2.1 N deprivation restrained the growth and physiological status
To monitor growth, single cells of A. coffeaeformis were separated in three petri dishes and observed using an inverted microscope every 24 h. The growth curve was shown in Fig. 1. The growth rate was 0.22 per day, the division was 0.32 per day, and the resulted doubling time was 3.15 days. The growth rate of A. coffeaeformis is comparable with those of the other known Amphora algae [18], which indicated that the condition in this experiment was suitable for this species.
As previously reported in other oleaginous microalgae [19], the lack of nitrogen inhibits the growth and significantly shortens the time required for A. coffeaeformis in the stable phase to enter the recession stage. The growth rate of A. coffeaeformis in f/2-N medium was 0.06 per day and the doubling time extended to 11.67 days (Fig. 1). For A. coffeaeformis culture in the stable phase, N deprivation restrained growth and attachment status. In Fig. 2, the A. coffeaeformis in normal f/2 medium was uniformly attached to the bottom of Petri dishes; in contrast, some cells fell off the substratum after 5 days of N deprivation. The stable phase of A. coffeaeformis under normal condition was maintained for more than 20 days, following which, some cell clusters fell off, were suspended in the medium, and died (recession stage). However, N deprivation shortened the stable phase of A. coffeaeformis to 7−8 days and the recession stage to 10 days.
The response of the photosynthetic performance to growth in nitrogen-rich and nitrogen-free media is particularly important for improving our understanding regarding the metabolic and physiological differences in diatoms under N deprivation [11]. The photosynthesis performance parameters can directly reflect the growth status of microalgae, providing detailed information regarding the cellular physiology of the algae under N deprivation [20]. Hence, the photosynthetic activity was examined during N deprivation to monitor the physiological status of A. coffeaeformis. The maximum PSII photochemical efficiency Fv/Fm and Etr characterize the physiological response of microalgal cells to changes in environmental conditions, such as nutrient starvation or photoinhibition [11]. In this study, Fv/Fm and Etr declined in N deprived medium (Fig. 3). Especially the character YII of A. coffeaeformis after 6 days’ N deprivation declined dramatically (0.46 to 0.19). Obviously, the physiological activity of A. coffeaeformis under N deprivation was inhibited owing to inhibition of photosynthetic activity.
2.2 N deprivation promoted lipid accumulation in A. coffeaeformis
The accumulation of lipids in microalgae under N deficiency has studied for decades. Studies have shown that the growth of microalgae (i.e., synthesis of protein and nucleic acids) is limited under N deficiency, although the process of carbon assimilation is always ongoing, which channelizes more carbon into lipid metabolism [10, 11]. As an important component of lipids, TAGs do not contain nitrogen. Therefore, despite the lack of nitrogen in microalgae under conditions supporting ongoing carbon assimilation, TAGs can accumulate rapidly in cells. Converti et al. [21] showed that when the nitrogen source in the culture medium was reduced by 75%, the total lipid content in Nannochloropsis oculata increased from 7.9–15.31%, while the total lipid content of Chlorella vulgaris increased from 5.9–16.41%. In the present study, the total lipid content of A. coffeaeformis in normal f/2 medium was 28.22% (TL/DW), which increased to 44.05% after 5 days of N deprivation, while the neutral lipid TAG content increased from 10.41% (TAG/DW) to 25.21% (TAG/DW) (Fig. 4). The lipid content in A. coffeaeformis was as high as that reported in other Amphora species.
Distinct difference between the fatty acid compositions of A. coffeaeformis in f/2 medium and under N deprivation was not observed (Table 1). Surprisingly, the fatty acid compositions of TAG in N-deprived A. coffeaeformis differed considerably from that of the control (Table 2). The saturated fatty acid contents increased dramatically from 57.94–81.21%, especially that of the long chain fatty acid C16:0, C18:0, and C20:0. Furthermore, the content of the unsaturated fatty acids decreased. These results indicated that the key enzymes in the TAG synthesis pathway may facilitate metabolism of saturated fatty acids and as a result, the unsaturated fatty acids flowed to the other lipids, with the exception of TAG.
