Proteome response of Dunaliella parva induced by nitrogen limitation

Highlights

• The changes of proteins induced by nitrogen limitation in Dunaliella parva were reported for the first time.

• Combined with our former transcriptome study, we discussed the possible mechanism of lipid accumulation induced by nitrogen limitation based on the results of this study.

• We suggested that perhaps lipid accumulation induced by nitrogen limitation was associated with the protein level changes of enzymes involved in starch metabolism, glycolysis, fatty acid metabolism and triacylglycerol metabolism.

Abstract

Nitrogen limitation is the most widely used method for directing metabolic flux to lipid biosynthesis in microalgae. Protein level changes induced by environmental stress could be investigated by proteomics analysis, which was a favourable approach for understanding the induction mechanisms. Although, we previously provided the results of transcriptome sequencing of Dunaliella parva under nitrogen limitation stress, the effects of nitrogen limitation on the microalgal proteome were not reported. The impact of nitrogen limitation on the proteome of green alga D. parva was investigated using iTRAQ quantitative proteomics technique. A total of 227 proteins were up-regulated, and 159 proteins were down-regulated under the nitrogen limitation condition. Functional analyses of differentially expressed proteins revealed that nitrogen limitation could induce protein level changes related to photosynthesis, stress response, lipid metabolism, carbohydrate metabolism and nitrogen metabolism. This study, together with our previous transcriptome sequencing of D. parva, therefore provides a foundation for understanding the regulatory mechanism of lipid biosynthesis induced by nitrogen limitation, which could further guide the genetic modification of lipid accumulation in D. parva.

Introduction

Biodiesel has recently attracted increasing attention as a renewable energy source because of its environmental benefits [1], [2]. Microalgal lipid was regarded as a promising feedstock of sustainable biodiesel production because of its higher growth rate and photosynthetic efficiency [3], [4]. Nevertheless, a major obstacle for microalgal biodiesel production has been a relatively high cost. It is important to increase lipid content in a cell for decrease of the production cost.

D. parva is a unicellular green alga. It can accumulate lipids and large amounts of carotenoids and adapt to hypersaline environment and other unfavourable environmental stresses [5], [6]. In addition, its lack of a cell wall favours genetic manipulation and easier product extraction [6]. These advantages have made D. parva appealing compared with model microalgae as feedstock of biodiesel production.

Nitrogen limitation has so far become the most commonly utilized method for directing metabolic fluxes to lipid biosynthesis in microalgae [7]. The lipid content of microalga Nannochloris sp. UTEX LB1999 grown in a nitrogen limitation condition (0.9 mM KNO₃) was 1.5-fold of that grown in a nitrogen sufficient condition (9.9 mM KNO₃) [8]. The biomass and lipid content of Neochloris oleoabundans grown in nitrogen limitation (3 mM NaNO₃) and nitrogen sufficient conditions (10 mM NaNO₃) were 1.85 g/L, 40% and 3.15 g/L, 16%, respectively [2]. The lipid content of Dunaliella salina CCAP1918 was measured at 44% for the nitrogen limitation condition (2 mM KNO₃), while only 38% for the nitrogen sufficient condition (20 mM KNO₃) [9]. However, the biomass of Dunaliella salina CCAP1918 grown in nitrogen limitation condition was only 27% of that grown in a nitrogen sufficient condition [9]. Under a nitrogen limitation condition, the microalga Scenedesmus sp. CCNM 1077 could accumulate 27.93% lipids but only 18.87% lipids under a nitrogen sufficient condition [10]. Our former study also found that nitrogen limitation resulted in an increase of lipid content from 25% to 40% in D. parva. However, the biomass of D. parva under nitrogen limitation was only approximately 61% of that grown under a nitrogen sufficient condition [6].

Therefore, D. parva is a candidate to explore the metabolic effect of nitrogen limitation on Dunaliella, allowing its comparison with the former explorations from other microalgae, and enabling a comprehensive and in-depth understanding of lipid accumulation in microalgae under a nitrogen limitation condition.

Many studies on nitrogen limitation are focused on the effect of nitrogen limitation on lipid content, lipid types and fatty acid composition. Moreover, past researches on the mechanism of response to nitrogen limitation provided adequate information at the metabolite, protein and mRNA levels in some model algae. Schmollinger et al. used Chlamydomonas reinhardtii for a multicomponent analysis of the nitrogen-deplete response (the analysis of transcriptome and proteome) [11]. Li et al. identified a mutant with reduced triacylglycerol accumulation following nitrogen deprivation in the model alga C. reinhardtii, and found that a galactoglycerolipid lipase was responsible for triacylglycerol accumulation following nitrogen deprivation [12]. In total, 259 proteins were identified by mass spectrometry in the lipid droplet of C. reinhardtii under the nitrogen limitation condition. Among these proteins, a major protein was named the major lipid droplet protein (MLDP) [13]. Repression of MLDP expression led to an increase in the lipid droplet size, but the triacylglycerol content was stable [13]. The lipid content, fatty acid composition and metabolic changes of Nannochloropsis oceanica IMET1 were studied under the nitrogen limitation condition [14]. The molecular mechanism of photosynthetic carbon partitioning into neutral lipids was studied, and a model of photosynthetic carbon partitioning towards triacylglycerol was suggested in Nannochloropsis oceanica under a nitrogen limitation condition [15]. These researches provided the much-valued help in studying the nitrogen limitation in D. parva. However, these studies were largely confined to the model microalgae, which greatly limited the application of the results. Only a few studies have reported the mechanism of response to nitrogen limitation at different levels in the non-modal algae [16], [17], [18].

Our previous study investigated the changes of the transcriptome in D. parva under the nitrogen limitation condition and identified many important pathways, genes and an important transcription factor gene wri1 associated with biofuel production [6]. Although we analyzed the changes of the transcriptome in D. parva under the nitrogen limitation condition, a comprehensive and in-depth understanding of the mechanism of response to nitrogen limitation is needed with an investigation of the changes in proteome. Proteomics analysis is an important method to investigate the changes of proteins under environment stress [19], [20]. Quantitative proteomics could provide a full understanding about the changes of proteins following environmental stress and could reveal a possible response mechanism [21], [22]. At present, iTRAQ (isobaric tags for relative and absolute quantitation) was a widely used quantitative proteomics method for relative quantification of proteins in up to eight samples simultaneously, which had higher sensitivity compared with protein staining of traditional two-dimensional gel electrophoresis [23], [24], [25]. The iTRAQ uses isobaric tags to label peptides of different samples followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to quantify the proteome [23], [24]. It was expected that quantitative proteomics analysis with improved sensitivity could investigate the changes of proteins resulting from the exposure to nitrogen limitation. While several proteomic studies had been reported in the microalgae [26], [27], [28], [29], [30], [31], there was limited information on D. parva.

The trends of changes in proteins were not in accordance with that of the changes in mRNAs at some extent because of the existence of post-transcriptional regulation. Therefore, the combination of transcriptome analysis and proteomics analysis may contribute to a comprehensive insight into the mechanism of response to nitrogen limitation in D. parva.