Chemical Genetics Approach Reveals Importance of cAMP and MAP Kinase Signaling to Lipid and Carotenoid Biosynthesis in Microalgae

Abstract

In this study, we attempted to understand signaling pathways behind lipid biosynthesis by employing a chemical genetics approach based on small molecule inhibitors. Specific signaling inhibitors of MAP kinase or modulators of cAMP signaling were selected to evaluate the functional roles of each of the key signaling pathways in three different microalgal species: Chlamydomonas reinhardtii, Chlorella vulgaris, and Haematococcus pluvialis. Our results clearly indicate that cAMP signaling pathways are indeed positively associated with microalgal lipid biosynthesis. In contrast, MAP kinase pathways in three microalgal species are all negatively implicated in both lipid and carotenoid biosynthesis.

Introduction

We are now facing the depletion of fossil fuels as well as serious environmental pollution owing to intensive utilization of petroleum resources. Therefore, bioenergy derived from biomass has attracted increased scientific and public attention, since it can provide fundamental solutions as a renewable and eco-friendly energy source. In particular, microalgal-based biofuels have great potential, because microalgae have a high rate of biomass productivity and high percentage of lipid content, which could be utilized as a biodiesel source. For example, biomass productivity using microalgae was estimated to be 50 times more than that using fast-growing territorial plants [9,19]. Owing to the competitive advantage of biofuel based on microalgal biomass, it is critical that we gain a better understanding of microalgal biology. However, our understanding of microalgal biology is in its infancy, requiring significantly more research and technical breakthroughs.

Every eukaryotic organism including microalgae should respond actively to various environmental cues to survive or adapt appropriately. To these ends, various signaling pathways upon external environmental stimuli are turned on or off with tremendous amplifications or inter-connections. Two well-conserved signaling cascades, MAP kinase and cAMP signaling pathways, have been known to play pivotal roles in many eukaryotic organisms. MAP kinase and cAMP signaling cascades function in a parallel way to transduce signals, controlling a wide variety of processes such as developments, differentiations, and metabolic processes in eukaryotic cells [5,10,14,15,18].

The MAP kinase signaling cascade normally consists of three enzymes: MAP kinase kinase kinase (MEKK or MAP 3K), MAP kinase kinase (MEK or MAP 2K), and MAP kinase (MAPK), which are activated in series. Upon responding to appropriate extracellular signals, the sequential activation of the MEKK-MEK-MAPK cascade via phosphorylation is allowed to occur and eventually leads to expression of a specific sets of genes. For example, in Saccharomyces cerevisiae, five MAPK pathways are known to regulate fundamental phenotypes such as mating, ascospore formation, invasive growth, cell wall integrity, and hyperosmoregulation [14].

On the other hand, cAMP is the key secondary messenger important in multiple biological processes, switching on/off a wide variety of downstream targets in many different organisms. Owing to its significance in cellular signaling, cellular levels of cAMP are tightly regulated by the activities of the adenylate cyclase and cAMP phosphodiesterase, which can synthesize or degrade cAMP, respectively. IBMX (3-isobutyl-1-methylxanthine) is a well-known chemical modulator of cAMP signaling, and it inhibits cAMP phosphodiesterase, thereby leading to an increase in cellular cAMP levels [12,21]. The downstream target of adenylate cyclase via cAMP is protein kinase A (PKA), which, in its resting state, exists as a tetramer consisting of two catalytic subunits and two regulatory subunits. Upon binding with cAMP, a conformational change in the regulatory subunit results in its disassociation from the catalytic subunit. The free catalytic subunits of PKA can then catalyze the phosphorylation of serine or threonine residues on target proteins, such as several different transcription factors [10].

Despite the central roles of MAP kinase and cAMP signaling pathway in regulating a wide variety of cellular functions, no information is available on the functions of these signaling cascades in microalgal species. Although several studies have described microalgal gene expression during nitrogen starvation or nitrogen stress response in order to understand lipid metabolism, there has been no study on microalgal signaling [2,13,23]. Therefore, it is obvious that we have very limited knowledge on the genes and signaling pathways in microalgae, particularly signaling pathways associated with lipid biosynthesis in microalgae.

