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It is known that adaptive evolution in permanently cold environments drives cold adaptation in enzymes. However, how the relatively high enzyme activities were achieved in cold environments prior to cold adaptation of enzymes is unclear. Here we report that an Antarctic strain of Chlorella vulgaris, called NJ-7, acquired the capability to grow at near 0 C temperatures and greatly enhanced freezing tolerance after systematic increases in abundance of enzymes/proteins and positive selection of certain genes. Having diverged from the temperate strain UTEX259 of the same species 2.5 (1.1–4.1) to 2.6 (1.0–4.5) Ma, NJ-7 retained the basic mesophilic characteristics and genome structures. Nitrate reductases in the two strains are highly similar in amino acid sequence and optimal temperature, but the NJ-7 one showed significantly higher abundance and activity. Quantitative proteomic analyses indicated that several cryoprotective proteins (LEA), many enzymes involved in carbon metabolism and a large number of other enzymes/proteins, were more abundant in NJ-7 than in UTEX259. Like nitrate reductase, most of these enzymes were not upregulated in response to cold stress. Thus, compensation of low specific activities by increased enzyme abundance appears to be an important strategy for early stage cold adaptation to Antarctica, but such enzymes are mostly not involved in cold acclimation upon transfer from favorable temperatures to near 0 C temperatures. Key words: Antarctica, cold adaptation, intraspecies divergence, omics, enzyme activity, Chlorella vulgaris. Introduction warm seasons (Convey et al. 2014). In contrast, microalgae, Antarctica is the coldest continent on the earth, with 99.8% of bacteria, and fungi not only grow on these ice-free sites but thearea(Burton-Johnson et al. 2016) covered by a sheet of ice also thrive in sea ice (Thomas and Dieckmann 2002), snow that averages about 2 km thick (Fretwell et al. 2013). The lowest fields (Davey et al. 2019), and ice-covered lakes (Karl et al. 1999; air temperature in Antarctica may reach 89.2 C(National Possmayer et al. 2016). Microbes from other continents could Polar Research Institute (Japan) 1991), but the ground surface be transported to Antarctica by atmospheric circulation temperatures on ice-free sites are in a range of 35 to 5 Cin (Mayol et al. 2017; C aliz et al. 2018), even though their contri- most time of a year (Guglielmin 2006). In Antarctica, animal bution to Antarctic microbial communities is limited (Archer and plant communities are naturally separated from those on et al. 2019). other continents (Convey and Stevens 2007; Fraser et al. 2012) In the geological history, Antarctica was formed from the and largely limited to ice-free sites that receive meltwater in breakup of the supercontinent Gondwana. About 34–33 Ma, The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any Open Access medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Mol. Biol. Evol. 37(3):849–863 doi:10.1093/molbev/msz273 Advance Access publication November 20, 2019 849 . Wang et al. doi:10.1093/molbev/msz273 MBE the atmospheric CO level significantly dropped, and the ice identical to that of C. vulgaris UTEX259, a strain initially 0 0 cover expanded rapidly on the continent to near-modern collected and isolated from Delft (52 00 N, 04 21 E), the dimensions or larger than present-day values; about 23 Ma, Netherlands (https://utex.org; last accessed November the Drake Passage opened between Antarctica and South 24, 2019); its ITS2-28S rRNA region differs from America (Galeotti et al. 2016; Lear and Lunt 2016). UTEX259 at five bases (supplementary fig. S1, Mesophilic microorganisms that experienced the quick cool- Supplementary Material online). They show similar ing period might evolve into psychrophilic or psychrotrophic growth at 20 C(fig. 1a), but only NJ-7 is able to grow species; on the other hand, those microorganisms trans- at 4 C(fig. 1b); after being frozen at 20 C, the surviv- ported from other continents in later periods might also de- ability