# Flowering Stage and Daytime Affect Scent Emission of _Malus ioeensis_ \"Prairie Rose\"
## 2 Results  
### Volatile Compounds Emission Characteristic of Different Flowering Stages  
#### 2.1.1 Identification and Comparison of Volatile Compounds of Flowers at Different Flowering Stages  
A total of 25 volatile compounds were identified from the four flowering stages of _M. ioensis_ \"Prairie Rose\" (Table 1, supplementary Figure S1), and the compounds released during these were significantly different (\(P<0.0001\)). Classification revealed that the main chemical categories of flower scent in the four flowering stages were terpen, benzenoids and aliphatics (Figure 1A), but the main volatile compounds were not identical in these flowering stages (Table 1). Concretely, the main compounds in the bud stage (S1) were 2-(4-methoxyphenylyl)ethanol (16.36%), methylheptenone (16.56%) and \(\alpha\)-cedrene (11.71%). The main compounds in the initial flowering stage (S2) were geranylacetone (10.70%) and methylheptenone (6.92%). The main compounds of the flowering stage (S3) was phenylethanol (10.60%), geranylacetone (9.64%) and methylheptenone (8.08%). The main compounds at the end of flowering (S4) were 2-(4-methoxyphenylyl)ethanol (16.36%), methylheptenone (11.72%), \(\alpha\)-cedrene (12.41%), geranylacetone (10.19%), and phenylethanol (7.51%). Interestingly, phenylethanol released the most at flowering stage. The main compounds in the bud stage and at the end of flowering were similar and their proportions were comparable (Table 1). There were eight common compounds in the four flowering stages (Figure 1B, Table 1). Although the numbers of compounds (18) and specific compounds (5) released during the bud stage were the highest, the amounts of compounds released during the initial flowering stage were the highest. From the bud stage to the end of flowering, the total amount of volatile compounds released showed an initial increase followed by a decrease (Figure 1C). \begin{table}
\begin{tabular}{c c c c c c c c c c c c c c c} \hline \hline \multirow{2}{*}{No.} & \multirow{2}{*}{Templenia} & \multirow{2}{*}{Components} & \multirow{2}{*}{CAS} &  
1.2 Difference Analysis of Flower Volatiles Based on Bray-Curtis Dissimilarity Analysis and Principal Component Analysis (PCA)  
Bray-Curtis dissimilarity analysis is often used to compare the degree of dissimilarity between two samples [26]. The larger the value, the greater the difference between two subjects (low similarity). For all identified compounds, the dissimilarity among the four flower stages ranged from 0.21 to 0.52 (Table 2). The floral scents in the initial and flowering stages were the most similar (dissimilarity 0.21), and the floral scents in the bud stage and at the end of flowering were also comparable (dissimilarity 0.27). To further analyze the contribution of different compounds to the floral variation among the four flowering stages, principal component analysis (PCA) was performed based on the 25 compounds (Table 1, Figure 2). The results show that the total contribution of the first principal component (PC1) and the second principal component (PC2) reached 93.0%. Compounds with high scores on PC 1 comprised \(\alpha\)-cedrene and 2-(4-methoxyphenyl)ethanol, which were obviously positively related to the bud stage and the end of flowering stage. Compounds that had high positive scores on PC 2 included phenylethanol and geranylacetone, which were highly positively correlated with the flowering stage. The four flowering stages were distributed in four coordinate quadrants without overlapping, which indicated that the compounds of floral scent differed throughout the whole flower development. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Flowering Stage** & **S1** & **S2** & **S3** & **S4** \\ \hline S1 & 0 & & & \\ S2 & 0.52 & 0 & & \\ S3 & 0.52 & 0.21 & 0 & \\ S4 & 0.27 & 0.48 & 0.41 & 0 \\ \hline \hline \end{tabular} Note: S1 bud stage; S2 initial flowering stage; S3 flowering stage; and S4 end of flowering stage.  
\end{table} 
Table 2: The Bray-Curtis dissimilarity index among different stages of _M. ioensis_ “Prairie Rose” flowers.