Chemical genetics is a recently developed approach to study the functional role(s) of gene(s) by using small molecules, which could bind specific proteins, mediating important signaling pathways [1,22]. Therefore, chemical genetics using small molecules of interest leads to the alteration of functions of particular signal transduction pathways in living organisms, and it is analogous to classic gene deletion study. Owing to the difficulties of loss-of-function genetic mutations in microalgae or the inefficiency of single gene deletions stemming from the presence of redundant genes, chemical genetics can act as a powerful and straightforward tool to understand cellular function(s) of signaling(s).

In this study, we first attempted to explore MAP kinase and cAMP signaling pathways in microalgal species. To understand whether these signaling pathways are conserved across different microalgal species, we chose three different microalgal species, Chlamydomonas reinhardtii, Chlorella vulgaris, and Haematococcus pluvialis, as target microorganisms for this study. With the help of chemical genetics using small molecular inhibitors, which are highly specific and highly selective for individual signaling pathways, we could reveal functional roles of individual signaling pathways in microalgae. Briefly, both cAMP and MAP kinase signaling are implicated in lipid biosynthesis in microalgae. Furthermore, MAP kinase signaling is highly conserved across microalgal species, since it is excluded as having a role in either lipid or carotenoid biosynthesis. Here, we report our novel discoveries on key signaling pathways in microalgae.

 

Materials and Methods

Microalgal Growth and Induction

Wild-type C. reinhardtii (CC-124) was used in this study. Unicellular green algae, C. vulgaris (UTEX# 0265) and H. pluvialis (UTEX# 2505), were obtained from the culture collection of algae at the University of Texas, Austin, TX, USA. The vegetative culture was carried out in Tris-acetate-phosphate (TAP) medium at 25℃. Air was bubbled into the flask culture, which was shaken at 200 rpm to ensure sufficient aeration. Continuous illumination was supplied at an average light intensity of 150 µE m-2 s-1. In the early exponential stage when lipid production is negligible, we transferred each of the microalgal suspensions into the appropriate inductive conditions as shown in Table 1. The MAP kinase inhibitor was purchased from Promega (U0126). IBMX (I5879) was purchased from Sigma-Aldrich. As a control, we included microalgal culture without any inhibitor or modulator. The dry cell weight was measured by filtering the algal suspension through a pre-dried and pre-weighed 0.45 µm cellulose nitrate membrane filter (Whatman, USA) and dried in an oven at 80℃ for 24 h.

Table 1.aGlucose, glucose addition; ROS, ROS stress driven by H2O2 addition; IBMX, cAMP signaling modulator; MAP, MAP kinase signaling inhibitor. bWe added the appropriate concentrations for each treatment as shown in this table. Concentrations used in this study were increased, denoted by increase in number (1 to 4).

Lipid Analysis

Algal lipid was stained with Nile red as described previously [7]. Subsequently, after adjusting the microalgal concentration to the same levels across treatments, relative quantifications of Nile red staining were performed by measuring the absorbance spectrophotometrically.

In addition, lipid extraction and subsequent GC analysis were performed by following Folch’s method [11,20]. Briefly, microalgal biomass was harvested by centrifugation (5,000 ×g, 10 min) and then washed twice with deionized water. The remaining cell pellet was freeze-dried and then subsequently used for lipid extraction and analysis. The total lipids were extracted from the 10 mg of lyophilized biomass with a chloroform-methanol (2:1 (v/v)) solvent mixture. Fatty acid methyl esters (FAMEs) were produced from the extracted lipid by transesterification reaction according to previous studies [11,20]. The FAMEs in the organic phase were analyzed by gas chromatography (HP5890; Agilent, USA) with a flame-ionized detector (FID) and INNOWAX capillary column (Agilent, USA; 30 m × 0.32 mm × 0.5 µm). Each FAME component was identified and quantified by comparing the retention times and peak areas with those of the FAME standard solutions.

Chlorophylls, Carotenoids, and Astaxanthin Analysis

Chlorophylls and carotenoids were measured spectrophotometrically according to the previous protocol [16]. For astaxanthin analysis, we followed the protocol for astaxanthin extraction and measurement exactly [6].

