Tropospheric ozone (O₃) is a global air pollutant that adversely affects plant growth and productivity. While the impacts of O₃ have previously been examined for some tropical commodity crops, no information is available for the pantropical crop, banana (Musa spp.). In this study, we exposed Australia’s major banana cultivar, Williams, to a range of [O₃] in open-top chambers. In addition, we examined 46 diverse Musa lines growing in a common garden for variation in traits that are hypothesized to shape responses to O₃: leaf mass per area, intrinsic water-use-efficiency, and total antioxidant capacity. Banana cv. Williams showed substantial susceptibility to O₃. Combined our results from open-top chambers and common garden conditions suggest a substantial risk of O₃ to banana production and food security throughout the tropics.

The ozone (O₃) susceptibility of cv. Williams was tested in nine independently controlled and monitored open-top chambers (OTC) built at the UK University of Exeter’s TropOz Research facility located at James Cook University’s Environmental Research Complex (ERC) on the Nguma-bada campus in far-north Queensland, Australia (www.tropoz.org). 

The plants (27 cv. Williams) were grown under O3 fumigation in OTCs for about three months. At the end of the O₃ fumigation period and when the plants were on average 97 cm in height, two leaves were collected from every plant, specifically the third most recently expanded and therefore newly mature leaf (new leaf) and the eighth-most recently expanded (old leaf) both new and old leaves having fully developed under O3 fumigation. From each leaf, two mid-lamina leaf sections ~300 cm² from both sides of the midrib were taken and measured for total fresh weight. After weighing, and scanning to determine area, one section was, wrapped in tinfoil, snap-frozen in liquid N₂ and stored at –20°C before freeze-drying for biochemical analyses; the other was dried at 70 °C to account for leaf mass lost to sampling.

At the end of the experiment, leaves, midrib, pseudostem, corm and small suckers were harvested separately, and dried in an oven at 70 °C until constant weight for biomass determination.

For all lamina samples collected from the OTC experiment, leaf mass per area (LMA) was calculated using the leaf dry mass (DM) obtained on freeze-dried samples and the leaf area (LA) determined by image analyser software (Image-J, NIH, Bethesda, Maryland, USA). LMA was calculated as LMA= DM/LA in units of g m⁻². Freeze-dried leaf samples were subsequently ground into fine powder (Rocklabs Bench Top Ring Mill) and stored in airtight vials until determination of leaf biochemistry and stable isotope concentrations.

Powdered leaf samples (~30 mg) were extracted in cold 50% acetone (Ritmejerytė et al. 2019). Total antioxidant capacity (TAC) was determined in the leaf extract by the ferric reducing antioxidant power (FRAP) assay. The assay was carried out according to Benzie and Strain (1996) with some modifications. Ascorbic acid was used as the standard and TAC was expressed as ascorbic acid equivalents (mg AAE g⁻¹ dry weight).

Total phenolic content (TPC) was measured in the same leaf extract by the Folin–Ciocalteau method (Cork and Krockenberger 1991; Singleton and Rossi 1965) with some modifications (Ritmejerytė et al. 2019). Gallic acid was used as a standard and TPC was expressed as Gallic acid equivalents (mg GAE g^–1 dry weight).

The carbon stable isotope ratio (δ¹³C, ‰) and weight percent (%C) were determined using a Costech Elemental Analyser fitted with a zero-blank auto-sampler coupled via a ConFloIV to a ThermoFinnigan DeltaVPLUS using Continuous-Flow Isotope Ratio Mass Spectrometry (EA-IRMS) at James Cook University’s Advanced Analytical Centre. Stable isotope results are reported as per mil (‰) deviations from the VPDB reference. Precisions (S.D.) on internal standards were better than 0.1‰  for δ¹³C. The iWUE was calculated from δ¹³C according to the equation of Farquhar et al. (1989).

Environmental variables such as air temperature (T), air relative humidity (RH), shortwave radiation and photosynthetically active radiation (PAR) were monitored using a single meteorological monitoring station (Campbell Scientific, Logan, UT, USA) established in the central OTC.

Hourly values of O₃ and meteorological conditions were measured for the DO₃SE (Deposition of O₃ for Stomatal Exchange) model. The DO₃SE model was used to estimate the O₃ flux into leaves.

References

• Benzie IF, Strain JJ (1996). The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Analytical biochemistry, 239(1), 70-76.
• Cork SJ, Krockenberger AK (1991). Methods and pitfalls of extracting condensed tannins and other phenolics from plants: insights from investigations on Eucalyptus leaves. Journal of chemical ecology, 17(1), 123-134.
• Farquhar GD, Ehleringer JR, Hubick KT (1989). Carbon isotope discrimination and photosynthesis. Annual review of plant biology, 40(1), 503-537.
• Ritmejerytė E, Boughton BA, Bayly MJ, Miller RE (2019). Divergent responses of above-and below-ground chemical defence to nitrogen and phosphorus supply in waratahs (Telopea speciosissima). Functional Plant Biology, 46(12), 1134-1145.
• Singleton VL, Rossi JA (1965). Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture, 16(3), 144-158.