of NJ-7 is much higher than that of UTEX259, and velop the cold-growth capability and freezing tolerance. the survivability can be enhanced by preconditioning (48- The main challenge faced by organisms in Antarctica is to h exposure) at 4 C(fig. 1). When cultured at 20 C, both keep cell activities at near 0 C temperatures and maintain strains show maximal photosynthetic activities at 30 C survivability under freezing conditions. The freezing tolerance (supplementary fig. S2, Supplementary Material online); depends on ice-binding proteins (Bar Dolev et al. 2016; Collins NJ-7 maintains higher photosynthetic activities than and Margesin 2019) and LEA (late embryogenesis abundant) UTEX259 at high temperatures. These physiological char- proteins (Shih et al. 2008; Liu et al. 2011; Wang et al. 2011). These acteristics indicate that NJ-7 is a mesophilic algal strain proteins prevent the growth/recrystallization of ice or freeze/ evolving toward a psychrotrophic one. The higher photo- thaw-induced inactivation of enzymes. Cell activities depend on synthetic activities of NJ-7 at high temperatures could be various biochemical reactions. Our understanding of biochem- either associated with or independent of the enhanced ical reactions in the cold is largely based on cold-adapted or freezing tolerance. psychrophilic enzymes (Morgan-Kiss et al. 2006). Compared with their mesophilic homologs, such enzymes feature signifi- Nuclear Genome Assembly and Synteny Analyses cantly lowered optimal temperatures and adaptive mutations To analyze the relationship between the two strains of the that destabilize the structures bearing the active site or the same species and the mechanism for NJ-7 to develop the overall structure (Feller and Gerday 2003; Santiago et al. cold-growth capability and freezing tolerance, we sequenced 2016). It has been shown that the optimal temperature or ther- the whole genomes of both strains. Transcriptomes were also mal stability of a mesophilic enzyme can be downshifted by site- sequenced to facilitate genome assembly and gene predic- directed mutagenesis (Saavedra et al. 2018; Liao et al. 2019)and tions. The assembled NJ-7 nuclear genome consisted of that the activity at a low temperature can be increased by di- 39.1 Mb in 753 scaffolds with an N50 size of 938 kb; the rected evolution (Zhao and Feng 2018). However, because UTEX259 nuclear genome consisted of 39.1 Mb in 780 scaf- adaptive mutations that cause coding differences are relatively folds with an N50 size of 498 kb. A total of 9,412 protein- rare (Jones et al. 2012) and coding differences toward cold ad- coding genes were predicted in NJ-7 and 9,439 in UTEX259; aptation must be even rarer, it remains to be answered whether 13,390 transcripts (isotigs) were identified in NJ-7, and 13,880 there is an alternative strategy to maintain relatively high en- identified in UTEX259 (features are summarized in table 1, zyme activities at near 0 C temperatures before enzymes are and detailed information for sequencing, assembly and anno- significantly cold-adapted. tation in supplementary tables S1–S6, Supplementary Without cold-growing microorganisms generated from Material online). mesophiles by experimental evolution, natural intraspecies The quality and gene coverage of genome assemblies divergence in the capability to grow at low temperatures were then assessed. First, completeness of genome assem- would provide very valuable materials for studies. Here, we blies was assessed by BUSCO (Simao et al. 2015). Of the analyzed an Antarctic strain and a temperate strain of 2,168 universal single-copy orthologs of the chlorophyta Chlorella vulgaris (green alga) at genomic, transcriptomic, gene set, 90.0% were found in the NJ-7 genome assembly as and proteomic levels. Our results indicate that systematic complete genes, and 90.3% found in UTEX259; 4.6% and elevation in abundance of enzymes could allow a mesophilic 4.3% were completely missing in NJ-7 and UTEX259 assem- newcomer to develop the capability to grow at near 0 C blies, respectively (supplementary table S7, Supplementary