Computational protocol: The Effect of Differential Growth Rates across Plants on Spectral Predictions of Physiological Parameters

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[…] Stomatal conductance (gs) and net CO2 assimilation rates (AN) of the youngest, fully-matured leaf of each plant were collected using a portable Li-6400 Infrared Gas Analyzer (Li-Cor Biosciences Inc., NE, USA). The open photosynthesis system was equipped with an external carbon dioxide source, in order to maintain the leaf chamber at a concentration of 400 µmol mol−1. Temperature and relative humidity range were 28°C and 30–55%, respectively, and the photosynthetic active radiation (PAR) rate was 1000 µmol photons m−2 s−1. Gas exchange measurements were taken inside the greenhouse.Foliage area was determined using a regression model that correlated the surface dimensions of a leaf with its actual area. After logging the width and length of 30 leaves, their pictures were then taken against a white background. The images were then made binary using The GIMP 2.6.11 software (http://www.gimp.org), so that the real areas could be derived from the sum of all relevant pixels. Eventually, a least squares linear regression model was found to best predict the actual leaf area from width and length (Image area  = 1.72× Squared area +12.1; R2 = 0.95; p<0.01). [...] At every time-point, an ASD Pro-FR Field Spectroradiometer (Analytical Spectral Devices Inc., CO, USA), equipped with an 1800-12S Integrating Sphere (Li-Cor Biosciences Inc., NE, USA), was used to log the signature of the oldest leaf in every fourth node of each vine, starting from the terminal bud. The system was set to measure mean radiance units (each measurement was averaged out of 20 scans) in the 350–2500 nm range, at 1 nm spectral resolution. Normalized values of leaf reflectance (ρ) were derived from complementary energy flux measurements of white-reference standards (BaSO4 tablets) and dark current, as suggested in the Integrating Sphere's manual. The reflectance data was used to exert several narrow-band, water stress-related indices: Normalized Difference Vegetation Index (NDVI; ; (ρ800 nm−ρ670 nm)/(ρ800 nm +ρ670 nm)), Water Index (WI; ; (ρ900 nm/ρ970 nm)), Normalized Difference Infrared Index (NDII; ; (ρ820 nm−ρ1650 nm)/(ρ820 nm +ρ1650 nm)), Moisture Stress Index (MSI; ; (ρ1600 nm/ρ820 nm)) and Structure Insensitive Pigment Index (SIPI; ; (ρ800 nm−ρ445 nm)/(ρ800 nm−ρ680 nm)). [...] All statistical calculations were performed using the SigmaPlot 12.0 software (Systat Software Inc., IL, USA). Differences between mean values were assessed using one-way Analysis of Variation (ANOVA) test, which was followed by Tukey's Honest Significance Difference (HSD) post-hoc test whenever necessary. Prior to those tests, assumptions of residuals' normality and homoscedasticity were checked and met using Shapiro-Wilk and Bartlett's tests, respectively. Least squares regressions were used in order to describe the continuous relationship between various variables. Results of all comparison tests and correlation analyses were considered significant at p<0.05. […]

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