 

Results

Understanding of Signaling Pathways Behind Lipid Biosynthesis in Chlamydomonas reinhardtii

In order to examine signaling pathways in microalgae, we first tested Chlamydomonas reinhardtii, since it has been extensively studied as a model organism. C. reinhardtii was grown in TAP medium for 2 days, and subsequently subjected to various inductive conditions for lipid biosynthesis. Since lipid biosynthesis does not fully occur in the early cellular stage, induction was strategically performed in the early exponential stage to differentiate lipid phenotypes clearly (Fig. 1A). Various small molecular inhibitors were tested in the hope of identifying specific signaling pathways related to microalgal lipid metabolism. In addition, several stress treatments were also included in order to facilitate sudden changes in environmental conditions, with the anticipation that these stresses could induce lipid biosynthesis. The identification of any specific stress condition resulting in lipid biosynthesis will help us to understand the regulatory or signaling mechanisms underpinning this pathway. Inductive conditions utilized in this study consisted of four treatments: glucose addition, ROS (reactive oxygen species), and various small molecular inhibitors or modulators against the MAP kinase or cAMP signaling pathways (Table 1).

Fig. 1.Lipid production in C. reinhardtii. (A) Growth of C. reinhardtii for the induction of lipid biosynthesis using small molecule inhibitors. Arrows at the bottom of the graph indicate the time point at which each of the species is subjected to inductive conditions. (B) Nile red staining with microalgal cells after induction using a small molecule inhibitor or modulator. Glucose, glucose addition; IBMX, cAMP signaling modulator; MAP, MAP kinase inhibitor; control, control without any treatment of small molecule inhibitor or modulator. Concentrations imposed are designated 1 to 4, according to the experimental design shown in Table 1. Red color indicates chlorophyll autofluorescence, whereas yellow color reflects Nile red fluorescence due to the specific staining of lipids. The 10 μm scale bars are shown below in the control. (C) Quantification and measurement of Nile red staining results of Fig. 1B. Concentrations imposed are designated 1 to 4, according to the experimental design shown in Table 1 (refer to Table 1). White bars indicate the control.

For two consecutive days after the induction, the lipid content for each treatment was thoroughly checked. In particular, the Nile red staining method was employed, because it is a well-established protocol for selectively staining or detecting microalgal lipid bodies (LBs) in living cells due to its bright yellow fluorescence [7]. Of the four different inductive treatments, some treatments could indeed alter the lipid content significantly by increasing the extent of its biosynthesis, compared with the control without any treatment. Glucose addition, cAMP modulator (IBMX) treatment, and MAP kinase inhibitor treatment increased the C. reinhardtii lipid content (Fig. 1B). To quantify these observations further, we analyzed the photoluminescence intensity of Nile red staining to estimate the relative amount of LBs. Consistent with our observation of Nile red staining under the microscope, we observed similar trends in the quantification of LBs (Fig. 1C). The similarity of the effect of the signaling inhibitor or modulator correlated precisely with the concentrations of each inhibitor or modulator (Figs. 1B and 1C). Our results clearly indicate that the MAP kinase signaling pathway(s) is(are) involved in C. reinhardtii lipid biosynthesis negatively, since the treatments of MAP kinase inhibitor resulted in increased LB formation. In the case of a cAMP signaling modulator, we used IBMX (3-isobutyl-1-methylxanthine), which raises intercellular cAMP by inhibiting the action of phosphodiesterase. Therefore, cAMP signaling must be positively associated with lipid biosynthesis in C. reinhardtii.

Examining Key Signaling Pathways Behind Lipid Biosynthesis in Chlorella vulgaris, in Comparison with Those in C. reinhardtii

After identifying the signaling pathways associated with lipid biosynthesis in C. reinhardtii, we sought to determine whether these signaling pathways are also involved in lipid biosynthesis in the other microalgae. If we could uncover the similar functional role(s) of signaling(s) in different microalgal species, it would be helpful to understand the evolutionary conservancy of signaling(s) across microalgal species. Therefore, as a next step, we selected Chlorella vulgaris to study, since it is the most famous and wellknown microalga with many potential benefits for people. We used the same small molecular inhibitors of interest proven to be effective in C. reinhardtii to find the important signaling pathway(s) in C. vulgaris. Selected inhibitors or modulators could disrupt or perturb specific signaling of the MAP kinase and cAMP pathways, respectively.

