## 3 Discussion  
### Synthesis of Astaxanthin and Fatty Acids under Blue Light Irradiation  
FPPS is a key enzyme in isoprenoid biosynthesis [55], providing the precursors (i.e., sesquiterpene) for carotenoids. Our results showed that the expression of _fpps_ was significantly regulated on the second day of blue light irradiation. Then, the flow of precursors of astaxanthin synthesis was reduced. Both PSY and PDS are the rate-limiting enzymes in the carotenoid biosynthesis pathway of _H. pluvialis_[56; 57]. Furthermore, both BKT and CRTR-B are involved in the synthesis of astaxanthin in _H. pluvialis_[58]. Therefore, these four enzymes are essential for the synthesis of astaxanthin in _H. pluvialis_. Previous studies showed that, compared with white light of 150 \(\mu\)mol photons m\({}^{-2}\)s\({}^{-1}\), blue light of \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Transcription Factor** & **B02 vs. W02** & **B07 vs. W07** & **B257 vs. B07** \\ \hline C2H2 & 45 & 30 & 44 \\ C3H & 20 & 20 & 16 \\ GNAT & – & 13 & 13 \\ HMG & 17 & 12 & 16 \\ Jumonji & 13 & 10 & – \\ MYB & 21 & – & 16 \\ MYB-related & 18 & 12 & 21 \\ SET & 13 & 17 & 16 \\ SNF2 & 21 & 41 & 38 \\ TRAF & 38 & 57 & 50 \\ zn-clus & 31 & 43 & 40 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Top 10 transcription factor (TF) families with number of genes significantly expressed in different pairwise comparisons of _Haematococcus pluvialis_. Symbol “–” indicates the TFs not included in the pairwise comparisons. 70 \(\mu\)mol photons m\({}^{-2}\)s\({}^{-1}\) significantly increased the expression of _psy_, _lcy_, _bkt_, and _crttR-B_ in _H. pluvialis_[9]. Our results showed that blue light reduced the expression of genes involved in astaxanthin synthesis in early stage of blue light irradiation, probably due to the different light intensities, suggesting that high-intensity blue light was not conducive for astaxanthin synthesis. It was expected that the carotenogenesis was correlated with the patterns of gene and protein expressions revealed in the current study. Future complementary studies are necessary to investigate the contents of astaxanthin and other important metabolites produced in _H. pluvialis_, i.e., carotenoids, chlorophylls, amino acids (i.e., glutamate), and lipids. Moreover, both the qRT-PCR and genetic knockout experiments are required to verify the expression patterns and functions of genes and proteins involved in astaxanthin biosynthesis pathway with the goals to explore the molecular mechanisms underlying the production of astaxanthin in _H. pluvialis_ treated with high-intensity blue light and SA. Previous studies showed that high light irradiation enhanced the synthesis of fatty acids in _H. pluvialis_, and the gene expression was significantly up-regulated with the increase in treatment time of high light irradiation [3; 29]. However, our results showed that on the second day of the treatment with blue light irradiation, both gene and protein expressions involved in the production of acetyl-CoA and NADPH were significantly down-regulated, and the genes and proteins directly involved in the fatty acid synthesis were also significantly down-regulated, ultimately leading to a decrease in fatty acid synthesis. However, no significant changes were observed in these genes on day 7 of the treatment with blue light irradiation. Compared with day 7, the shorter treatment time (day 2) of blue light irradiation showed a greater effect on the production of astaxanthin of _H. pluvialis_, specifically resulting in the significant down-regulation of genes involved in astaxanthin synthesis (Figure 3). These results were consistent with those of the changes in gene and protein expressions involved in the biosynthesis of fatty acids (Figures 10 and 11). Because astaxanthin is esterified with fatty acids, the content of fatty acids is regulated with the change of astaxanthin content [59]. ROS burst in _H. pluvialis_ was observed at the beginning of the treatment of light irradiation [44]. It is important for the cells of _H.