RNA-­‐seq Analysis of the Haptophyte Emiliania huxleyi Reveals Novel Oleosin-­‐like Proteins Related to ProducFon of Neutral Lipids for Biofuels Gordon V. Wolfe, Dept. Biological Sciences, California State University, Chico Adarsh Jose, NSF Engineering Research Center for Biorenewable Chemicals (CBiRC), Iowa State University Results Background 1 Phosphate limitaFon and bicarbonate feeding sFmulate PULCA and LB producFon. Darkness sFmulates lipid catabolism. +BC L-­‐D 1.E+06 1.E+05 4.E-­‐04 3.E-­‐04 2.E-­‐04 60 PO4 deple9on light dark 300 250 200 150 100 50 0 1516 1516+BC 3 0 1 2 3 4 5 6 7 8 9 10 11 Neutral lipid (µg) PO4 (uM) 5.E-­‐04 1.E-­‐04 0 1 2 3 4 5 6 7 8 9 10 11 17 15 13 11 9 7 5 3 1 -­‐1 6.E-­‐04 # cells NL / cell (arbitrary) 1.E+07 Cells mL-­‐1 Emiliania huxleyi (Ehux), a coccolithophorid alga, is ubiquitous in the world’s oceans and forms massive blooms visible from space (photos above). Famous as one of the dominant calcifying organisms on the planet, Ehux also well known for its unusual neutral lipids. Instead of triacylglycerides (TAGs), it produces a series of Poly-­‐trans-­‐Unsaturated Long-­‐Chain (C37-­‐39) Alkenes, Alkenones, and Alkenoates (PULCA, fig. 1). Because the ra9o of di-­‐ and tri-­‐unsaturated C37 methyl alkenones (Uk’37) is a func9on of growth temperature [1], they are a widely used paleo-­‐thermometric tool. Some strains also produce a set of C31-­‐33 alkenes (HCs, fig. 1). PULCA, like TAGs, appear to play a role in cellular energy storage, accumula9ng under N or P limita9on to up to 20% of cell C in sta9onary phase [2, 3], and decreasing under energy-­‐depleted growth condi9ons (prolonged darkness) [3, 4]. By staining with the fluorescent lipophilic dye Nile Red, Eltgroth et al. [5] showed Ehux, like many other algae, package their neutral lipid into cytoplasmic vesicles or lipid bodies (LBs) that are extruded from plas9ds to the cytoplasm. Discussion 50 40 4 5 6 Size (µm) 7 Fig. 3: Cells increase in size increase from 3.5 to 4.5 µm a|er division stops due to phosphate limita9on (leY; curves are Gaussian fits) and LBs increase in size. Photos: cells in sta9onary phase, showing neutral lipid fluorescence from Nile Red staining. LBs accumulate in light (middle) and decrease in dark (right). C31:X HC C33:X HC C29 std C31-­‐33 HCs 30 PULCAs 20 10 0 0 1 2 3 4 5 6 7 8 9 10 11 day Fig. 1. Cells grew exponen9ally over 8 days un9l PO4 deple9on
0 1 2 3 4 5 6 7 8 9 10 11 day Fig. 2. Neutral lipid increased a|er bicarbonate dosing, and decreased in darkness (red symbols) Right: GC trace showing C31-­‐33 HCs, internal standard, and C37-­‐39 PULCAs
2 RNA-­‐seq reads detect the majority of genes. We detected 30,399 proteins as transcripts, 78% of which showed significant (p<0.05) change for at least one 9me point. These were grouped into 10 clusters by STEM analysis. The Emiliania huxleyi CCMP 1516 genome has been sequenced by the DOE JGI [6], offering an opportunity to use a transcriptomics approach to inves9gate genes associated with PULCA biosynthesis. Here we describe the experimental setup and one result, the discovery of a novel set of proteins possibly associated with LB packaging and mobiliza9on. 25
reduced PIDs RNA-­‐seq was performed on the 24 samples using Illumina HiSeq 2500. 100-­‐bp paired-­‐end reads were filtered (quality value >25) using the Fastx-­‐toolkit (h@p://hannonlab.cshl.edu/fastx_toolkit/ ); ~94% of the short reads were retained. RNA-­‐Seq by Expecta9on Maximiza9on (RSEM) was used to map the filtered reads to the reference transcript set to obtain read count es9mates (in Fragments per Kb transcript per Million Reads, FPKM) for each sample. Because JGI’s reduced transcript models have high redundancy (~2% sequences with 100% iden9ty and ~9% sequences with >99% iden9ty), we clustered the transcript model set using cd-­‐hit-­‐est [7] and only retained sequences with <99% iden9ty (30,340 transcripts) as the reference set. ~60% of the short reads mapped to the reference set; the remaining unmapped reads were assembled using Trinity Transcriptome Assembler [8] into 32,160 de-­‐novo assembled con9gs. Data analysis – Expression levels were log2-­‐transformed rela9ve to expression at day 2, then screened for sta9s9cally-­‐significant differen9al expression (t-­‐test, p < 0.05) and at least a 2-­‐fold increase or decrease on one or more days. The Short-­‐Term Expression Miner (STEM) [9] was used to cluster genes by expression profile using the K-­‐means algorithm. myosin mRNA splicing factor 20