temperatures, representing an early stage adaptive mecha- Material online). Numbers of predicted genes in C. vulgaris nism in Antarctic environments. Remarkably, most enzymes strains NJ-7 and UTEX259 are close to those of C. variabilis with higher levels in the Antarctic strain are not upregulated NC64A and Coccomyxa subellipsoidea C-169. Second, the upon transfer from 20 to 4 C. genome assemblies were evaluated by aligning with assem- bled transcripts (Sanger-based fosmid-end sequences also used for assessing NJ-7). For NJ-7, about 97.3% of the Results fosmid-end sequences were mapped to the genome as- Physiological Divergence between C. vulgaris NJ-7 and sembly, and 93.5% of the transcripts mapped over at least UTEX259 90% of their length. Of 956 pairs of fosmid-end sequences, Chlorella vulgaris NJ-7 was isolated from rock samples 790 pairs were mapped to the same scaffold, and the av- collected near a transitory pond 5 km away from the erage distance was 34.7 kb, almost identical to the esti- 0 0 Zhongshan Station (69 22 S–76 22 E) in Antarctica (Hu mated average insert size of fosmid clones (35.0 kb). et al. 2008). Its 18S rRNA-ITS1-5.8S rRNA region is To the UTEX259 genome assembly, about 93.4% of the 850 . Early Stage Adaptation of a Mesophilic Green Alga to Antarctica doi:10.1093/molbev/msz273 MBE FIG.1. Comparisons of the cold-growth capability and freezing tolerance between NJ-7 and UTEX259. (a) Growth of NJ-7 and UTEX259 at 20 C and survivability of the two strains after being frozen at 20 C (inset). (b) Growth of NJ-7 and UTEX259 at 4 C and survivability of the two strains grown at 20 C, pretreated at 4 C (48 h) and frozen at 20 C (inset). Data are means 6 SD from three biological replicates. Table 1. Genome and Transcriptome Statistics of NJ-7 and UTEX259. Comparison of Organelle Genomes and Estimation of Features NJ-7 UTEX259 Divergence Time The relationship between the two strains was also shown with Nuclear genome size (Mb) 39.08 39.13 Genomic G1C content (%) 61.68 61.68 their organelle genomes and divergence time. Except for two Number of scaffolds 753 780 genomic inversions and differences in psbA and psbC (with or Scaffold N50 (bp) 937,767 497,853 without an intron that contains an endonuclease gene), the Repeated sequences (%) 6.2 5.9 chloroplast genomes of NJ-7 and UTEX259 are identical in Number of protein-coding genes 9,412 9,439 gene content and arrangement (supplementary fig. S5 and Genes with transcript support (%) 84.92 86.48 Genes with homology support (%) 87.78 86.16 table S9, Supplementary Material online). The two chloroplast Average protein length (aa) 507 515 genomes closely resemble that of C. vulgaris C-27 [a strain Average exon length (bp) 178 180 0 0 isolated from Sendai (38 16 N, 141 02 E), Japan (NIES-2170, Average intron length (bp) 216 218 http://mcc.nies.go.jp/; last accessed November 24, 2019)] but Average number of exons per gene 8.54 8.57 differ from those of other Chlorella strains in gene arrange- Number of isotigs 13,390 13,880 Syntenic regions (%) 99.37 97.68 ment (supplementary fig. S6, Supplementary Material online). The mitochondrial genomes of NJ-7 and UTEX259 are even more similar to each other (supplementary fig. S7 and table S9, transcripts were mapped over at least 90% of their length. Supplementary Material online). Organelle genomes of NJ-7 Single-base mismatch and insertion/deletion frequencies are smaller than that of UTEX259 due to the reduced non- of the two genome assemblies were <1.0 base/10 kb (com- coding regions. pared with sequences generated by Sanger sequencing). Divergence times between NJ-7, UTEX259, other green al- These results indicated the high coverage and high quality gae, and higher plants were estimated based on sequences of of the genome assemblies. 