Accumulation of phytoene and astaxanthin and related genes expression in Haematococcus pluvialis under sodium acetate stress

Phytoene and astaxanthin are 2 important carotenoids in the green alga Haematococcus pluvialis. Under environmental stress, the synthesis of phytoene in H. pluvialis increases significantly, and phytoene is converted to astaxanthin through enzymatic catalysis. This paper analyzes the relationship between astaxanthin and phytoene accumulation in carotenoid synthesis pathways under different concentrations of sodium acetate (NaAc) by high-performance liquid chromatography. The highest concentrations of phytoene and astaxanthin were observed at the NaAc concentration of 6 g l−1 on the 12th day of induction. The highest astaxanthin concentration achieved was 2.26 ± 0.28%. Therefore, we concluded that 6 g l−1 NaAc and induction for 12 d provided the optimal inducing conditions for astaxanthin accumulation in H. pluvialis. psy, pds, lcyB, β-carotene ketolase crtw, and crtz, which are genes related to phytoene and astaxanthin synthesis, were cloned and studied at the transcriptional level. crtw and crtz were continuously up-regulated since the first day of induction, while psy, pds, and lcyB were continuously up-regulated starting on the 3rd day of induction. These findings are important for enhancing our understanding of the mechanism of accumulation of phytoene and astaxanthin in H. pluvialis and provide a foundation for identifying the induction conditions necessary for optimizing astaxanthin production and increasing astaxanthin yields.


INTRODUCTION
Astaxanthin is a red carotenoid pigment with a high commercial value that has attracted considerable attention for its biological properties, such as its antioxidant, anti-inflammatory, and anti-cancer activities, as well as its coloring for fish (Fujii 2015). Green microalgae are the main natural source of astaxanthin, and also provide ideal models for studying the regulation of ketocarotenoid synthesis (Linden 1999, Grünewald et al. 2001. The unicellular green alga Haematococcus pluvialis is mass cultivated and produced at an industrial scale . The life cycle of H. pluvialis is divided into 2 stages: the green stage and the immobile stage (Boussiba 2000). Astaxanthin is accumulated during the process of transformation from the green motile vegetative cells to the aplanospore stage (Fábregas et al. 2000). Additional work is needed to enhance our understanding of astaxanthin biosynthesis, the accumulation and degradation of carotenoids, and the role of the expression of carotenogenic genes in these processes.
In autotrophic plants and bacteria, carotenoids are important components of photosynthetic membranes and can absorb and transmit light energy. Therefore, when the synthesis of these carotenoids is inhibited, the photosynthesis and growth of plants do not proceed at normal rates (Jackson et al. 2008, Poojary et al. 2016. Carotenoids can be produced during the growth and metabolism of H. pluvialis. Phytoene is an important carotenoid precursor of astaxanthin (Harker & Young 1995), as it is the first carotenoid produced during astaxanthin synthesis. Moreover, the accumulation of phytoene is closely tied to astaxanthin production. Here, we study the relationship between astaxanthin and phytoene under induction conditions to enhance our understanding of the accumulation process of astaxanthin and identify the most optimal induction conditions for astaxanthin synthesis.
Phytoene synthase (PSY) and phytoene desaturase (PDS) are the first 2 enzymes that play fundamental roles in the carotenoid biosynthetic pathway. Phytoene is synthesized from 2 geranylgeranyl diphosphate molecules under the action of PSY, followed by the conversion of phytoene into ζ-carotene by PDS. The formation of β-carotene from lycopene is catalyzed by lycopene cyclase. Astaxanthin biosynthesis follows a general carotenoid biosynthesis pathway up to β-carotene, and astaxanthin is produced with βcarotene as precursor by the action of β-carotene ketolase (CRTW) and β-carotene hydroxylase (CRTZ) (Lotan & Hirschberg 1995, Fraser et al. 1998. Sodium acetate (NaAc) might stimulate the accumulation of astaxanthin in H. pluvialis (Raman & Ravi 2011). Gao et al. (2012) analyzed the transcriptional expression of ipi-1, psy, pds, crtR-B, lyc, ipi-2, bkt, and crtO from H. pluvialis inducted by NaAc in the absence of other stimuli. Given that NaAc is an important inducer of astaxanthin synthesis, we focus on its effects on the transcriptional levels of 5 key genes involved in astaxanthin synthesis -crtw, crtz, psy, pds, and lcyB -to provide a foundation for furthering our understanding of the mechanisms underlying astaxanthin synthesis.
This study uses NaAc as the carbon source to induce red cysts and compares the accumulation of phytoene and astaxanthin under different concentrations of NaAc by high-performance liquid chromatography (HPLC). The transcriptional expression of carotenogenic genes during astaxanthin biosynthesis was analyzed using qRT-PCR. This research will provide a foundation for deepening our understanding of astaxanthin accumulation in H. pluvialis and its optimal induction conditions.

