Computational protocol: Plant responses to heterogeneous salinity: growth of the halophyte Atriplex nummularia is determined by the root-weighted mean salinity of the root zone

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[…] Plants were sampled on days 0 and 21 after the commencement of treatments for the determination of shoot and root DM. Stem length was measured with a ruler on days 0 and 21 to determine the extension rate during the treatment period. In order to assess any differences in root DM between sides, the two sides of each root system were harvested separately. Young fully expanded leaves (one for plants in uniform treatments and two for heterogeneous treatments, i.e. one leaf from each side of the plant) were collected between 11.00h and 11.30h for subsequent analyses of ions and total soluble sugars. These leaf tissues were snap-frozen in liquid N2, stored at –80 °C, freeze-dried, and then stored at –20°C. All remaining shoots were oven-dried at 60°C. For root tissues, a subsample of the roots for each treatment was taken to determine root surface area per unit root DM, so that total root surface area could be estimated from total root DM. Roots were scanned for surface area using a WinRhizo root scanner (Regent Instruments Inc., Quebec, Canada) with a resolution that would have captured main and fine lateral roots but not root hairs. These root subsamples, and all remaining roots, were oven dried at 60 °C to determine DM. [...] Statistical analyses were conducted using Genstat for Windows 10th Edition (Genstat software, VSN International, Hemel Hempsted, UK). Analysis of variance (ANOVA) was used to identify overall significant differences between treatments and between sides within treatments, depending on the data set. When significant differences were found, mean separations were calculated using Duncan’s multiple range test. Unless otherwise stated, the significance level was P ≤ 0.05. Linear or polynomial (quadratic or cubic) regression analyses were performed with SigmaPlot 11.0 (Systat Software Inc., Version 11.0, Chicago, IL, USA). Cubic regression curves were fitted to growth (stem extension rate and shoot ethanol-insoluble DM) and stomatal conductance data because these best describe the growth pattern expected for a dicotyledonous halophyte in response to increasing salinity, incorporating an optimum at moderate salinities (cf. ) with decreases in growth at salinities above the optimum (cf. the compound discount curve of ). In contrast, visual inspection of the relationships between leaf Na+ or shoot water potential against external salinity showed that simple linear and quadratic relationships, respectively, could be fitted to these data (see the Results). […]

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