As shown in Fig. 2A, C. vulgaris was grown until the early exponential stage, at which lipid biosynthesis does not fully occur, and then subjected to the above-mentioned conditions for testing lipid biosynthesis. For two consecutive days after the induction, we monitored the shifts in lipid content according to different inductive or suppressive treatments for lipid biosynthesis. The lipid content varied significantly depending on each treatment. Similar to C. reinhardtii, glucose addition could increase the lipid content in C. vulgaris, compared with treatment without glucose supplementation. MAP kinase inhibitors and cAMP modulators also increased the C. vulgaris lipid content (Fig. 2B). To confirm our observation using Nile red staining, we performed photoluminescence analysis, and we were able to obtain similar results to those of this staining (Fig. 2C). Moreover, our data indicate that lipid biosynthesis of C. vulgaris varies according to the concentrations of each treatment (Figs. 2B and 2C). Therefore, our results clearly indicate that the MAP kinase or cAMP signaling pathway is involved in C. vulgaris lipid biosynthesis negatively or positively. To further confirm our observations, we next performed GC analysis using both C. reinhardtii and C. vulgaris biomass grown with or without IBMX as a cAMP signaling modulator. Again, we could successfully demonstrate that IMBX treatment affects lipid production positively, supporting our results based on Nile red staining (Tables 2 and 3).

Fig. 2.Lipid production in C. vulgaris. The same experimental procedures for C. reinhardtii shown in Fig. 1 were applied to C. vulgaris. (A) Growth of C. vulgaris for the induction of lipid using a small molecule inhibitor or modulator. Arrows at the bottom of the graph indicate the time point at which each of the species is subjected to inductive conditions. (B) Nile red staining with microalgal cells after induction using a small molecule inhibitor or modulator. The 10 μm scale bars are shown below in the control. (C) Quantification and measurement of Nile red staining of Fig. 2B. Concentrations imposed are designated 1 to 4, according to the experimental design shown in Table 1 (refer to Table 1). White bars indicate the control.

Table 2.W/O, without IBMX treatment; W, with IBMX treatment.

Table 3.Fatty acid composition of C. vulgaris and C. reinhardtii in the presence or absence of IBMX treatment (mg FAME/mg biomass).

Chlorophyll is Sensitive to Exogenous ROS Treatments Both in C. reinhardtii and C. vulgaris

Generally, photosynthesis is tightly linked with ROS generation, since oxygen is one of the major final products of photosynthesis. Because there is a possibility that microalgal cAMP or MAP kinase signaling affects photosynthesis, thereby altering microalgal lipid production, it would also be interesting to determine whether ROS treatment has additional influence on photosynthesis and, in turn, lipid production. We discovered that ROS treatment of the cultures of C. reinhardtii and C. vulgaris could decrease the chlorophyll content significantly. Chlorophyll is a natural pigment found in microalgae as well as green plants, and it is mainly responsible for reddish autofluorescence, when exposed to UV light. The red autofluorescence resulting from endogenous chlorophylls could be consistently observed across different microalgal species (Figs. 1B and 2B). However, when we introduced exogenous ROS into the cultures of C. reinhardtii and C. vulgaris, chlorophyll autofluorescence levels in microalgal cells declined sharply. Moreover, the decline in chlorophyll levels became apparent as the ROS concentration increased. Specifically, more than 0.1% H2O2 caused a sharp decline in chlorophyll content (data not shown). To further verify the phenomenon of chlorophyll degradation upon ROS treatment, chlorophyll was also extracted and quantified (data not shown). Our results indicate that microalgal chlorophyll is sensitive to exogenous ROS addition. Although the ROS effect on chlorophyll degradation was confirmed, lipid production was not detectable, owing to the deadly effect of ROS tested in the study.

