# Flowering Stage and Daytime Affect Scent Emission of _Malus ioeensis_ \"Prairie Rose\"
## 2 Results
### Diurnal and Nocturnal Release Rhythm of M. ioensis \"Prairie Rose\"  
#### 2.2.1 Identification and Comparison of Volatile Compounds at Different Times  
Table 1 and supplementary Figure S2 show the composition and release rate of _M. ioensis_ \"Prairie Rose\" flowers during the day and night. A total of 15 compounds were detected in the five time periods, and the composition of each time was significantly different (\(P<0.0001\)). According to the classification of compounds (Figure 3A), except for 1:00-3:00 and 15:00-17:00 (benzenoids and aliphatics), the chemical categories at the three other times were mainly benzenoids, terpen, and aliphatics, but the main compounds were not identical in five time periods. The main compounds released from 1:00-3:00 was methyl hexadecanoate; the main compounds released from 6:00-8:00 were \(\alpha\)-pinene (19.89%), methylheptenone (14.65%) 2-(4-methoxyphenyl)ethanol (9.16%) and \(\alpha\)-cedrene (9.10%); the main compounds released from 10:00-12:00 were 2-(4-methoxyphenyl)ethanol (21.99%), methylheptenone (11.49%), dodecane (9.65%) and phenylethanol (8.67%); the main compounds released from 15:00-17:00 were methylheptenone (31.55%) and 2-(4-methoxyphenyl)ethanol (7.84%); and the main compounds released from 20:00-22:00 were \(\alpha\)-pinene (28.54%) and 2-(4-methoxyphenyl)ethanol (4-methoxyphenyl) ethanol (8.09%). The aliphatic and benzenoids content were significant higher in the daytime than at night. In particular, phenylethanol was released only during the day time (Table 1). There were only two common compounds among the five time periods (Figure 3B), and the types of flower volatiles released during the daytime were significantly higher than those released at night (\(P<0.0001\)). Over the nocturnal, diurnal, nocturnal period, the amount of flower volatiles released first increased and then decreased, and the release reached a peak between 10 am and 15 pm (Figure 3C), which is consistent with the pollination biological characteristics of _Malus_ flowers. Figure 2: Principal component analysis (PCA) biplot based on the volatile compounds released from _M. ioensis_ “Prairie Rose” flowers during flowering, showing correlations with volatile compounds (numbers correspond to those in Table 1). S1 bud stage; S2 initial flowering stage; S3 flowering stage; and S4 end of flowering stage. 2.2 Difference Analysis of Volatiles in Different Flower Parts Based on Bray-Curtis Dissimilarity Analysis and Principal Component Analysis  
Bray-Curtis dissimilarity analysis showed that the difference in the volatile compounds at different times ranged from 0.44 to 0.65 (Table 3), which indicates that the floral release compounds of _M. ioensis_ \"Prairie Rose\" changed constantly during the day and night, but approximately half of the compounds in an adjacent time period were similar. To further analyze the contribution of different compounds to the differences in floral scent release over time, we performed PCA (Figure 4) based on 15 compounds (Table 1). The results showed that the total contribution of PC1 and PC2 reached 80%. \(\alpha\)-pinene had high positive scores on PC 1, which were highly positively correlated with the 1:00-3:00 and 20:00-22:00 time periods. Compounds with high negative scores on PC 2 contained 2-(4-methoxyphenylyl)ethanol, which was highly positively related to the 10:00-12:00 time period. The PCA biplot did not overlap among the five periods indicated that the compounds of floral scent differed throughout the day and night. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Times of Day** & **N1** & **D2** & **D3** & **D4** & **N5** \\ \hline N1 & 0 & & & & \\ D2 & 0.65 & 0 & & & \\ D3 & 0.61 & 0.49 & 0 & & \\ D4 & 0.63 & 0.55 & 0.47 & 0 & \\ N5 & 0.60 & 0.44 & 0.63 & 0.59 & 0 \\ \hline \hline \end{tabular} Note: N1 1:00-3:00, D2 6:00–8:00, D3 10:00–12:00, D4 15:00–17:00 and N5 20:00–22:00.  
\end{table} 
Table 3: The Bray-Curtis dissimilarity index of _M. ioensis_ “Prairie Rose” floral scent during the day and night.