Table 1
Fatty acid composition of A. coffeaeformis following N deprivation.
Fatty acids | control | -N (5 d) |
12:00 | 0.17 | 0.165 |
13:00 | 0.1 | 0.09 |
14:00 | 10.02 | 10.04 |
14:01 | 0.15 | 0.13 |
14:01t | 2.17 | 2.13 |
14:01t | 4.57 | 4.31 |
15:00 | 2.81 | 2.66 |
16:00 | 23.48 | 23.41 |
16:01 | 24.28 | 25.31 |
16:01t | 0.62 | 0.61 |
16:02 | 1.24 | 1.32 |
16:03 | 2.27 | 2.32 |
18:00 | 1.57 | 1.245 |
18:01 | 2.34 | 2.32 |
18:01t | 1.3 | 1.175 |
18:02 | 2.76 | 2.715 |
18:03 | 0.06 | 0.06 |
19:01 | 0.76 | 0.79 |
18:03 | 0.15 | 0.15 |
20:00 | 0.07 | 0.07 |
20:03 | 0.24 | 0.25 |
20:04 | 8.06 | 7.95 |
20:05 | 8.67 | 8.775 |
24:00 | 0.97 | 0.96 |
24:01 | 0.56 | 0.06 |
Table 2
Fatty acid composition in TAGs with significant changes following N deprivation.
Fatty acids | Normal | -N (5 d) |
14:0 | 7.52 | 4.99 |
16:0 | 30.72 | 33.08 |
16:1 | 23.17 | 7.3 |
16:1t | 2.77 | 5.51 |
18:0 | 10.29 | 29.14 |
18:1 | 3.92 | 6.81 |
18:1t | 1.97 | 1.47 |
18:2 | 1.82 | 5 |
18:3 | 0.34 | 4.56 |
20:0 | 1.06 | 3.66 |
2.3 High-throughput analysis of A. coffeaeformis under N deprivation
To obtain in-depth knowledge regarding the mechanisms, comprehensive analyses such as analysis of gene expression should be used. Therefore, RNA-seq analysis was used in this study to assess differences in gene expression between the N-deprived A. coffeaeformis and the control group. In this regard, we hypothesized that the molecular mechanism underlying the effect of N deprivation on lipid accumulation in A. coffeaeformis could be determined by studying the changes in gene expression. Considering the lack of reported genome sequences, the RNA-seq analysis was performed using the P. tricornutum genome as the reference. Approximately 55 million transcripts were sequenced in each sample, and more than 80% transcripts mapped to the P. tricornutum genome, and the high similarity to the transcripts of other microalgae species indicated the validity of the data.
In this study, the comprehensive transcriptome data of A. coffeaeformis under N deprivation was compared with that under normal conditions, and the raw data is available at http://www.ncbi.nlm.nih.gov/bioproject/753251. To identify functional categories of unigenes, KEGG analysis was performed in accordance of all 110,454 unigenes of A. coffeaeformis. And the analyses suggested that the putative functional roles of each transcript obtained were involved in diverse cellular functions, i.e., general metabolism, organismal systems, genetic information processing, environmental information processing, and cellular processes (Fig. 5). The category of amino acid metabolism accounted for 2,877 unigenes, whereas lipid metabolisms contained 1,273 transcripts. Moreover, transcripts associated with genetic information processing also constituted a large fraction in the unigenes, which demonstrated the importance of the transcriptional regulation under N deprivation.
2.4 DEGs in response to N-deprived conditions
To evaluate the effect of N-deprived condition on A. coffeaeformis transcripts, DEGs that showed over two-fold changes in their expression compared with the control were selected for further analysis, are shown in the volcano plot (Additional file 1: Fig S1). In total, 591 genes were found to be up-regulated and 1,021 genes were down-regulated, implying induced extensive regulatory reprogramming. And in particular, many genes were widely down-regulated, which may illustrate that most physiological activities were repressed under the conditions of the lack of nitrogen in the environment.