Microalgal MAP Kinase Signaling Regulates Carotenoid Biosynthesis in a Negative Manner, and It Is Well-Conserved Across Different Microalgal Species, C. reinhardtii and C. vulgaris

Interestingly, we noticed remarkable phenotypes of color change from green to reddish brown in the algal suspensions, changes that were only visible following treatment with certain amounts of MAP kinase inhibitor (Fig. 3A). The change in color to reddish brown became apparent at 92 µM of MAP kinase inhibitor, and it became more evident upon the 460 µM treatment (Fig. 3A). Notably, both C. reinhardtii and C. vulgaris cells could respond to the MAP kinase inhibitor similarly, since the MAP kinase inhibitor exerted the same effect of increasing the reddish color of microalgal cultures (Fig. 3A). Chlorophyll and carotenoid are two major components of microalgal pigments and are responsible for the green and red colors, respectively. We followed an extraction and measurement protocol [8,20] specifically designed for microalgal chlorophyll and carotenoid to estimate the levels of pigmentation upon addition of the MAP kinase inhibitor. We observed significant up-regulation of carotenoid with respect to the chlorophyll content following treatment with the MAP kinase inhibitor, particularly at concentrations above 92 µM. Both C. reinhardtii and C. vulgaris exhibited similar behavior in response to the MAP kinase inhibitor as a stimulant for carotenoid biosynthesis (Fig. 3B). Our results, therefore, demonstrated that the MAP kinase signaling pathway in C. reinhardtii and C vulgaris is associated with carotenoid biosynthesis in a negative manner.

Fig. 3.MAP kinase signaling in carotenoid production. (A) Photos of C. vulgaris were taken after treatment with MAP kinase inhibitors for 2 days. MAP kinase inhibitor concentrations imposed are designated 1 to 4, according to the experimental design shown in Table 1. Control indicates the microalgal culture without any inhibitor. (B) Quantification and measurement of the extent of carotenoid production. Relative carotenoid content is presented as carotenoid/chlorophyll ratios.

Haematococcus pluvialis MAP Kinase Signaling Regulates Lipid As Well As Carotenoid Biosynthesis in a Negative Manner but Astaxanthin Production Positively

Microalga H. pluvialis is of particular interest owing to their capacity to produce the highly valued keto-carotenoid, astaxanthin [17]. Because MAP kinase signaling in C. reinhardtii and C. vulgaris is involved in carotenoid production, we speculated that MAP kinase signaling might also be conserved in H. pluvialis. To test our hypothesis, we performed MAP kinase inhibition analysis using H. pluvialis. H. pluvialis biomass was allowed to grow for 2 days and then subjected to MAP kinase inhibitory conditions by supplementing an appropriate amount of inhibitor (Fig. 4A). Likewise, lipid production significantly increased, since both Nile red staining and photoluminescence analysis consistently demonstrated enhanced lipid production upon MAP kinase inhibitor treatment. Above 92 µM, the MAP kinase inhibitor began to yield noticeable yellow staining, and at 460 µM, the MAP kinase inhibitor led to intensive yellow fluorescence due to the increased lipid content (Fig. 4B). H. pluvialis lipid biosynthesis was correlated with the concentration of MAP kinase inhibitor (Fig. 4C).

Fig. 4.Influence of the MAP kinase inhibitor on the lipid production of H. pluvialis. (A) A MAP kinase inhibitor was added in order to induce lipid biosynthesis. Arrows at the bottom of the graph indicate the time point at which each of the species was subjected to inductive conditions using the MAP kinase inhibitor. (B) Nile red staining with H. pluvialis cells after induction using small molecule inhibitors. The 10 μm scale bars are shown below in the control. (C) Quantification and measurement of Nile red staining of Fig. 4B.

Next, we tested whether the disruption of MAP kinase signaling could cause similar morphological differences in enhanced carotenoid biosynthesis in H. pluvialis. Consistent with our hypothesis, we detected elevated carotenoid production only after a certain amount of MAP kinase inhibitor treatment (more than 92 µM MAP kinase inhibitor) (Fig. 5A). To determine the levels of carotenoid production, we again used the relative carotenoid amount per chlorophyll (carotenoids/chlorophyll) as an indicator for pigment alteration. The disruption of the MAP kinase signaling pathway via MAP kinase inhibitor resulted in up to approximately 5-fold increases in carotenoid content when compared with controls without any treatment (Figs. 5B and 5C). These results strongly support the hypothesis that the H. pluvialis MAP kinase signaling pathway is functionally conserved across the microalgal species, affecting carotenoid biosynthesis in a negative manner.