36 chloroplast genes, with three fossil calibrations as previ- Similarities between nuclear genomes of the two strains ously reported (Herron et al. 2009). Broad-scale analyses were were evaluated with synteny and collinearity analyses. performed with four combinations of species/strains (fig. 2a; Scaffolds longer than 10 kb, which covered over 97% of supplementary fig. S8a–c, Supplementary Material online). It each genome, were used to generate the syntenic dot plot is deduced that NJ-7 diverged from UTEX259 about 2.5 (95% (supplementary fig. S3, Supplementary Material online). The confidence interval 1.1–4.1) Ma to 2.6 (1.0–4.5) Ma, much result indicated that syntenic regions covered 99.37% of the later than the divergence between NJ-7/UTEX259 and NC64A NJ-7 scaffolds and 97.68% of the UTEX259 scaffolds. The lon- [153.4 (69.2–247.1) Ma], also later than the opening of Drake gest 12 scaffolds (with a total length of 18.1 Mb) of NJ-7 and Passage (23 Ma). The structure of the chronogram in the corresponding scaffolds of UTEX259 were used to gener- figure 2a is consistent with the dendrograms based on amino ate the dual synteny plot (supplementary fig. S4, acid and nucleotide sequences of 1,080 nuclear genes (sup- Supplementary Material online). Only one large-scale geno- plementary fig. S9a and b, Supplementary Material online). In mic rearrangement was identified. The percentage of collin- addition to the broad-scale chronograms, we also con- earity between NJ-7 and UTEX259 was 97.33%; amino acid structed a fine-scale chronogram for Chlorella species, in sequence identity between their reciprocal best blast hits was which the divergence time between UTEX259 and NJ-7 94.68%, significantly higher than those between C. vulgaris (and C-27) was estimated to be 2.9 (0.6–6.4) Ma (fig. 2b). In and other green algae (supplementary table S8, light of the mesophilic characteristics of NJ-7 and the esti- Supplementary Material online). mated time for its divergence from UTEX259, the ancestor of 851 . Wang et al. doi:10.1093/molbev/msz273 MBE FIG.2. Chronograms showing estimated divergence times for green algae and higher plants. Divergence times were estimated using BEAST based on 36 chloroplast genes. Branch lengths are proportional to the absolute ages of nodes (scale on x-axis in million years). Numbers to the right or left of nodes are the ages of nodes and 95% confidence intervals (in parentheses). (a) One of the broad-scale chronograms. A, B, and C indicate the nodes to which time constrains were applied based on fossil records. The other three chronograms (with different combinations of species/strains) are shown in supplementary figure S8, Supplementary Material online. (b) The fine-scale chronogram. The divergence time between Chlorella variabilis and C. vulgaris estimated from the broad-scale analysis, in particular 157.0 Ma (73.6–245.1 Ma), was used to calibrate the fine-scale analysis (indicated by an arrow) but slightly changed after the computations. Of the four divergence times between C. variabilis and C. vulgaris, 157.0 Ma (73.6–245.1 Ma) from (a) is close to the average. NJ-7 was probably transported to the Antarctic continent With the genomic data, we first searched for genetic changes from a temperate region. that may be associated with cold adaptation. NJ-7 shows much less nonsynonymous substitutions per site and gene duplications than UTEX259 (supplementary table S10, Genomic Evolution toward Cold Adaptation of NJ-7 Supplementary Material online), but the two strains are com- Cold adaptation is an evolutionary process toward the capa- parable in numbers of positively selected genes (supplemen- bilities to grow at near 0 C temperatures and survive under tary excel S1, Supplementary Material online) and alternative freezing conditions. This process depends on accumulation of splicing events (supplementary excel S1, Supplementary adaptive mutations and leads to divergence between strains. 