KEGG pathway enrichment analysis of the DEGs was performed for demonstrating the putative functional roles of the identified DEGs in further detail. KEGG analysis for DEGs classified them as being associated with different functional categories, in which multiple genes were significantly up-regulated or down-regulated, suggesting possible mechanisms involved in the response to N deprivation. For example, genes mediating metabolism of nitrogenous compounds and ribosome biogenesis were up-regulated, while genes encoding enzymes required for photosynthesis, biosynthesis of unsaturated fatty acids, carbon fixation, glycolysis and gluconeogenesis, porphyrin and chlorophyll metabolism were generally down-regulated under N deprivation (Fig. 6). GO analysis suggested similar patterns to the KEGG analysis. Hence, it can be speculated that certain biochemical processes might be involved in adaptation to the lack of nitrogen.
Genes with log2-fold differences in transcripts between the control group and N-deprived A. coffeaeformis were considered global changes in major categories of genes involved in various pathways, which reflected general transcriptional responses to N deprivation [17], are shown in Fig. 7. The fold changes in the expression of some genes encoding enzymes involved in A. coffeaeformis metabolisms following N deprivation were summarized in Additional file 2 (Table S1).
2.5 Transcripts involved in nitrogen assimilation increased
Nitrogen element is basic component constituting nucleic acids, proteins, and other nitrogen-containing compounds, the main effect of nitrogen deprivation is the reduction in nitrogen availability and eventually cause the synthesis of nitrogenous compounds hindered [16, 17]. Therefore, there is no doubt that the transcription levels of genes in A. coffeaeformis involved in nitrogen assimilation and metabolism were significantly affected under nitrogen deprivation (Additional file 2: Table S1a). For example, the transcripts encoding three nitrate reductase (NADH-nitrate reductase, EC 1.7.1.1; assimilatory NAD(P)H-nitrate reductase, EC 1.7.1.2; assimilatory NADPH-nitrate reductase, EC 1.7.1.3) that catalyzes the reaction of nitrate to nitrite were increased with 3.4-fold changes. We also observed that the transcript of glutamine synthase (EC 6.3.1.2), which plays an important role in the effective utilization of nitrogen sources and nitrogen metabolism, increased significantly by 2.1-fold. Meanwhile, significant increases were also found in three transcripts encoding ammonium transporters present in microalgae, which are known to be activated by N deprivation, thereby transporting ammonium ions across the cell membrane [17, 22]. In addition, some critical nitrogen metabolism enzymes, such as NADH-glutamate synthase (EC 1.4.1.14) and ferredoxin-glutamate synthase (EC 1.4.7.1) also showed significant increases (1.5-fold and 1.6-fold, respectively) in transcription levels. Hence, it can be speculated that N deficiency induces a steady-state response, including the activation of the glutamine synthesis pathway and the increase in the ability of cells to utilize trace nitrogen resources, as well as the possible redistribution of intracellular nitrogen [17, 23]. In summary, these results indicated that N deprivation caused many changes in metabolic pathways, especially in amino acid and nitrogen metabolism, which may be a stress response requiring significant changes in gene expression.
2.6 Transcripts of photosynthesis-related genes decreased
As mentioned earlier, decreased availability of nitrogen element lowered growth rates, photosynthetic efficiency and had consequences on cell production. In particular, for photosynthetic cells, inhibition or stop of photosynthesis means stagnation of cell growth or death. In fact, the transcripts encoding proteins related to photosynthesis were investigated under nitrogen deficiency, and the levels of most transcripts were decreased (Additional file 2: Table S1b), which implied the stagnation of photosynthesis. Notably, ferredoxin-NADP+ reductase (EC 1.18.1.2), an enzyme that catalyzes the last electron transfer (from photosystem I to NADPH) during photosynthesis, was also observed the decreased transcription levels (4.8-fold). In accordance with theoretical expectations, the down-regulation of transcripts of light harvesting complexes may reduce photosynthetic rates of the cells under the conditions that do not consider changes in other levels [17, 24].