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Fig. 6. STEM cluster analysis. Clusters are ordered by number of PIDs. Clusters 1-­‐6 show rela9vely li@le change except on d4, but clusters 7 & 9, or 5 & 3, show large increases or decreases following nutrient limita9on and bicarbonate dosing. 10
detected PIDs 5
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Methods Cell collecFon, RNA & lipid extracFon – Cells were collected on days 2, 4, 6, 9, 10, and 11 by centrifuga9on (RNA) or filtra9on onto GF/F (lipid). RNA was extracted immediately with Trizol (Life Technologies), and quality was checked by BioAnalyzer (Agilent). Neutral lipids were extracted with 1:1 hexane:chloroform and analyzed by GC-­‐MS with C29 fa@y acid internal standard. 40745
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total PIDs Fig. 4. PID sta9s9cs for genome (top 2 bars) and RNA-­‐seq (bo@om two bars). Culturing – We designed a 9me-­‐series experiment with E. huxleyi CCMP 1516 (genome strain). Cells were grown in triplicate 0.5-­‐1 L volumes (Sigma Sea Salts) under 80 µmol m-­‐2 s-­‐1 illumina9on at 16 °C in a 16:8 hr L:D cycle. We added f/2 nutrients with excess nitrate, trace metals, and vitamins to induce phosphate limita9on by day 8, when we added NaHCO3 (10 mM) to boost lipid produc9on, and split cultures on day 9 to con9nuous darkness to s9mulate lipid catabolism. Cultures were monitored daily for cell counts (hemocytometer), chlorophyll fluorescence, and neutral lipid (Nile Red). Phosphate was measured colorimetrically. 35
FPKM
Fig. 1: C37 PULCA structure. Top: poly-­‐C7-­‐trans unsaturated backbone. Two central alkene bonds are C14 and C21 from the carbonyl end, while sites of addi9onal desatura9on are shown by dashed double bonds. C38 and C39 analogs are extended at the methyl end. Terminal groups include methyl and ethyl alkenones, alkenoates, or alkenes. Bo\om: C31-­‐33 alkenes (HCs). # protein IDs (PIDs) 20,000 25,000 30,000 35,000 40,000 PULCA are poten9ally advantageous as biofuels compared to TAGs, in that these poly-­‐
trans unsaturated pure hydrocarbons have no glycerol, and are also much more resistant to photooxida9on than cis-­‐unsaturated fa@y acids [11]. Ehux has been shown to yield extremely high energy values via pyrolysis [12], and these lipids are star9ng to be explored as poten9al biofuels [13]. While a previous LB proteomics screen (Wolfe, in prepara9on) found other LB structural proteins such as PAP-­‐fibrillin homologs, the oleosin-­‐like proteins were missed because of their low mass (6-­‐12 kDa). Although these proteins are less hydrophobic and lack the proline knot mo9f, the hypothesis that they may coat LBs and help maintain or traffic them, as shown below, is supported by observa9ons that LBs grow larger when cells become phosphate limited (fig. 3), exactly when these proteins show reduced transcrip9on. 1
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Fig. 5. Examples of expression levels, significance, and pa@erns for 4 PIDs. Error bars show standard devia9on from mean (n=3). ExponenFal growth: •  high nutrients, low neutral lipid •  high ‘oleosin-­‐like’ protein expression maintains small LBs StaFonary phase: •  low nutrients, increased neutral lipid •  ‘oleosin-­‐like’ protein expression decreases, allows LBs to coalesce Acknowledgements 3 A novel set of oleosin-­‐like proteins that are strongly down-­‐regulated. As expected, proteins for P acquisi9on are among the most highly up-­‐regulated during P limita9on. In the two most highly down-­‐expressed clusters (3 & 5), we observed a set of proteins that, although unique to Emiliania, somewhat resemble oleosins, <25 kDa proteins that coat LBs in plant seeds and chlorophytes [10], and regulate LB trafficking and mobiliza9on. We thank Mark Brown, ISU, for GC-­‐MS analysis of lipids, and Jennifer Placek, VWR, for Moxi cell sizer analysis. This work was supported by NSF grant EFRI-­‐0938157. References 1.
2.
Fig. 8: Heat map of log2-­‐fold expression (rela9ve to day 2) of oleosin-­‐like PIDs detected in clusters 3 and 5, ordered by abundance and expression pa@ern. Two PIDs (452782 and 119000) appear to go down in dark, while one (436790) shows different expression pa@ern. All have BLAST hits to Oleosin-­‐B6 (B11, B13) or Pollen_coat_protein. Fig. 7: Heat map of log2-­‐fold expression (rela9ve to day 2) of phosphate transporters and other P-­‐related genes detected in cluster 9, ordered by abundance. 3.