852 . Early Stage Adaptation of a Mesophilic Green Alga to Antarctica doi:10.1093/molbev/msz273 MBE Material online). Positive selections involved regulation of quality of RNA-seq data. Expression levels assessed with RT- RNA conformation, desaturation of membrane lipids, energy qPCR correlated well (R ¼ 0.903) with those obtained from metabolism, anabolic and catabolic reactions, metabolite the RNA-seq analysis (supplementary fig. S11, Supplementary transport, protein processing and translocation, intracellular Material online). The RNA-seq and proteomic data were also movement,transcription,DNA repair,etc.For an example, used to analyze the differential expression between two the sphingolipid delta(4)-desaturase (NJ- strains at 20 or 4 C (supplementary excels S2 and S3, 7.evm.TU.scaffold00053.17) was positively selected. Supplementary Material online), but the differences between Sphingolipids are enriched in the outer leaflet of plasma protein abundance in two strains were analyzed by generat- membrane in plants, and the long chain base of sphingolipids, ing a new NJ-7/UTEX259 protein database, with which orthol- called sphinganine, can be desaturated by sphingolipid ogous proteins were identified based on those identical delta(4)-desaturase and sphingolipid delta(8)-desaturase trypsin-digested peptides. The RNA-seq and proteomic anal- (Luttgeharm et al. 2016). The delta(8)-desaturase plays a ma- yses were highly reproducible between biological replicates as jor role in desaturation of long chain base and cold tolerance shown with Pearson’s correlation coefficients (supplementary in higher plants (Zhou et al. 2016) but is not found in fig. S12, Supplementary Material online). Numbers of differ- Chlorella, Chlamydomonas,and Volvox. The positive selection entially expressed genes in four data sets are shown in sup- of sphingolipid delta(4)-desaturase in NJ-7 probably reflects plementary table S17, Supplementary Material online. Genes the critical role of sphingolipid desaturation in cold adapta- up- or downregulated in NJ-7 or UTEX259 after transfer from tion. Of genes with alternative splicing, there are seven related 20 to 4 C are related to cold acclimation, whereas those with to DNA repair in NJ-7 but only one in UTEX259 (supplemen- altered expression in NJ-7 relative to their homologs in tary table S11, Supplementary Material online). These alter- UTEX259 are related to strain divergence. native splicing events in NJ-7 might have contributed to Using upregulated genes as the example, we first analyzed adaptation to the strong UV radiation in Antarctica (Liao overlaps between different data sets. The results are summa- and Frederick 2005). rized in figure 3, from which four implications can be derived: Unique genes (supplementary excel S1, Supplementary 1) Strain divergence is less dependent on regulation at mRNA Material online), high-copy-number genes (supplementary level than cold acclimation. During cold acclimation, over fig. S10 and table S12, Supplementary Material online), and 70% of genes [215/(215þ 84)] upregulated in NJ-7 at the expanded gene families (supplementary tables S13–S15, protein level are concurrently upregulated at the mRNA level Supplementary Material online) were also analyzed, but no (see fig. 3a-I); relative to their homologs in UTEX259, only apparent connection to cold adaptation was identified. Gene 28.1% of genes [113/(113þ 299)] with higher protein levels in duplication has been shown to be a rapid mechanism for NJ-7 also show higher mRNA levels (at 4 C, see fig. 3b-II); 2) adaptation to stressful or novel environmental conditions most proteins [193/(193þ 106)] upregulated in NJ-7 during (Kondrashov 2012). We identified genes with multiple copies cold acclimation are also upregulated in UTEX259 (see fig. 3c- in NJ-7 and UTEX259 and analyzed the relationship between I); 3) most proteins [312/(312þ 100)] with higher abundance gene expression and copy numbers. Only one gene (MFS in NJ-7 than in UTEX259 at 4 C show higher abundance at transporter) with higher copy number in UTEX259 showed 20 C(see fig. 3c-III); and 4) most proteins [374/(374þ 38)] increased expression relative to its homolog in NJ-7 (supple- with higher abundance in NJ-7 than in UTEX259 at 4 Care mentary fig. S10 and table S12, Supplementary Material not upregulated during cold acclimation (see fig. 3c-II). online). For downregulated genes, overlaps between data sets of “NJ-7/UTEX259” exhibit a similar pattern (supplementary fig. Cold Adaptation and Cold Acclimation of NJ-7 Based S13b, c-II, and