Origin version 2021

The drawings and statistical analysis were performed using MS Excel and Origin version 2021 (Origin Lab., Hampton, VA, USA). All data are presented as the arithmetic mean ± standard deviation. For the calculation of the statistical significance, two-sample t-tests (two-tailed) were performed (alpha level 0.05). An analysis of variance was performed to test for equal variances prior to t-test analysis.

Origin version 2021 (Origin Lab., Hampton, MA, USA) was used to perform statistical analysis and drawing. All data are presented as the mean ± SD (Standard Deviation). Comparisons between groups were conducted using one-way ANOVA or Student’s t-test. Moreover, UPLC-MS/MS analyses were performed, where metabolites with a variable important in projection (VIP) ≥1 and fold change (FC) ≥2 or FC ≤0.5 were considered as criteria for screening potential biomarkers. The VIP values were extracted from the orthogonal partial least-squares discriminant analysis (OPLS-DA) results, which also contain score plots and permutation plots. These results were generated using the R package MetaboAnalystR (https://www.metaboanalyst.ca/docs/RTutorial.xhtml, accessed on 21 October 2020). Significantly different metabolites were analyzed based on VIP values and Log₂FC. A p-value < 0.05 was considered statistically significant.

Origin (Version 2021, Origin Lab Corporation, Northampton, MA, USA) was used for data analysis, plotting, and statistical evaluation. Raw data analysis was performed using MITlaos software (MATLAB R2016a, Natick, MA, USA). All data were tested in triplicate, represented as the mean ± standard deviation (SD) and statistically analyzed using one-way ANOVA in SPSS (version 24.0, SPSS Inc., Chicago, IL, USA) with a 95% confidence level. p < 0.05 was considered as significant difference.

The growth characteristics of summer maize under different drought conditions were analyzed by taking the difference between the drought-stressed and no drought-stressed summer maize indexes. The drought treatment group data minus the mean value of the control group data was used to represent the variation of each index. Difference data were shown in mean + standard deviation. The figures were drawn in Origin version 2021 (OriginLab Inc., Hampton, MA, USA).

Data were checked for normal distribution with the one-sample Kolmogorov–Smirnov test and for homogeneity of variances with Levene’s test. One-way analysis of variance (ANOVA) and the Duncan test were used to determine the differences among different groups of samples. When normal distribution and/or homoscedasticity were not achieved, data were subjected to the Kruskall–Wallis non-parametric test. Statistical analyses were performed using SPSS version 20.0 (SPSS Inc, Chicago, IL, USA) with a significance level of p < 0.05. Microsoft Excel 2019 and Origin version 2021 (OriginLab, Northampton, MA, USA) were used to generate additional plots.

Data were imported into SIMCA version 17.0.2 (Satorius AG, Goettingen, Germany) and multivariate statistical analysis including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed. The unit variance scaling was applied to normalize the intensities of lipid features prior to analytical model fitting. The quality of PLS-DA model was evaluated by the values of explanatory parameter R² and predictive parameter Q². To avoid overfitting of analytical mode, thousand permutations test was carried out on the model. For univariate statistical analysis, we used Student’s t-test to obtain p-value. Additionally, the receiver operating characteristic (ROC) curve was used for the discovery of biomarkers. For heatmap analysis, data was normalized by the area of internal standard for each lipid species, log-transformed and auto-scaled using Metaboanalyst version 5.0, “http://www.metaboanalyst.ca (accessed on 1 December 2022)”, and data visualization was conducted using Microsoft office Excel 2016. Differences in the biomarkers were compared using Origin, Version 2021 (Origin Lab Corporation, Northampton, MA, USA)

Statistical analysis was performed using Origin Version 2021 (OriginLab Corporation, Northampton, MA, United States, Version 9.8.0.200). Results are presented as mean values with corresponding confidence interval (CI) at 95% confidence level calculated from triplicate analyses (Cumming et al., 2007 (link)).