Extraction of carotenoids
Carotenoids were extracted by acetone (Dere et al. 1998). After induction, 100 ml of algal cells were harvested by centrifugation and stored at −80°C for 2−3 h. Next, the algal sludge was freeze-dried and weighed, and the powder was poured into a 10 ml centrifuge tube. Three ml of acetone was added and ultrasonically extracted for 15 min on ice, followed by centrifugation. The supernatant was then transferred to a 5 ml volumetric with the addition of up to 5 ml of acetate. The solution was filtered through a 0.22 μm filter membrane as samples were tested.

Preparation of standard curve
Phytoene and astaxanthin standards were purchased from MD Bio (MD Biosciences). Phytoene and astaxanthin were dissolved by acetone to make the final concentrations either 2, 4, 6, 8, or 10 μg ml −1 . Five different concentrations of standards (20 μl) were injected into the HPLC, and the linear regression equation for the standard curve was obtained by plotting the amount of the standard compound injected against the peak area.

RNA isolation and cDNA synthesization
Algal samples with different concentrations of NaAc were harvested by centrifugation and ground into a fine powder using a mortar and pestle. Total RNA was then extracted using Plant RNA Kit (Omega Bio-Tek). Genome DNA was digested by RNase-Free DNase I Set (Omega Bio-Tek). The cDNA was prepared by using PrimeScript™ RT reagent Kit (TaKaRa).

qRT-PCR analysis
The primers used for amplification of crtz, crtw, psy, pds, and lcyB were all synthesized (Sangon) ( Table 1) according to the sequences obtained from the transcriptome library constructed in our laboratory. The large subunit of ribulose 1, 5-diphosphate carboxylase/oxygenase gene was used as the internal standard. PCR products were then quantified continuously with the BIOER LineGene 9640 (Bioer Technology) using TB Green Premix Ex Taq II (TaKaRa) according to the manufacturer's instructions. The PCR amplification profile was 95°C for 10 min followed by 40 cycles of 95°C for 15 s, and 53°C for 60 s. The 2 −Ct method was used to analyze the transcript abundance of relative gene expressions (Livak & Schmittgen 2001).

Statistical analysis
Graphing and statistical analysis were performed using Origin 8.5 (OriginLab). Student t-tests were used for comparing the cell dry weight of H. pluvialis and the accumulation of astaxanthin and phytoene, under different concentration of NaAc and over time. Pearson correlation analysis was done to evaluate the correlation between accumulation of astaxanthin and phytoene. A p-value of < 0.05 was considered to be significant.