c-III, Supplementary Material online) to those on Altered Gene Expression for upregulated genes (fig. 3b, c-II, and c-III), but the number Unlike cold adaptation, cold acclimation is a physiological of proteins downregulated during cold acclimation (supple- process that relieves the cold stress in temperature- mentary fig. S13a, Supplementary Material online) is much fluctuating environments and prepares for the freezing con- less than that of proteins upregulated (fig. 3a), and most ditions (Morgan-Kiss et al. 2006). It depends on the regulation proteins downregulated in NJ-7 and UTEX259 during cold of gene expression and metabolic pathways. To which extent acclimation do not overlap (supplementary fig. S13c-I, genes/proteins regulated in cold acclimation are involved in Supplementary Material online). cold adaptation has not been addressed. Divergence between the two strains was driven by adap- To understand how NJ-7 adapted to the Antarctica, we tation to environments and genetic drifts. Adaptation of NJ-7 further analyzed mRNA and protein profiles in NJ-7 and to the cold environment is one of the most important factors. UTEX259 cultured at 20 C with or without treatment at Of the 38 upregulated proteins (fig. 3; supplementary table 4 C, and identified differential gene expression in the same S18, Supplementary Material online) involved in both strain strain at mRNA (RNA-seq) (supplementary table S16 and divergence and cold acclimation, sphingolipid delta(4)- excel S2, Supplementary Material online) and protein (prote- desaturase was positively selected (supplementary excel S1, omic analysis) levels (supplementary excel S3, Supplementary Supplementary Material online). Other proteins in the list are Material online). Real-time quantitative polymerase chain re- involved in freezing tolerance (cryoprotective protein), cold action (RT-qPCR) analysis was conducted on eight genes in stress response and tolerance, cell division, carbon metabo- cells grown at 20 C and exposed to 4 Cto evaluate the lism, etc. As we pointed out above, many more proteins with 853 . Wang et al. doi:10.1093/molbev/msz273 MBE FIG.3. Venn diagrams showing overlaps between genes upregulated in RNA-seq and proteomic analyses or between proteomic data sets. Upregulation at protein level is defined as a fold change in protein abundance 1.3 (P-value < 0.05); upregulation at mRNA level is defined as a fold change 2.0 (P-value < 0.05). NJ-7 4 C/20 C, UTEX259 4 C/20 C: genes upregulated at mRNA or protein level in NJ-7 or UTEX259 during cold acclimation; 4 C NJ-7/UTEX259, 20 C NJ-7/UTEX259: genes with higher expression in NJ-7 than in UTEX259 (strain divergence) at 4 Cor 20 C. Prot., proteomic data; RNA, RNA-seq data. (a) The overlap between genes upregulated in RNA-seq and proteomic analyses for cold acclimation of NJ-7 (I) or UTEX259 (II). There are 215 genes upregulated at both mRNA and protein levels in NJ-7 at 4 C relative to 20 C, 216 genes at both levels in UTEX259. (b) The overlap between genes upregulated in RNA-seq and proteomic data for strain divergence at 20 C (I) or 4 C (II). At 20 C, 138 genes show increased expression at both levels in NJ-7 relative to UTEX259; at 4 C, 113 genes. (c) Overlaps between proteomic data sets showing 193 genes upregulated at the protein level in both NJ-7 and UTEX259 during cold acclimation (I), 312 genes upregulated in NJ-7 relative to UTEX259 at both 20 and 4 C (III), but only 38 genes upregulated in NJ-7 in both cold acclimation and strain divergence (II). higher abundance in NJ-7 are not upregulated during cold extracted predicted NJ-7/UTEX259 proteins based on their acclimation. Increases in abundance of these proteins may similarities to gene products of the two GO categories in play an important role in the maintenance of cellular activ- AmiGO 2 (http://amigo.geneontology.org/amigo;last ities at near 0 C temperatures and the greatly enhanced accessed November 24, 2019). GSEA showed positive enrich- freezing tolerance. ments for these two groups of proteins in cold acclimation To identify functions enhanced in NJ-7 relative to (supplementary