forms reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂), superoxide (O₂⁻), and hydroxyl (OH˙) radicals (Ainsworth et al., 2012). O₃ and its ROS derivatives can initially attack the cell membranes with primary targets being membrane lipids, susceptible amino acids in membrane proteins, and a variety of organic metabolites contained in the cell wall (Fiscus, Booker & Burkey, 2005). Due to these reactions within the cell membrane, O₃ may produce new reaction products causing further cellular changes including oxidation of membrane lipids, alterations of protein function, and formation of ozonolysis products (Emberson et al., 2018; Fiscus, Booker & Burkey, 2005). This metabolic dysfunction and possible cell death causes a reduction in leaf photosynthesis and stomatal conductance, increased respiration rates, accelerated leaf senescence and visible injury (Ainsworth et al., 2012; Emberson et al., 2018). These physiological changes ultimately reduce plant growth, yield, and biomass accumulation (Emberson et al., 2018).

The reductions of biomass accumulation and yield represent integrated responses, useful in O₃ risk assessments (Mills et al., 2018). Although leaf visible injury has commonly been used as an O₃ response indicator, it is biologically less significant than the integrated measure of impacts on growth (Feng et al., 2014). The reduction in photosynthesis is also a functional O₃ response variable that corresponds with the O₃ impact on plant productivity (Li et al., 2016); however, the response of biomass has received more attention, as growth integrates internal and external processes that operate at different plant organizational levels (Uddling et al., 2004). These response variables in relation to O₃ exposure metrics have been used to examine O₃ effects on many plant species across a range of studies (Büker et al., 2015; Calatayud et al., 2011; Li et al., 2016; Zhang et al., 2012).

Different O₃ metrics have been developed for assessing O₃ risk (Emberson et al., 2018). At present, the most commonly used O₃ metrics are the concentration-based AOT₄₀ (accumulated O₃ concentrations over a threshold of 40 ppb) and the flux-based POD_y (Phytotoxic O₃ Dose-the accumulated stomatal O₃ flux above a threshold of y) (Shang et al., 2017). The AOT₄₀ metric only considers the O₃ concentrations in air that are above the 40 ppb threshold during daylight hours (Assis et al., 2015). However, as O₃ uptake is mediated through the stomata, POD_y which quantifies the effective stomatal flux of O₃ entering the leaves, and considers the biological and climatic factors influencing g_s, is a more rational metric than AOT₄₀ (Assis et al., 2015; Peng et al., 2019). Studies also have indicated that POD_y represents a better predictor than AOT₄₀ to estimate the negative O₃ impacts on plants (Büker et al., 2015; Karlsson et al., 2007; Mills et al., 2011). Therefore, the development of O₃ flux-response relationships for specific species in different climatic conditions is a central step for assessing the O₃ risk at regional, national, and global scales (Feng et al., 2015).

There are many studies about the adverse effects of O₃ on C₃ plant species (e.g., wheat, rice, soybean). However, relatively little work has been done on C₄ species (Li et al., 2022, 2023; McGrath et al., 2015). Sorghum (Sorghum bicolor), a C₄ tropical crop, is the fifth most important cereal crop in the world and has a high potential for bioenergy and ethanol production (Regassa & Wortmann, 2014; Yuan et al., 2008). Sorghum is highly adaptable and can be cultivated on marginal land where water deficit stress, alkalinity, salinity, and other limitations exist (Regassa & Wortmann, 2014; Yuan et al., 2008). Sorghum originated in Africa and is widely planted in tropical and sub-tropical regions (Hossain et al., 2022). At present, the tropics are experiencing high levels of O₃ pollution due to the combined pressures of an increase in population, industrialization, and energy consumption (Kunhikrishnan et al., 2004; Leventidou et al., 2018). Though some studies have investigated the effects of O₃ on sorghum in temperate environments (Li et al., 2021, 2022), studies under tropical conditions with local cultivars are still absent. As regional variation in the O₃ susceptibility of crop cultivars is often present due to differences in breeding strategies and different climatic conditions (Hayes et al., 2020b), it is essential to investigate the impact of O₃ pollution on sorghum under tropical climatic conditions.