The decreased transcripts of genes encoding photosynthetic proteins was consistent with expectations, as many previous reports also showed these results [25, 26], which indicates the assimilation of nitrogen has profound effects on photosynthetic metabolism [27]. Meanwhile, the decreases of physiological parameters associated with photosynthesis, such as Fv/Fm and Etr (Fig. 3), are also consistent with the down-regulation of transcripts of light harvesting complexes. These physiological parameters significantly decreased, indicating that the decrease of photosynthetic protein under nitrogen-deficiency condition may impair the PSII reaction centers, and eventually resulted in the decrease of both chlorophyll fluorescence yield and photochemical activity of PSII reaction centers [17, 28].
2.7 Carbon assimilation and carbon fluxes towards TAG accumulation increased
There are three carbon fixation pathways for photosynthetic organisms: 1) Calvin-Benson-Bassham (CBB) cycle, 2) C4-dicarboxylic acid cycle, and 3) Crassulacean acid metabolism [29, 30]. To our best of knowledge, so far, only the CBB cycle of microalgae has been reported [31]. However, it was found that two genes encoding phosphoenol-pyruvate carboxylase (PEPC, EC 4.1.1.31) were up-regulated (1.53-fold and 1.46-fold, respectively) in the genome of A. coffeaeformis under N deprivation, which catalyzed the synthesis of OAA from phosphoenolpyruvate (PEP) (Additional file 2: Table S1c). In fact, PEPC is usually involved in carbon fixation as a key enzyme in C4-dicarboxylic acid cycle [24]. Hence, it can be speculated that the C4 photosynthetic pathway exist in A. coffeaeformis. PEPC catalyzes an irreversible carboxylation in the presence of HCO3− and Me2+, the increase of the transcripts of these two PEPCs observed in this study may suggest that it can absorb inorganic carbon and possesses a CO2 concentration mechanism [17, 32].
Pyruvate-phosphate dikinase (PPDK, EC 2.7.9.1) is also a key enzyme in C4-dicarboxylic acid cycle, which catalyzes the synthesis of PEP from pyruvate, and its expression significantly affects the accumulation of photosynthetic products in C4 plants [33]. The transcripts of two PPDK genes in A. coffeaeformis under N deprivation were significantly decreased (1.7-fold and 2.2-fold, respectively), implicating that the decrease of photosynthetic rate [17]. Hence, it can be predicted that the reduction of PPDK level reduces the consumption of pyruvate, thereby enables more pyruvate to synthesize acyl-CoA, the precursor of fatty acid, while the excessively expressed PEPCs can use the available PEP for carbon fixation. Moreover, PPDK is also involved in PPDK-mediated gluconeogenesis [34], which indicates that the decreases of PPDKs expression may inhibit the activation of gluconeogenesis.
Notably, the significant increased transcripts of a gene encoding NADP+-malic enzyme (ME, EC 1.1.1.40) was also observed. It is speculated that this gene may be involved in the carbon fixation pathway in A. coffeaeformis, and is localized in the chloroplast. ME catalyzes the irreversible decarboxylation of malate to pyruvate in photosynthetic cells with the formation of NADPH from NADP+, which is the rate-limiting step of fatty acid biosynthesis [17]. In fact, many previous reports have confirmed the promoting effect of ME overexpression on fatty acid synthesis. For example, the overexpression of endogenous NADP-dependent ME in N. salina enhanced the lipid production, and the report also analyzed the total carbon concentration and NADPH/NADP+ ratio, which were found to be enhanced in the transformants [35]. Hence, the up-regulation of ME in A. coffeaeformis under N deprivation may increase the NADPH production, thereby providing both reducing power and cofactors for reactions catalyzed by enzymes involved in fatty acid synthesis such as ACCase, fatty acid synthase, and eventually leading to increases in TAG accumulation [17]. Similarly, overexpression of ME gene from P. tricornutum in C. pyrenoidosa reportedly showed higher ME enzymatic activity, which subsequently promoted fatty acid synthesis, and the neutral lipid content was significantly increased by up to 3.2-fold [17]. Taken together, the dramatic increase in ME transcription observed in this study under N deprivation could contribute substantially to the accumulation of neutral lipids in A. coffeaeformis.