Effect of NaAc concentration on dry weight accumulation of Haematococcus pluvialis
During the induction of Haematococcus pluvialis, the dry weight of the cells in the different concentration groups of NaAc increased to varying degrees (Fig. 1). Medium concentrations (4−5 g l −1 ) of NaAc in the early stage (0 th −4 th day) increased the weight of the algal cells significantly (p < 0.05), and high concentrations (6 g l −1 ) of NaAc reduced the dry weight of the algae slightly from 0.138 to 0.136 g l −1 . From the 8 th to the 12 th day, 3−6 g l −1 concentrations of NaAc all began to promote a steady increase in cell dry weight. On the 16 th day, the 4 groups at different NaAc concentrations had similar dry weights, which were much higher than that of the group without NaAc (p < 0.05). Standard phytoene and astaxanthin were detected by HPLC (Fig. 2). Since the phytoene standard purchased contains 30% cis-phytoene, there were 2 peaks. The first peak corresponded to trans-phytoene and the second peak to cis-phytoene. The peak times of trans-and cis-phytoene were 13.216 and 13.598 min, respectively. The peak time of the astaxanthin standard was 9.584 min.
The standard solutions of the gradient concentrations 2, 4, 6, 8, and 10 μg l −1 were injected into the sampler using an injection needle. The relevant data, peak time and peak area were then recorded. For example, the peak times of peaks I and II are shown in Fig. 2a, while the peak areas of the standards for the gradient concentrations 2, 4, 6, 8, and 10 μg l −1 were calculated to make Fig. 3a. The regression equation of the trans-phytoene is y 1 = 5.488x + 0.2686, R 2 = 0.9996 > 0.735, which has a significant correlation with the concentration of the standard; the regression equation of the cis-phytoene is y 2 = 1.8124x − 0.2384, R 2 = 0.975 > 0.735, which has a significant correlation with the concentration of the standard. Referring to the peak time in Fig. 2b, the peak area of the standard of gradient concentrations of 2, 4, 6, 8, and 10 μg l −1 were calculated to make Fig. 3b. The regression equation of the astaxanthin is y = 4.5161x + 0.6189, R 2 = 0.999 > 0.735, which also has a significant correlation with the concentration of the standard.

Determination of phytoene and astaxanthin in H. pluvialis by HPLC
The samples consisted of carotenoids obtained from H. pluvialis following induction by NaAc and high light. The phytoene and astaxanthin in the samples were determined based on the retention time and peak shape of the standard sample. The peak at 12.926 min corresponded to trans-phytoene, and the peak at 13.541 min to cis-phytoene, and the peak at 9.572 min to astaxanthin (Fig. 4).

Effect of NaAc concentration on phytoene accumulation in H. pluvialis
Phytoene accumulation differed when different concentrations of NaAc were added to the culture medium (Fig. 5). As the NaAc concentration in creased during the early stage (4−8 d), the accumulation of phytoene gradually increased; in contrast, during the later stage (12−16 d), trends in accumulation began to diverge. Phytoene accumulation in the 3 g l −1 NaAc culture continued to in crease and was significantly lower than that in the other NaAc groups during the early stage (p < 0.05); however, phytoene accumulation was higher in the 3 g l −1 NaAc group compared with that in the 4−5 g l −1 NaAc groups on the 16 th day (p < 0.05). The accumulation of phytoene in the 5 g l −1 NaAc group rapidly reached its maximum during the early stage (8 d), which was higher than that in the 3 and 4 g l −1 NaAc groups, but began to decrease in the later stage, with the lowest content of phytoene occurring on the 12 th −16 th day. The 6 g l −1 NaAc group showed the most significant increase in phy- In contrast, the peak of phytoene was too low to be integrated during the 16 d of induction for the group without NaAc.

Effect of NaAc concentration on astaxanthin accumulation in H. pluvialis
The accumulation of astaxanthin at different concentrations of NaAc reached its maximum on the 12 th day (Fig. 6). During the middle phase of induction, astaxanthin accumulation increased stably as NaAc concentration increased (p < 0.05). At an NaAc concentration of 6 g l −1 , the concentration of astaxanthin reached a maximum.

Relationship between astaxanthin and phytoene accumulation in H. pluvialis
To ascertain the relationship between the accumulation of phytoene and astaxanthin, the concentrations of the 2 substances were analyzed together. The accumulation of phytoene and astaxanthin both increased gradually as the concentration of NaAc in creased during the early stage (4−8 d) (Figs. 5 & 6). On the 8 th day, there was a noticeable positive correlation be tween them. Thus, we selected the accumulation of astaxanthin and phytoene de tected on the 8 th day to study the relationship at differ-ent concentrations of NaAc (Fig. 7a). Asta xan thin and phytoene both reached their highest levels when the concentration of NaAc was 6 g l −1 (p < 0.05). Thus, we speculated that 6 g l −1 was the optimal concentration for induction. The relationship between astaxanthin and phytoene under different induction times at an NaAc concentration of 6 g l −1 is shown in Fig. 7b. The astaxanthin and phytoene content increased continuously with in duction time from the 4 th day to the 12 th day. During the late induction period (12 th −16 th day), the contents of astaxanthin and phytoene decreased. Asta xanthin and phytoene both reached their highest levels when the induction period was 12 d (p < 0.05). Thus, a period of 12 d was considered to be the optimal time for in ducing phytoene and astaxanthin production.