Fig. 5.Influence of a MAP kinase inhibitor on carotenoid (A-C) or astaxanthin (D-F) production in H. pluvialis. (A) Photos of microalgal suspensions were taken after treatment with MAP kinase inhibitors for 2 days. The MAP kinase inhibitor concentrations imposed were designated 2 to 4, according to the experimental design shown in Table 1. Control indicates the microalgal culture without any inhibitor. MAP kinase inhibitor concentrations designated 2, 3, and 4 were adjusted to be 9.2 μM, 92 μM, and 460 μM, respectively. (B) Quantification and measurement of the extent of H. pluvialis carotenoid production. Relative carotenoid content is presented as carotenoid/chlorophyll ratios. (C) Microscopic images of H. pluvialis cells of Fig. 5A. Brown pigmentation was observed as a result of a certain amount of MAP kinase inhibitor (designated as treatments 3 and 4) due to increased carotenoid production. The 10 μm scale bars are shown below in the control. (D) Photos of microalgal suspensions were taken after treatment with MAP kinase inhibitors for 20 days. (E) Quantification and measurement of the extent of H. pluvialis astaxanthin production during long-term incubation. (F) Microscopic images of H. pluvialis cells of Fig. 5D. Astaxanthin production showed the opposite trend with carotenoid production, as more reddish cells could be observed in the absence of or at a lower concentration of MAP kinase inhibitor. The 10 μm scale bars are shown below in the control.

One striking feature of the H. pluvialis MAP kinase signaling pathway is its negative effect on astaxanthin production, which contrasts with our observations during carotenoid biosynthesis. As presented in Fig. 5A, carotenoid biosynthesis increased drastically following a short period of incubation with the MAP kinase inhibitor. However, this activity was prevented when we incubated the sample for an extended period of time (20 days), since we observed much higher carotenoid levels, mainly due to increased astaxanthin content, particularly in the control or in the sample subjected to a low amount of MAP kinase inhibitor treatment (9.2 µM) (Fig. 5D). To confirm our observation of H. pluvialis astaxanthin biosynthesis upon addition of the MAP kinase signaling inhibitor, we repeated our experiment again and obtained consistent results (data not shown). The standard protocol for astaxanthin extraction was employed and reduced astaxanthin production following treatment with the MAP kinase signaling inhibitor, a result that was further verified by analyzing the relative content of astaxanthin or by examination of microscopic images (Figs. 5C and 5F). Interestingly, regardless of the presence of MAP kinase inhibitor, H. pluvialis cells quickly transformed into green palmelloids and red aplanospores during the short and long periods of MAP kinase inhibitory incubation, respectively. These phenomena might be due to the fact that the inductive experimental conditions caused basal stress in H. pluvialis cells. However, the patterns for carotenoid and astaxanthin production upon addition of MAP kinase inhibitor were also consistently observed in microscopic images (Figs. 5C and 5F). Our results indicate that MAP kinase signaling in H. pluvialis is positively associated with astaxanthin production.

 

Discussion

Microalgae have been recognized as one of the most important microorganisms for biodiesel production. Therefore, microalgal biodiesel production has been the subject of extensive research recently. Owing to the significant impact of microalgal biofuel, it will be crucial to advance our knowledge of microalgal lipid metabolism. Although there has been substantial progress in clarifying the physiological conditions of some genes encoding enzymes necessary for microalgal lipid production, we still do not understand the details of how overall lipid metabolic pathways are regulated in microalgal cells.

Like other eukaryotic organisms, microalgae must recognize and respond to various environmental cues for their own survival or adaptation. It is clear that a wide variety of signal transduction activities in microalgal cells are involved in processing environmental cues and directing appropriate reactions to their environment. Lipid serves as a reservoir of energy in many living organisms, and consequently tight regulation of its biosynthesis is mandatory to coordinate proper response of microalgal cells to environmental cues such as nitrogen depletion. Therefore, it is likely that microalgal lipid biosynthesis is regulated via signaling pathways, which have remained unclear to date, in spite of its tremendous significance.