fig. S15, Supplementary Material online). UTEX259, we performed gene set enrichment analysis We also identified 152 transcription factors (TFs) in NJ-7 (GSEA) of differentially expressed genes based on Gene and 163 TFs in UTEX259 and analyzed their differential ex- Ontology (GO) categories (supplementary fig. S14, pression across temperatures and strains (supplementary ex- Supplementary Material online). NJ-7 (supplementary fig. cel S4, Supplementary Material online). The differential S14a and b, Supplementary Material online) and UTEX259 expression of TFs may lead to the differences in transcription (supplementary fig. S14c and d, Supplementary Material on- of target genes. Several TFs with identified recognition motifs line) show positive enrichments for many GO terms in cold (for orthologs) in C. variabilis NC64A were used to test this acclimation but only one to several GO terms in strain diver- possibility, but none of them showed a change in expression gence (supplementary fig. S14e, Supplementary Material on- correlated with that of a potential target gene. line). At 4 C, only the GO category “chromatin” is enriched in NJ-7 compared with UTEX259. However, this analysis would Elevated Abundance of LEA Proteins in NJ-7 not identify positive enrichments for functional categories LEA proteins were first identified in cotton and so named not in the GO list or pathways with only one or two rate- because they accumulate during the late maturation stages of limiting reactions enhanced. In addition, many NJ-7/UTEX259 seed development (Dure et al. 1981). Proteins in this family genes involved in cold acclimation show the highest similarity play important roles in stress tolerance in bacteria, fungi, to homologs of other green algae in public databases, but plants, and animals (Shih et al. 2008). In particular, they can these algal homologs have not been assigned to the GO cat- enhance freezing tolerance as cryoprotectants (Honjoh et al. egory of “cold acclimation” or “response to cold.” We 2001; Sasaki et al. 2014). In this study, we systematically 854 . Early Stage Adaptation of a Mesophilic Green Alga to Antarctica doi:10.1093/molbev/msz273 MBE FIG.4. Physical locations of LEA protein genes, predicted cellular locations of LEA proteins and their phylogenetic relationship. LEA proteins/genes are almost identical to each other between counterparts in NJ-7 and UTEX259, but hiC6-5 is only found in NJ-7. (a) LEA protein genes in assembled scaffolds of NJ-7. Arrows indicate orientations of genes. (b) Unrooted phylogenetic tree of NJ-7 LEA proteins with the predicted cellular locations indicated. The scale bar shows expected substitutions per site. Genes clustered on the genome and the phylogenetic tree are highlighted with corresponding colors. identified LEA protein genes in NJ-7 and UTEX259 by motif respectively treated as one protein, plus CvLEA3, CvLEA5, and secondary structure searches, Pfam domain searches, and CvLEA10, CvLEA11, and CvLEA14) with higher abundance similarity (to known LEA proteins) searches (supplementary in NJ-7 than in UTEX259 (fold change 1.3, P-value table S19, Supplementary Material online). Compared with < 0.05) at both 20 and 4 C, three additional LEA proteins other green algae, C. vulgaris strains possess more LEA pro- (CvLEA2, CvLEA13, and hiC12) with higher abundance in NJ-7 teins (supplementary table S20, Supplementary Material on- at 20 C(fig. 5). All of their encoding genes, except CvLEA2 line). Except hiC6-5 that is not found in UTEX259, all LEA and CvLEA14, also showed higher mRNA levels in NJ-7 (fold protein genes are very similarly arranged in the two genomes. change 2.0, P-value < 0.05) under the corresponding con- In NJ-7, CvLEA1/CvLEA2/CvLEA3/CvLEA4, CvLEA7/CvLEA8, ditions. GSEA of LEA proteins differentially expressed between CvLEA12/CvLEA11/Ccor1-1/Ccor2-1/Ccor1-2/Ccor2-2, and NJ-7 and UTEX259 showed significant positive enrichments in hiC6-1/hiC6-2/hiC6-3/hiC6-4/hiC6-5 are organized in four NJ-7 at both 20 and 4 C(supplementary fig. S16a, gene clusters (fig. 4a); genes from the same genomic cluster