Therefore, we carried out this study to evaluate the effects of a range of O₃ exposures on sorghum using a cultivar grown in tropical Australia. Our objectives were (1) to examine the effects of O₃ on sorghum biomass under various O₃ concentrations and (2) to assess the leaf morphological trait responses to O₃ to understand mechanisms of impact and/or tolerance.

Materials and Methods

The effects of O₃ on Sorghum bicolor were examined in open top chambers (OTC) built at the UK University of Exeter’s TropOz Research facility (www.tropoz.org) located at James Cook University’s Environmental Research Complex (ERC) on the Nguma-bada campus in northeast Queensland, Australia. The experiment was conducted using nine independently controlled and monitored OTCs employing a gradient design (daytime mean O₃ concentrations ranged between 20 and 97 ppb, Table 1). This gradient-design offers a better approach than replicating a limited number of exposure points, especially when attempting to identify a possibly nonlinear treatment response (Kreyling et al., 2018). Details of the OTC experimental set up are described in Farha et al. (2023). However, in brief, pure O₃ generated on site (AirSep Onyx Plus O₂ generator and OZ-T4600 O₃ generator; Oxyzone International, Somersby, Australia) was provided between 08:00 and 17:00 h to OTC’s (internal volume 22.2 m³) receiving continuous ventilation with activated carbon filtered air (AirFlow-VC; Airpure Australia North St Marys, North St Marys, NSW, Australia). An ultraviolet (UV) absorption O₃ analyser (Model 205, 2B technology, Boulder, CO, USA) was used to continuously monitor O₃ concentrations in each chamber. At the same time environmental variables including air temperature (T), relative air humidity (RH), and photosynthetically active radiation (PAR) were recorded in the central OTC using a single meteorological monitoring station (Campbell Scientific, Logan, UT, USA) with an assumption that environmental conditions other than O₃ concentrations were the same between OTC’s.

Stomatal conductance measurements and DO₃SE model parameterization

At various points throughout the experiment, stomatal conductance to water vapor (g_s) was measured on the newest fully expanded mature leaf using an SC-1 Leaf Porometer (Decagon Devices, Pullman, WA, USA). Point measurements of g_s on both abaxial and adaxial leaf surfaces were conducted over a range of conditions for a total of 270 measurements of g_s. These data were used to parameterize the Deposition of O₃ for Stomatal Exchange (DO₃SE) model (www.sei.org/tools/do3se-deposition-ozone-stomatal-exchange/) based on an empirical model of g_s (Emberson et al., 2000; Jarvis, 1976), allowing for the calculation of the O₃ flux-based metric POD₆ (phytotoxic O₃ dose-the accumulated stomatal O₃ flux above a threshold of 6 nmol O₃ m⁻² projected leaf area (PLA) s⁻¹) according to Convention on Long-range Transboundary Air Pollution (CLRTAP) (2017).

Prior to fitting DO₃SE empirical parameters (Table 2), all g_s data were transformed to relative stomatal conductance (RSC, g_s/g_max), with the maximum stomatal conductance (g_max) calculated as the measurement’s 95th percentile of abaxial and adaxial data and final g_s computed on a projected leaf area basis (PLA) using the abaxial: adaxial g_s ratio. Parameterization of DO₃SE was carried out according to Holder & Hayes (2022) using the plotting of RSC against light, leaf temperature and VPD independently. In order to fit the physiologically relevant curves, the x-axis was divided into parts and for each part, the 90th percentiles of the observed g_s data were calculated (Fig. 1). To convert the g_max to g_maxO₃, a conversion factor of 0.663 was used. The DO₃SE model estimates the stomatal O₃ uptake using the hourly mean values of O₃ and meteorological conditions from the experiment (Table 2, Data set S1).

Table 2:

Stomatal conductance model parameterization of Sorghum bicolor cv HAT150843.