In order to understand and characterize the metabolic pathways in microalgae, so far, we have depended heavily on gene disruption as a key experimental procedure. However, the major problem with mutagenesis as a method for evaluation is the presence of multiple redundant pathways as well as technical difficulties in microalgal gene manipulations. To overcome these challenges, we employed chemical genetics, since it provides a way to bypass the above-mentioned difficulties and an alternative solution for understanding the otherwise elusive microalgal signal transduction activities [1,22]. From the study of their chemical genetics, it is possible to conclude that the biochemical reagents highly selective for an enzyme in the signaling pathways must be essential to understanding the microalgal pathways leading to phenotypes of interest. Although genetic engineering could eventually facilitate biodiesel production, it is certain that chemical genetics will give us significant clues about which genes should be manipulated or altered for desirable phenotypes of human interest.

Therefore, in this study, we suggest putative functional roles of several signaling pathways in microalgae and demonstrate their influences on lipid biosynthesis via chemical genetics (Figs. 1, 2, and 4). GC analysis also supports the same conclusion, though there were plenty of short fatty acid chains such as C4:0 (Table 3). It is likely that the young microalgal cells subjected to short-term inductive conditions might have enough time to completely biosynthesize the long fatty acid chains. Instead, microalgal cells accumulated short fatty acids as well. However, overall, it must be true that the signaling pathways identified in this study have a significant influence on microalgal lipid biosynthesis.

To the best of our knowledge, this is the first study of microalgal signaling pathways associated with lipid metabolism and the development of lipid bodies in microalgae. Moreover, we did not limit our study to a single microalgal species, instead performing comparative genomics using three different microalgal species: C. reinhardtii, C. vulgaris, and H. pluvialis. In this regard, we believe that our results are of considerable importance in that we first characterized signaling pathways across different microalgal species, which will benefit rapidly expanding microalgal research. Overall, a summary of signaling pathways found in this work is illustrated in Fig. 6. Our results clearly reveal that signaling pathways in microalgal species are well-conserved despite microalgal speciation. MAP kinase and cAMP signaling have negative and positive impacts on microalgal lipid production in both C. reinhardtii and C. vulgaris. Because we observed very similar effects of MAP kinase and cAMP signaling in spite of the evolutionary divergence between C. reinhardtii and C. vulgaris, we propose that the functional roles of both signaling pathways in microalgae are well-conserved across many divergent microalgal species.

Fig. 6.A model for major signaling pathways regulating carotenoid and lipid biosynthesis in microalgae. (A) Signal transduction cascades in C. reinhardtii. MAP kinase and cAMP signaling pathways are implicated in carotenoid and lipid biosynthesis negatively. (B) Signal transduction events in C. vulgaris for carotenoid and lipid biosynthesis are depicted. (C) Schematic presentation of H. pluvialis MAP kinase signaling. MAP kinase signaling in H. pluvialis is negatively involved in carotenoid and lipid production, whereas it is positively associated with astaxanthin production.

Remarkably, we observed intriguing morphologies of microalgae, which could be unexpectedly discovered in the treatments of inhibitors as a chemical genetic approach. We demonstrated that the alteration of MAP kinase signal transduction has been simultaneously linked to microalgal carotenoid biosynthesis, other than lipid production. These observations are consistent across all microalgal species tested in this study: C. reinhardtii, C. vulgaris, and H. pluvialis. Therefore, it is conceivable that MAP kinase signaling in microalgae is evolutionarily well-preserved in the regulation of carotenoid biosynthesis in a negative manner (Figs. 3 and 5). Because both lipid and carotenoid can be produced at a later stage of growth, microalgal MAP kinase signaling(s) might be associated with cellular development in the stationary phase or with the biosynthesis of multiple secondary metabolites, including carotenoids. Microalgal MAP kinase signaling(s) might function as a “master switch,” turning on/off a wide variety of metabolic pathways, thereby rendering microalgal cells capable of switching from a stage of vigorous growth to stationary survival. Further investigation is necessary to precisely determine the microalgal MAP kinase signaling pathway that directly regulates both lipid and carotenoid biosynthesis.

