Originpro 2023

All MS data was processed using Xcalibur
(version 4.3), OriginPro 2023, and Python. Titration data were fit
with a sequential binding model with slight changes made to previously
described methods.^{45 (link)} Notably, to calculate
mole fractions as a function of the total Ca²⁺ concentration
(rather than the free ligand concentration), equilibrium populations
were calculated from kinetic simulations of the system. K_D values are reported as mean ± s.d., where standard
deviations were derived from an estimated covariance matrix and are
plotted as error bars in Figure 3d. P-values were determined using
a two-sampled t test in OriginPro 2023. The K_D values for Ca²⁺ binding were not
determined for the truncations.

The relative percentage area of each IR band in the region of 1500 to 1700 cm⁻¹ was determined by the second-derivative transformation of raw absorption spectra to resolve overlapping bands using OriginPro 2023 (Origin Lab, MA, Northampton, USA) [62 (link)]. Raw spectral data were first normalized, smoothed (SG, 5-points), second derivative-transformed (SG, 7-points), multiplied by (−1) to invert second derivative IR bands, and finally baseline-corrected. Then, absorbance spectra were fitted using full width at half maximum (FWHM) Gaussian band profiles using the Multipeak option (Figure 1). In the fitting process, the height and width of selected IR bands varied until the best fit with the experimental curve (Chi-square tolerance value of 1 × 10⁻⁹, <400 iterations) was found. The goodness of the fit was determined by observing the F-statistics and values of Chi-square [63 (link)]. In order to test the statistical significance of the relative percentage area of each IR band deconvoluted, one-factor analysis of variance (ANOVA) was performed using OriginPro 2023 (Origin Lab, Northampton, MA, USA). The mean values were compared using Tukey’s test at the 5% significance level.

All analyses were performed in three independent replications for each sample. The contents of phenolic compounds, arbutin hydroquinone content and antioxidant activity were expressed as the mean ± standard deviation. The acquired findings were subjected to statistical analyses with the use of Statistica v13.1 (StatSoft Inc., Tulsa, OK, USA). Significant differences between types of highbush blueberry leaves based on the tested parameters were obtained through a one-way analysis of variance (ANOVA), followed by the Tukey multiple comparison test. The multivariate statistical analysis was performed using cluster analysis (CA), combined with a heat map with the use of Statistica v13.1 (StatSoft Inc., Tulsa, OK, USA) and principal component analysis (PCA) with the use of OriginPro 2023 (OriginLab Corporation, Northampton, MA, USA). Correlations between tested parameters were established using Pearson’s correlation test and presented as a triangle heat map with the use of OriginPro 2023 (OriginLab Corporation, Northampton, MA, USA).