# Effects of Glyoxylic Acid on Metabolism and Ripening of 'Rocha' Pears Treated with 1-MCP
## 3 Results
### Multivariate Analysis Reveals Distinct Clustering of GLA-Treated and CTRL Samples  
A PLS-DA model was developed to evaluate the impact of exogenous GLA treatment on 'Rocha' pear ripening under the effect of 1-MCP. Figure 1 presents the PLS-DA scoresmap and the corresponding VIP scores, where the first two components account for nearly 60% of the observed variability.  
The PLS-DA scores map reveals two well-defined clusters separating the GLA-treated from the CTRL samples (Figure 1). Notably, the CTRL samples from day 0 and day 7 are relatively close, indicating that these fruits maintained their measured parameters with lower variability over time, remaining similar to each other. However, after 15 days, the CTRL samples, while still in the same cluster as the other CTRL samples, were noticeably distanced from the T0 and T7 samples. In contrast, the GLA-treated pears exhibited differentiation over time and from the CTRL samples, particularly between day 7 and day 15, suggesting metabolic modifications driven by the treatment.  
The VIP scores evidence the most relevant variables (measured parameters) that contribute most to the differentiation between the CTRL and GLA-treated samples. From the VIP scores (those higher than 1), it can be observed that the GLA treatment impacted the volatile emission pattern of pears, the respiration rate (CO\({}_{2}\)), the ACC substrate concentration, the TA, the hue angle, and the ACO enzyme activity. These are the contributing variables that played the most significant role in distinguishing the CTRL samples from the GLA-treated samples.  
Additionally, a heatmap was generated to allow for a more detailed analysis of the treatment's impact and highlight specific trends and similarities among samples (Figure 2).  
Combining the VIP scores with the heatmaps obtained from each experiment (Figure 2), it can be corroborated that the GLA treatment mainly impacted the above-mentioned variables. The heatmap analysis demonstrates that the application of GLA had quite an impact on various compounds and physical characteristics, as significant color changes (from blue to red, and vice versa) in the heatmap between the CTRL and GLA conditions can be observed for some parameters, especially after 7 and 15 days. The GLA treatment significantly influenced the production of VOCs. Specifically, hexanal, 2-hexenal, butyl, and hexyl acetate showed increased levels (red intensity) in GLA-treated pears, while other VOCs, such as 2-ethyl-1-hexanol, limonene, and ethanol, exhibited varied or reduced responses to GLA treatment. Additionally, the heatmap shows that exogenous  
Figure 1: PLSDA scores map and VIP scores of ‘Rocha’ pear characteristics after treatment with GLA (T7_GLA and T15_GLA) and their respective controls (T0_CT; T7_CT and T15_CT) across 15 days of shelf-life (T0; T7 and T15).  
GLA increased ACO enzyme activity, while the CTRL samples exhibited a higher ACC concentration until day 7, followed by a decrease after 15 days of shelf-life. Also, based on the color intensity from the heatmap, the GLA-treated samples showed the highest ethylene and TA concentrations at all timepoints compared to the CTRL samples. Furthermore, there was a decrease in sucrose concentration (blue shades in the heatmap) and an increase in CO2 production (more intense red shades in the heatmap) in GLA-treated pears compared to the CTRL.