DOI: 10.5061/dryad.fbg79cp26
title: Examining ozone susceptibility in the genus Musa (bananas)

description: 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.
description: The ozone
 (O<sub>3</sub>) 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
 O<sub>3</sub> 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 cm<sup>2</sup> 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 N<sub>2</sub> 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<sup>−2</sup>. 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<sup>−1</sup> 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<sup>–1</sup> dry weight). The carbon stable isotope ratio (δ<sup>13</sup>C, ‰) 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 δ<sup>13</sup>C. The iWUE was calculated from δ<sup>13</sup>C 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 O<sub>3</sub> and meteorological conditions were measured for the DO<sub>3</sub>SE (Deposition of O<sub>3</sub> for Stomatal Exchange) model. The DO<sub>3</sub>SE model was used to estimate the O<sub>3</sub> 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. 
description: # Examining ozone susceptibility in the genus Musa (bananas)
 [https://doi.org/10.5061/dryad.fbg79cp26](https://doi.org/10.5061/dryad.fbg79cp26) The ozone (O<sub>3</sub>) 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](https://tropoz.org/)). The plants (27 cv. Williams) were grown under O<sub>3</sub> fumigation in OTCs for about three months. At the end of the O<sub>3</sub> fumigation period, 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 O<sub>3</sub> fumigation from every plant for the determination of leaf functional traits such as leaf mass per area (LMA), intrinsic water-use-efficiency (iWUE), total antioxidant capacity (TAC), and total phenolic contents (TPC). 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](https://en.wikipedia.org/wiki/Bethesda,_Maryland), USA). LMA was calculated as LMA= DM/LA in units of g m<sup>−2</sup>. TAC was determined in the leaf extract by the ferric reducing antioxidant power (FRAP) assay. TAC was expressed as ascorbic acid equivalents (mg AAE g<sup>−1</sup> dry weight). Ascorbic acid was used as the standard. TPC was measured in the same leaf extract by the Folin–Ciocalteau method with some modifications. Gallic acid was used as a standard and TPC was expressed as Gallic acid equivalents (mg GAE g<sup>–1</sup> dry weight). The carbon stable isotope ratio (δ<sup>13</sup>C, ‰) was  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. The δ<sup>13</sup>C values were used to calculate the iWUE. 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. Values (dataset 1) represent OTC of three plants (n=3). 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 O<sub>3</sub> concentration and meteorological conditions were measured for the DO<sub>3</sub>SE (Deposition of O<sub>3</sub> for Stomatal Exchange) model. The DO<sub>3</sub>SE model was used to estimate the O<sub>3 </sub>flux into leaves. ## Description of the data and file structure Dataset was uploaded in three different excel sheets, Data1, Data2 and Data3 with their metadata (Data1\_Metadata, Data2\_Metadata, and Data3\_Metadata). Metadata sheets represents the parameter names, description, and units. Data1 sheet contains open top chamber averages data. Data2 sheet contains environmental variables during experimental period. Data3 sheet contains hourly values of O<sub>3</sub> concentration and meteorological conditions that were measured for the DO<sub>3</sub>SE (Deposition of O<sub>3</sub> for Stomatal Exchange) model. ## Sharing/Access information Data was produced from our own experimental 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](https://tropoz.org/)). <br> ## Code/Software