# Effects of Climate Conditions before Harvest Date on Edamame Metabolome
## 2 Results
### Annual Fluctuation between Meteorological Conditions and Edamame Metabolome
#### 2.4.2 Annual Fluctuation of Edamame Metabolome  
Similar to climate conditions, the amounts of metabolites in edamame also showed annual variations. To clarify the correlation between the edamame metabolome and the climate conditions before the harvest date, we performed principal component analysis (PCA) (Figure 5). The 2013 and 2016 clusters and other clusters were separated along PC1 (Figure 5A). In PC2, no difference between years was observed (Supplementary Figure S7), but in PC3, the data for 2018 were separated in a positive direction from those of others (Figure 5A). From the loading plot, it was found that sunshine duration, average temperature, and the amounts of some amino acids, such as Ala, Tyr, and Lys, had positive effects on cluster separation in 2013 and 2016 from others, while citric acid, isocitric acid, \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Compounds** & **AT\_1** & **AT\_3** & **AT\_5** & **AT\_7** & **AT\_9** \\ \hline Lys & **0.67** & 0.64 & 0.59 & 0.60 & 0.61 \\ Hydroxy Pro & 0.35 & 0.40 & **0.41** & 0.39 & **0.41** \\ Gly & **0.33** & 0.28 & 0.27 & 0.30 & 0.31 \\ Pro & **0.30** & 0.29 & 0.27 & 0.27 & **0.30** \\ Tyr & 0.16 & 0.17 & 0.25 & 0.33 & **0.41** \\ \hline GDP & \(-\)0.27 & \(-\)0.30 & \(-\)0.36 & \(-\)0.45 & \(-\)**0.48** \\ Citrate & \(-\)0.19 & \(-\)0.24 & \(-\)0.35 & \(-\)0.47 & \(-\)**0.51** \\ Spermie & \(-\)0.23 & \(-\)0.29 & \(-\)0.36 & \(-\)0.41 & \(-\)**0.43** \\ Fumarate & \(-\)0.33 & \(-\)0.30 & \(-\)0.33 & \(-\)**0.36** & \(-\)0.35 \\ NAD & \(-\)0.16 & \(-\)0.20 & \(-\)0.32 & \(-\)0.46 & \(-\)**0.50** \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Top five compounds that showed a correlation or inverse correlation with average temperature (AT). The ranking was determined by the sum of the correlation coefficients from AT_1 to AT_9. Bold numbers indicate the highest correlation or inverse correlation coefficient between AT_1 and AT_9. AT_x means the AT amount accumulated x days before harvest. Figure 4: Annual fluctuation of the accumulated sunshine duration and average temperature during the 5 days before harvesting, shown as SD_5 and AT_5, respectively. GDP, ADP, etc., were found to have a negative impact on the clusters of 2013 and 2016 (Figure 5B). Rainfall, along with guanosine and cytidine, influenced the positive direction of PC3 (Figure 5B). The annual fluctuations in sunshine duration and average temperature matched those expected from the graphs of average values (Figure 4 and Supplement Figures S3 and S4); however, the rainfall contribution to variance was only isolated by PCA. The positive correlation between the sunlight duration and average temperature for the number of amino acids, such as Ala, and the negative correlation for the number of organic acids, such as citric acid, and nucleotides, such as GDP, agreed with the results of the correlation analysis (Tables 1 and 2). The 2018 cluster suggested correlations with rainfall and the amount of nucleosides such as guanosine and cytidine. Furthermore, hierarchical cluster analysis (HCA) was performed to clarify the relationship between weather conditions and the content of compounds in edamame (Figure 6). The cluster at the bottom of this figure (from Phe to SD_3) contained multiple amino acids, including Lys and tyrosine (Tyr), as well as the average temperature, sunshine duration, and accumulated values (Figure 6). This result indicated a strong association between these metabolites and climate conditions. The large central cluster in Figure 5 (from RF_1 to HydroxyPro) included rainfall values and metabolites such as GABA and choline. Many organic acids and nucleotides, such as citric acid and GDP, were identified in clusters separated in the upper group. These metabolites did not show an association with any climate conditions. However, the metabolites clustered in this group indicated inverse correlations to the sunshine duration and average temperature (Tables 1 and 2, Figure 5).