The g_max is the maximum conductance, f_min is the fraction of g_max at minimum stomatal conductance (g_min). L_m is the effective leaf blade width, f_temp, f_light, f_VPD are the functions of g_s response to air temperature (T, °C), photosynthetically active radiation at the leaf surface (PAR, μmol m⁻² s⁻¹), and vapour pressure deficit (VPD, kPa), respectively.

Parameter		Unit	Sorghum bicolor cv HAT150843
gmax		mmol O3 m−2 s−1	290
Lm		m	0.02
fmin		Fraction	0.13
fPAR		Unitless	0.007
fVPD	VPDmin	kPa	4.50
	VPDmax	kPa	2.60
ftemp	Tmin	°C	14
	Topt	°C	31
	Tmax	°C	48

DOI: 10.7717/peerj.18844/table-2

Results

Sorghum (Sorghum bicolor cv HAT150843) was exposed to nine different concentrations of O₃ in OTCs. Over the experimental period, the average values of daytime (i.e., between 8 am and 5 pm) O₃ concentration ranged between 20.7 ± 4.8 and 97 ± 39.3 ppb (Table 1) and the average daytime air temperature, PAR, and RH were 28.1 ± 3.2 °C, 1137.7 ± 670.3 μmol m⁻² s⁻¹, and 57.5 ± 12.9%, respectively (Data set S1). Parameterization of g_s functions in the DO₃SE model (Table 2, Fig. 1) were carried out in accordance with Holder & Hayes (2022), with calculated POD₆ ranging between 0.1 and 44.9 mmol m⁻² (Table 1).

Biomass accumulation in sorghum was found to be not significantly impacted by exposure to O₃. Specifically, there was no significant change in above ground (leaves and stems) biomass (Adj-R² = −0.14, P = 0.86), below ground biomass (Adj-R² = −0.01, P = 0.38) or grain biomass (Adj-R² = −0.14, P = 0.85) with increasing O₃ flux metric, POD₆ (Fig. 2).

Our quadratic model provided an estimate of the optimal time (t_opt) for maximizing chlorophyll concentration and a rate parameter beta (β) as well as the mean and standard deviation of each parameter across all chambers (Table 3). Parameter beta indicates how quickly chlorophyll concentration decreases as time moves away from t_opt. The maximum chlorophyll concentration showed a slight but significant decline with increasing POD₆ (Adj-R² = 0.39, P < 0.05) (Fig. 3)_. However, leaf morphological traits such as LMA, SD, and SPL were not affected by O₃ flux in sorghum. The LMA across the O₃ exposure ranged between 44.8 and 48.8 g m⁻² (Adj-R² = −0.07, P = 0.52), SD ranged between 173 and 206 mm⁻² (Adj-R² = −0.10, P = 0.62) and SPL ranged between 19.4 and 20.6 µm (Adj-R² = −0.13, P = 0.80) (Data set S1).

In addition, we tested for correlations among leaf traits in sorghum. There was a strong correlation between A_net and log of g_s as measured by the portable photosynthesis analyzer (Adj-R² = 0.88, P < 0.001), but the slope of the relationship was not affected by increasing POD₆ (Fig. 4). Between the morphological traits the Pearson’s correlation test showed a positive correlation between LMA and SD (r = 0.41, P < 0.05) and a negative correlation between SD and SPL (r = −0.41, P < 0.05).

Discussion

Many studies have highlighted the interspecific and cultivar-level variability in negative impacts of O₃ on plant biomass and yields in C₃ crops (Emberson et al., 2018; Wittig et al., 2009). However, while C₄ plants are generally more O₃ tolerant there is also evidence that elevated O₃ has a variable impact on C₄ plants (Li et al., 2022) with some species such as switchgrass (Li et al., 2019) and sorghum (Hayes et al., 2020a; Li et al., 2021) showing a low suceptibility to O₃ exposure. In the first experiment for sorghum grown under tropical conditions, we found that in the cultivar examined (cv. HAT150843) there was no significant change in biomass or yield with increasing O₃ flux, although maximum chlorophyll concentration was found to be reduced with increasing POD₆. As sorghum has evolved in the tropics, environmental adaptations to cope with environmental stresses such as high temperature, drought, and light intensity (Regassa & Wortmann, 2014) have resulted in high water-use efficiency and antioxidant capacity, which may confer a capacity to also cope with the oxidative stress as a result of O₃ exposure (Li et al., 2021).