Fructose-1,6-bisphosphatase (FBP, EC 3.1.3.11) catalyzes the conversion of fructose-1,6-biphosphate to fructose-6-phosphate, which is a key enzyme in CBB cycle and gluconeogenesis. A. coffeaeformis appears to possess at least three isoforms of FBPs, and transcript levels of genes encoding these three isoforms declined under N deprivation, 3.4-, 1.3- and 2-fold, respectively, indicating that CBB cycle and gluconeogenesis were markedly inhibited and carbon flux was re-directed towards TAG accumulation under N deprivation. Notably, the transcript abundance of pyruvate carboxylase (PC, EC 6.4.1.1), a tricarboxylic acid (TCA) cycle enzyme identified in the A. coffeaeformis genome, decreased 3-fold. In fact, the PC catalyzed reaction is the main supplementary reaction for the supply of OAA in the TCA cycle, and the reaction is activated when acetyl-CoA is abundant. Therefore, the down-regulation of PC transcript level may indicate the decrease of acetyl CoA flowing into TCA cycle, while a large amount of acetyl-CoA was used to synthesize TAG. In addition, the significantly differences of transcripts encoding two potentially rate-limiting enzymes of the TCA cycle, citrate synthase (EC 2.3.3.1) and isocitrate dehydrogenase (EC 1.1.1.42), were not observed. However, the transcript levels of genes encoding two succinate dehydrogenases (SDH, EC 1.3.5.1), another key enzyme in TCA cycle, were significantly decreased (2.2-fold and 1.1-fold, respectively), implying that the metabolic flux through the TCA cycle could be reduced under N deprivation. In summary, the transcriptional changes in carbon assimilation of A. coffeaeformis under N deprivation may substantially increase carbon influxes, thereby providing a rich source of substrate for TAG production.
2.8 Increased GPAT levels promoted TAG biosynthesis
In general, oil-bearing microalgae significantly accumulated neutral lipids after nitrogen induction. Hence, to further determine the precise mechanism via which the microalga up-regulated gene expression under N deprivation, genes involved in TAG and fatty acid biosynthesis were studied. Notably, N deprivation increased the transcript levels of genes associated with TAG biosynthesis. In particular, the expression of glycerol-3-phosphate O-acyltransferase (GPAT, EC 2.3.1.15) that catalyze the first committed step of TAG biosynthesis increased (1.3-fold); thus, the increase in their mRNA abundance under N deprivation may have increased TAG levels.
Notably, transcripts of gene encoding lysophosphatidic acid-acyltransferase (LPAAT, EC 2.3.1.51) that catalyzes the second step of TAG biosynthesis decreased (1.2-fold). Interestingly, the significantly differences of transcripts encoding diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) that catalyzes the final committed step of TAG biosynthesis, were not observed; Meanwhile, the mRNA levels of another enzyme, responsible for the last step of TAG biosynthesis, phospholipid: diacylglycerol acyltransferase (PDAT, EC 2.3.1.158), decreased 1.7-fold. These findings suggest that the overexpression of GPAT seems to be a key factor in TAG biosynthesis of A. coffeaeformis. In fact, some previous reports also showed the same phenomenon. For example, only transcripts of GPAT1 and GPAT2 were increased among fatty acid and TAG biosynthesis genes under TAG accumulation conditions in Cyanidioschyzon merolae [36]. Similarly, overexpression of GPAT1 and GPAT2 in C. merolae resulted in up to a 56.1-fold increases in seed oil content [37]. Taken together, the reaction catalyzed by the ER-localized GPAT is a rate-limiting step for TAG synthesis in A. coffeaeformis, and would be a potential target for improvement of TAG productivity in microalgae.