Prahl, F.G., G.V. Wolfe, and M.A. Sparrow. 2003. Physiological impacts on alkenone paleothermometry. Paleoceanogr. 18: 1025. 4.
Epstein, B.L., S. D'Hondt, and P.E. Hargraves. 2001. The possible metabolic role of C37 alkenones in Emiliania huxleyi. Org. Geochem. 32: 867. 5.
Eltgroth, M.L., R.L. Watwood, and G.V. Wolfe. 2005. Produc9on and cellular localiza9on of long-­‐chain neutral lipids in the haptophyte algae Isochrysis galbana and Emiliania huxleyi. J. Phycol. 41: 1000. 6.
Read B.A., J. Kegel, M.J. Klute, A. Kuo et al. 2013. Pan genome of the phytoplankton Emiliania underpins its global distribu9on. Nature 499: 209. 7.
Li W. & A. Godzik. 2006. Cd-­‐hit: a fast program for clustering and comparing large sets of protein or nucleo9de sequences. Bioinforma9cs 22: 1658. 8.
Grabherr M.G., B.J. Haas, M. Yassour et al. 2011. Full-­‐length transcriptome assembly from RNA-­‐Seq data without a reference genome. Nature Biotechnology 29: 644. 9.
Ernst, J. & Z. Bar-­‐Joseph. 2006. STEM: a tool for the analysis of short 9me series gene expression data. BMC Bioinforma9cs 7: 191. 4 Ehux has a large set of highly similar oleosin-­‐like proteins, which are structurally disFnct from known oleosins in plants and algae. They are less hydrophobic, lack the proline knot mo9f, and are alanine-­‐proline-­‐arginine-­‐serine-­‐rich. 373518
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Fig. 9: E. huxleyi ‘oleosin-­‐like’ protein alignment. Most are near 12 kDa, but a few have inser9ons or dele9ons (above). Right: Jotan-­‐Hein alignment, with cluster-­‐iden9fied PIDs in green or red. 436790, which is structurally different, has very different expression pa@ern. 340.0
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PID 350586 351461 351793 354021 354409 354649 436790 356718 357507 357715 358275 362174 362703 363055 364259 364589 366057 367380 367503 369279 370464 371421 373518 317798 373841 374243 scaffold MW (kD) 7 12.72 11 13 25 27 28 48 11.80 51 59 61 12.77 67 126 135 140 163 168 12.71 204 255 261 358 6.38 455 12.81 600 1701 12.68 2201 13.53 2676 5373 Ala% Pro% Arg% Ser% Aliph. GRAVY 23.4% 13.7% 9.7% 8.1% 89.3 -­‐0.042 27.4% 8.5% 4.3% 8.5% 87.2 -­‐0.091 23.4% 12.9% 9.7% 8.9% 87.7 -­‐0.082 22.8% 12.2% 8.9% 8.9% 91.5 -­‐0.041 34.8% 16.7% 22.6% 13.7% 9.1% 7.6% 9.7% 8.1% 83.5 0.079 86.9 -­‐0.109 24.2% 13.7% 22.3% 13.1% 9.7% 8.1% 9.2% 8.5% 87.7 -­‐0.056 89.6 -­‐0.067 Fig. 10. LeY: ProtParam analysis. Right: Hydropathy analysis shows hydrophobic N-­‐
terminus, with hydrophilic C-­‐terminus. Below: I-­‐TASSER models (
h@p://
zhanglab.ccmb.med.umic
h.edu/I-­‐TASSER/) show globular structures. 375318
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Prahl, F.G., and S.G. Wakeham. 1987. Calibra9on of unsatura9on pa@erns in long-­‐
chain ketone composi9ons for paleotemperature assessment. Nature 330: 367. Epstein, B.L., S. D'Hondt, J.G. Quinn, J. Zhang, and P.E. Hargraves. 1998. The effect of dissolved nutrient concentra9ons on alkenone-­‐based temperature es9mates. Paleoceanogr. Curr. 13: 122. 10.  Huang, N.-­‐L., M.-­‐D. Huang, et al. 2013. Oleosin of subcellular lipid droplets evolved in green algae. Plant Physiology 161: 1862. 354649 369279
11.  Rontani, J.-­‐F., P. Cuny, V. Grossi, and B. Beker. 1997. Stability of long-­‐chain alkenones in senescing cells of Emiliania huxleyi: effect of photochemical and aerobic microbial degrada9on on the alkenone unsatura9on ra9o (Uk'37). Organic Geochem. 26: 503. 12.  Wu, Q., J. Dai, Y. Shiraiwa, G. Sheng, and J. Fu. 1999. A renewable energy source — hydrocarbon gases resul9ng from pyrolysis of the marine nanoplanktonic alga Emiliania huxleyi. J. Appl. Phycol. 11:137. 13.  O’Neil, G. W., C. A. Carmichael, et al. 2012. Beyond fa@y acid methyl esters: expanding the renewable carbon profile with alkenones from Isochrysis sp. Energy & Fuels 26: 2434.