Leaf mass per area is a good indicator of plant functional type that can explain the differences in O₃ susceptibility among species (Feng et al., 2018), whereby leaves with high LMA tend to be less susceptible to O₃ (Li et al., 2016). However, there can be an impact of O₃ on LMA itself, as demonstrated by Shang et al. (2017) who found a significant negative relationship between LMA and O₃ metrics in C₃ poplar clones. Our results show no significant relationship between LMA and O₃ flux in sorghum, consistent with a recent study of maize (Peng et al., 2020) and other C₄ bioenergetic crop species (i.e., switchgrass, sorghum, maize, and miscanthus) (Li et al., 2022). This difference may be the result of the unique leaf anatomical features of C₄ species, including mesophyll cells surrounding bundle sheath cells in which the photosynthetic carbon reduction cycle occurs (Montes et al., 2022).

As O₃ uptake from the atmosphere occurs through leaf stomata, stomatal properties play an important role in shaping plant responses to O₃, at the same time that O₃ exposure can impact stomata through disrupting leaf development. Previous work in other species has reported that O₃ exposure increases SD (Fusaro et al., 2016; Paoletti & Grulke, 2005). In our study, neither SD nor SPL were found to be significantly correlated with O₃ flux, POD₆, with similar findings reported in switchgrass with respect to foliar anatomy (Li et al., 2019). However, in our study on sorghum, there was a negative correlation between SD and SPL, and a positive correlation between SD and LMA. Across various species, SD shows a negative relationship with SPL (Franks & Beerling, 2009; Hetherington & Woodward, 2003; Roth-Nebelsick et al., 2012), with plants with a higher density of small stomata able to open and close them more rapidly in response to changes in leaf water status (Hetherington & Woodward, 2003). This has been shown to contribute in varying degrees to drought tolerance in grasses (Xu & Zhou, 2008) and olive cultivars (Bosabalidis & Kofidis, 2002). It may also contribute to tolerance of O₃ induced stress. However, more studies on a range of sorghum genotypes would be required to understand the relationship among leaf traits across the breadth of genetic variation observed within the species.

As might be expected we found a strong correlation between A_net and log of g_s as measured by leaf-level gas-exchange in sorghum, however the proportionality between the two did not respond to increasing O₃ exposure. This is perhaps inconsistent with a recent study in which this proportionality was shown to have responded to increasing O₃ concentrations (Cernusak, Farha & Cheesman, 2021), and provides yet another indication that the physiology of sorghum was largely resistant to impacts of O₃ across the range of exposure that we applied in our experiment. In this context, it is interesting that we could detect an influence of increasing level of O₃ exposure on the maximum chlorophyll concentrations observed in leaves of the experimental plants (Fig. 3). However, it seems that the approximately 10% decline in maximum chlorophyll concentration from lowest to highest O₃ exposures was not of large enough magnitude to markedly impact leaf-level gas exchange (Fig. 4).

Conclusion

Based on plant biomass, leaf-level gas exchange and the morphological trait responses studied, our results showed little significant effect of O₃ on grain sorghum. Thus, it can be concluded that sorghum is not susceptible to elevated O₃, even when grown under tropical field conditions. Hence, sorghum could be an advantageous crop in tropical regions with high levels of O₃ pollution to help combat tropical food insecurity challenges (Hayes et al., 2020a). However, it should be noted that in this study, only one genotype was examined. To more broadly understand O₃ responses, additional experiments with more genotypes, and a broader range of functional traits and conditions are required.

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