Transcriptional-level analysis of carotenogenic genes
The transcriptional levels of genes associated with general carotenogenesis and specific astaxanthin biosynthesis were quantified by qRT-PCR and compared with the transcription of these same genes in green motile cells (Fig. 8). These genes included crtw, crtz, psy, pds, and lcyB. The transcriptional levels of crtw and crtz were both up-regulated since the first day of induction (p < 0.05). The transcripts of crtw of the 5 and 6 g l −1 NaAc groups reached their highest levels on the 9 th day (p < 0.05). The groups at the other 3 NaAc concentrations in creased until the 12 th day. However, the crtz transcripts of 0 and 3 g l −1 reached their highest levels on the 3 rd day after exposure to the inductive conditions, and then reversed. After the 3 rd day, all of the groups maintained a sus-tained increase until the 12 th day, except for the transcripts crtz of the 5 g l −1 NaAc group, which reached its highest level on the 9 th day (p < 0.05). The transcriptional level of psy was up-regulated on the first day of induction, and re duced immediately on the 2 nd day (p < 0.05). The psy transcripts of the 0, 3, and 4 g l −1 groups began to increase from the 3 rd to the 12 th day, and reached their highest level on the 12 th day (p < 0.05). The psy transcripts of the 5 and 6 g l −1 NaAc groups reached their highest levels on the 9 th day. The transcriptional levels of pds and lcyB showed a decreasing trend during the first 2 d (p < 0.05). They then began to in crease starting on the 3 rd day of induction, with the transcripts of the 4 and 6 g l −1 NaAc groups reaching their highest levels on the 12 th day (p < 0.05). Overall, all 5 genes were highly transcribed under NaAc concentrations of 4 and 6 g l −1 on the 12 th day. The optimal induction concentration of NaAc and induction time for maximizing the production of astaxanthin and phytoene in H. pluvialis was 6 g l −1 and 12 d, respectively.