Since H. pluvialis astaxanthin ((3S-3’S)-dihydroxy-β,β-carotene-4,4’-dione) is a ketocarotenoid pigment of biotechnological importance in the nutraceutical, pharmaceutical, and food industries [17], we hypothesized that astaxanthin biosynthesis can also be regulated by MAP kinase signaling in a negative manner, similar to the production of other microalgal carotenoids. However, it turned out that the MAP kinase pathway in H. pluvialis has an opposite role in astaxanthin biosynthesis, although most of the phenotypes observed indicate well-conserved functional roles of microalgal MAP kinase in the production of a secondary metabolite, carotenoid (Figs. 5D, 5E, and 5F). A possible explanation for this phenotype could be that H. pluvialis metabolic pathways leading to either carotenoid or astaxanthin biosynthesis might compete against each other. Presumably, once the H. pluvialis metabolic flux is directed to carotenoid biosynthesis, it will be difficult to be redirected to astaxanthin biosynthesis. It remains to be determined whether reduced astaxanthin biosynthesis via the down-regulation of MAP kinase signaling in H. pluvialis is directly linked to increased carotenoid biosynthesis. Because light is the most important factor in H. pluvialis astaxanthin biosynthesis [6], we propose that MAP kinase signaling could be interconnected with putative light signaling in H. pluvialis. In filamentous fungi, light signaling is mediated by the velvet gene [3]. We believe that there is a counterpart or analog of the fungal velvet gene in microalgae that might transmit signals to facilitate astaxanthin biosynthesis with or without connection to MAP kinase signal transduction.

In addition, glucose addition has a significant effect on LB formation, as its addition promoted lipid biosynthesis. The data imply that there should be hitherto unidentified microalgal carbon-nutrient signaling transduction. Nutrient signaling associated with glucose utilization should be positively related to microalgal lipid biosynthesis. We also investigated whether exogenous application of protein phosphatase inhibitors would induce or repress microalgal lipid biosynthesis, because protein phosphatase is important in multiple eukaryotic cellular developments [4]. We used okadaic acid, which preferentially inhibits protein phosphatase activity, but failed to observe any difference in microalgal lipid biosynthesis (data not shown). It is therefore possible that protein phosphatase 2A might have no relationship with microalgal lipid biosynthesis. Alternatively, we could not eliminate the possibility that it might not be possible to detect microalgal lipid production within the range of concentrations of protein phosphatase 2A inhibitor used in this study.

As with MAP kinase and cAMP signaling in eukaryotic cells, MAP kinase and cAMP signaling in microalgae supposedly have regulatory roles in gene transcription by activating multiple transcriptional factors, which must be responsible for the several phenotypes observed in this study. However, we still have limited knowledge of genes that act downstream of both signaling pathways. To better understand the effects of both MAP kinase and cAMP signaling regulation on transcriptional factors and subsequent gene transcriptions, it will be important to investigate genome-wide mRNA transcription using microarrays or mRNA sequencing. Therefore, we are planning to perform further study on microalgal signaling pathways with genome-wide mRNA or proteomic analysis to pinpoint how each signaling pathway could influence carotenoid or lipid production in microalgae.

In summary, we attempted to unravel the complex regulatory mechanisms via signal transduction activities associated with microalgal lipid biosy nthesis. Chemical genetics could aid in expanding our knowledge on important microalgal signaling pathways in either lipid or carotenoid biosynthesis. The signal transduction pathways identified were those of MAP kinase and cAMP. Furthermore, we extended our findings by investigating the functional roles of signaling in different microalgal systems, highlighting the evolutionary conservancy or distinct speciation in microalgal signaling pathways. We believe that we were successful in obtaining remarkable experimental data for future application of microalgae in biodiesel production or commercial applications for value-added products such as carotenoid or astaxanthin. Our findings will greatly facilitate future efforts aimed at practical applications of biodiesel or carotenoid production using microalgae.