Origin 2022

Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were carried out to examine the separation of individual samples and the overall difference in metabolites between the two groups. Statistical significance (p value) was determined by univariate analysis (t test). The screening criteria for the differentially abundant metabolites (DAMs) were variable importance in projection (VIP) value > 1, p value < 0.05 and fold change (FC) ≥ 2 or FC ≤ 0.5 [29 (link),30 (link)]. The functions and metabolic pathways of the DAMs were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The metabolic pathways were considered enriched when the ratio satisfied x/n > y/n and were statistically significant when the p value < 0.05. The fruit physiological and quality index data were all analyzed with SPSS 20.0 software and Microsoft Office Excel 2016. One-way analysis of variance (ANOVA) and Tukey’s test were performed to detect possible differences among the different substrate treatments, and significant differences were expressed as p < 0.05. Correlation analysis and PCA of physiological and quality indexes were performed with Origin 2022 (Origin Lab Inc., Northampton, MA, USA).

The phosphatase assay was conducted with the EnzCheck Phosphatase Assay Kit (Molecular Probes, Eugene, OR, USA). The 100 μL reaction mixture in the 96-well black plate for the measurement of phosphatase activity consisted of a 100 μM 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) substrate, 1 mM metal such as Mg²⁺, Zn²⁺, Mn²⁺, or Ca²⁺, 100 mM NaCl, 25 mM Tris-HCl pH 7.0, and a 10 μM protein sample. The reaction was started by adding the protein, and the results were measured with the Filter Max F3 (Molecular Devices, San Jose, CA, USA) as soon as possible after adding the protein to the reaction mixture. The interval of measurement was 3 min. The amount of the product, 6,8-difluoro-4-methylumbelliferyl (DiFMU), in the solution was determined photometrically at 460 nm. The graph for the phosphatase assay was modified using Origin 2022 (OriginLab, Northampton, MA, USA). The data were fitted to the quadratic equation to calculate a Kd value between Mg²⁺ and Sav2152, as follows: $$\mathrm{V}={\mathrm{V}}_{\mathrm{m}\mathrm{a}\mathrm{x}}\times \frac{\left[{\mathrm{P}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]+\left[{\mathrm{A}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]+\mathrm{K}\mathrm{d}-\sqrt{{\left(\left[{\mathrm{P}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]+\left[{\mathrm{A}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]+\mathrm{K}\mathrm{d}\right)}^2-4\times \left[{\mathrm{P}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]\times \left[{\mathrm{A}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]\times \mathrm{K}\mathrm{d}}}{2\left[{\mathrm{P}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]}$$
where V represents the initial reaction velocity, V_max represents the maximum initial reaction velocity, P_total represents the total amount of protein, A_total represents the total amount of Mg²⁺ ion, and Kd represents a dissociation constant.

Statistical analyses were conducted using Origin 2022 (OriginLab Corporation, Northampton, MA, USA) and SPSS 22.0 (IBM SPSS, Chicago, IL, USA) software. Significant differences between the control and treated cultures were analyzed using a one-way ANOVA test followed by the LSD test or the Games-Howell test. Values were considered significant at p < 0.05.

The sequencing data were analyzed using the Majorbio Platform, Shanghai. Using Chromas, NCBI Blast,¹ and Mega v11.0 to analyze gene sequences. All the high-quality recovered sequences were blasted in the NCBI database. Mothur software was used to group sequences with 97% similarity into the same operational classification unit (OTU). The phylogenetic tree of hzsB gene sequence was constructed with the NJ (neighbor-joining) method through Mega v11.0. An Origin 2022 (OriginLab Corp., Northampton. MA, United States) were used to plot the contents of ammonium, nitrate nitrogen and the abundance of anammox bacteria. One-way analysis of variance (ANOVA) was used to analyze soil samples in SPSS25.0.

Data analysis was performed using pCLAMP (Axon Instruments, Foster City, CA, USA), Origin 2022 (OriginLab Corp, Northampton, MA, USA), Excel (Microsoft, Redmond, WA, USA) and GraphPad Prism (GraphPad Software, San Diego, CA, USA). Statistical significance was determined using Student’s t-test. The values were presented as mean ± standard error of the mean (SEM) and were considered statistically significant when the p-value was less than 0.05. Statistically significant differences were assessed using the paired t-test at *p < 0.05, **p < 0.01 or ***p < 0.001.