DISCUSSION
Haematococcus pluvialis has been shown to accumulate large amounts of carotenoids when exposed to stress, such as nitrogen and phosphate limitation, salt stress, and high light intensities (Johnson & Schroeder 1996). Astaxanthin content or cell density of H. pluvialis under nitrogen starvations was significantly lower than that under conditions with sufficient nitrogen (Jinxun et al. 2016). Although astaxanthin accumulation is usually conducted by high light condition (Scibilia et al. 2015), excess light energy may accelerate the generation of reactive oxygen species (ROS), resulting in photoinhibition (Zhang et al. 2011). Conditions contributing to astaxanthin accumulation in H. pluvialis include high light intensity (175 μmol photons m −2 s −1 ), nitrogen limitation, phosphate or sulfate starvation, and salt stress (0.8% NaCl) (Boussiba et al. 1992). We selected a light intensity of 172 μmol photons m −2 s −1 without nitrogen to induce red cysts in the present study. Previous work has shown that when NaAc acts as the carbon source, astaxanthin accumulation in H. pluvialis is accelerated (Orosa et al. 2001). Exogenous NaAc enhances astaxanthin accumulation and photoprotection capacity in H. pluvialis at the non-motile stage (Zhang et al. 2018). In our study, phytoene and astaxanthin accumulated little in the group without NaAc, suggesting that NaAc plays an important role in carotenoid synthesis. The accumulation of phytoene showed different trends in the early induction phase and in the later induction phase. During the initial stage of induction, the concentration of phytoene increased as the concentration of NaAc increased, indicating that phytoene, the upstream meta bolite of caro tenoids, was primarily synthesized during this period. Phytoene is consumed as a precursor of astaxanthin in the later induction period; thus, the concentration of phytoene showed different trends under different concentrations of NaAc. High concentrations of NaAc in H. pluvialis can in crease the accumulation of both phyto ene and asta xanthin. The optimal induction concentration of NaAc was inferred to be 6 g l −1 and the induction time 12 d.
Related studies have found that high concentrations of NaAc are not conducive to cell growth. Although high salinity can accelerate the formation of cysts in H. pluvialis, excessive salinity can promote cell death (Tao et al. 2015). In our study, medium concentrations (3−5 g l −1 ) of NaAc during the early induction stage increased the weight of the algal cells, while high concentrations (e.g. 6 g l −1 ) of NaAc reduced the dry weight of algal cells during the initial induction stage, which promoted cell death. However, over medium timeframes (8 th day), a high concentration (e.g. 6 g l −1 ) of NaAc promoted increases in dry weight. The explanation for this pattern might be that the high concentration of NaAc induced algal cells to produce thicker cyst walls as a self-protective mechanism, which might have increased cell mass rather than cell growth (He et al. 2018). The effect of NaAc on astaxanthin accumulation is always integrated with increased biomass production (Göksan et al. 2010). Astaxanthin is known to accumulate in the thick-walled cyst cells of H. pluvialis (Sarada et al. 2006). Thus, we infer that the optimal induction concentration of NaAc is 6 g l −1 , and the induction time is 12 d.
Carotenogenic genes, such as bkt and chy, along with psy, pds, and lcy, were all up-regulated under stresses, such as high light intensity, salinity, acetate addition, and nutrient depletion. These carotenogenic gene transcripts have even been detected in 3 mo old cysts (Vidhyavathi et al. 2007(Vidhyavathi et al. , 2008. When green cells were transformed to red cysts under stress, total carotenoids in the cells increased and ketocaro ten oids were preferentially formed (Huang et al. 2006). This pattern was coupled with the upregulation of the genes for β-carotene ketolase and hydroxylase (Grüne wald et al. 2000, Steinbrenner & Linden 2001). The bkt and crtz genes are not expressed in the absence of stress (Lemoine & Schoefs 2010). In this study, crtw and crtz were continually up-regulated under the stress of high light and NaAc, probably because they are 2 key enzymes involved in astaxanthin synthesis. PSY plays an important role in the first major reaction of the carotenoid biosynthetic pathway (Cárdenas et Fig. 7. Relationship between astaxanthin and phytoene accumulation in Haematococcus pluvialis: (a) in different sodium acetate concentrations, and (b) under different detection times. All data show mean ± SD. Three independent experiments conducted (n = 3). *p < 0.05 al. 2012). The expression of psy under different environmental stresses showed that the expression of psy could be detected after 4 h of induction and reached its highest level after 1−2 d under the simultaneous application of NaAc, ferrous sulfate, and high light intensity (Jia et al. 2014). psy is overexpressed in lettuce cells under salt stress, suggesting that changes in psy expression depend on the relative degrees of external stress. We found that psy was up-regulated on the 1 st day of induction. Although its transcriptional level was lower the 2 nd day, on the 3 rd day, the transcription level of psy began to rise again. The transcriptional level of psy increased as the concentration of NaAc increased, which is consistent with patterns in the accumulation of phytoene and astaxanthin. Previous studies have primarily focused on the transcriptional level of pds and found that mRNA levels of pds are highest when astaxanthin accumulates (Giuliano et al. 1993, Steinbrenner & Linden 2001. Trends in the transcription of pds were below their initial value during the first 2 d, at which point it began to be up-regulated. Grünewald et al. (2000) have demonstrated that pds is up-regulated at the mRNA level during accumulation of secondary carotenoids in H. pluvialis. The down-regulation of psy and pds might be caused by environmental changes. Changes in lcyB under induction conditions are inconsistent with changes in psy and pds. Studies have shown that a certain range of salt concentrations can up-or down-regulate the expression of lcyB, and the expression of lcyB in leaves of different processed tomato varieties increases as the concentration of salt increases (Zhang et al. 2014). These differences might stem from the fact that the genotypes of different varieties vary in salt tolerance (Liang et al. 2009). For example, RNAi technology has been used to inhibit the expression of lcyB and lycopene content of transgenic tomatoes has been found to be significantly increased. In addition, under intense light and nitrogen-deficiency conditions, there was no significant increase in lcyB expression in wildtype H. pluvialis when cells changed from green to red (Wei & Madeline 2002). In our study, the expression of lcyB, like other carotenogenic genes, increased as the concentration of NaAc increased and its expression appeared to exhibit certain synergy with other carotenogenic genes.