Supplementary Material online). Because LEA proteins are are also clustered in the phylogenetic tree (fig. 4b). In not listed as a GO category, and only one from NJ-7 (NJ- C. variabilis NC64A, there are genes homologous to CvLEA1 7.evm.TU.scaffold00034.28) shows similarity to an LEA pro- (1 copy) and hiC6 (1 copy), but no homologs to CvLEA7/ tein in the GO category “response to cold” (including cold CvLEA8 or Ccor1/Ccor2. Homologs to Chlorella LEA genes acclimation), the positive enrichment would not be identified are not found in other green algae. Apparently, these gene by the GSEA based on GO (supplementary fig. S14, clusters in C. vulgaris, except for the occurrence of hiC6-5, had Supplementary Material online).The increased abundance been formed by gene duplication before the divergence be- of so many LEA proteins would greatly enhance the freezing tween NJ-7 and UTEX259. The encoded LEA proteins are tolerance of NJ-7. On the other hand, because LEA proteins predicted to be located in the cytoplasm, nucleus, or organ- can reduce cellular peroxides and protect enzyme activities elles (fig. 4b) to provide cryoprotection of enzymes/proteins under stresses, they may promote tolerance against high tem- in different cellular compartments, but the predicted loca- perature and other stresses (Zhang et al. 2014; Wang et al. tions need to be confirmed with experiments in the future. 2017). In NJ-7, such effects of LEA proteins may indirectly Most LEA proteins in the two strains differ from each other by promote photosynthetic activities at high temperatures (sup- <3% amino acid residues (supplementary table S21, plementary fig. S2, Supplementary Material online). Supplementary Material online). As shown in the fine-scale chronogram (fig. 2b), NJ-7 di- Based on proteomic analyses, we identified seven LEA pro- verged from another temperate strain C-27 of C. vulgaris 1.9 teins (five isoforms of hiC6 and two versions of Ccor2 were (0.3–4.2) Ma. C-27 also accumulates LEA proteins upon 855 . Wang et al. doi:10.1093/molbev/msz273 MBE FIG.5. Differential expression of LEA protein genes in NJ-7 and UTEX259 at 20 or 4 C based on RNA-seq and proteomic analyses. ND, not detected. Different copies of hiC6 could not be differentiated from each other at protein and mRNA levels; therefore they were treated as one gene. Different copies of Ccor1 and Ccor2 could be differentiated at mRNA level, but the two copies of Ccor2 could not be differentiated at protein level. Data are means of three biological replicates, P-values are given in supplementary excels S2 and S3, Supplementary Material online. Blue and red asterisks indicate significantly higher mRNA levels (fold change 2.0, P-value < 0.05) in NJ-7 and UTEX259, respectively. exposure to cold treatment (3 C) (Honjoh et al. 1995; Joh (fig. 6b). However, the NR activity in NJ-7 was significantly et al. 1995) and shows cold-induced freezing tolerance higher than that in UTEX259 at all the temperatures tested. (Hatano et al. 1976). It is deduced that the ancestor of NJ-7 The activity of NJ-7 NR at 4 C was approximately equal to had possessed cold-inducible freezing tolerance before arrival that of UTEX259 NR at 25–30 C. This implied that NJ-7 at Antarctica, but the tolerance of NJ-7 was further developed probably has higher abundance of NR than UTEX259. to a higher level with much less dependence on cold induc- Western blot analysis confirmed this idea (fig. 6b). The ratio tion (fig. 1). Even so, many LEA protein genes in NJ-7 of NR abundance in NJ-7 and UTEX259 (cultured at 20 C) remained to be cold-regulated. hiC6, hiC12, CvLEA1, CvLEA2, was 2.546 0.62, very close to the ratio of NR activity, CvLEA7,and CvLEA13 in NJ-7 showed weaker responses to 2.706 0.89. In the proteomic analysis data, the abundance cold than in UTEX259; CvLEA14 showed similar responses in of NR in NJ-7 was shown to be 1.366 0.08-fold of that in the two strains; the two Ccor2 genes and CvLEA3, CvLEA5, UTEX259 at 20 C and 1.376 0.07-fold at 4 C(fig. 6c). CvLEA9,and CvLEA11 even showed stronger responses in NJ-7 Compared with Western blot and enzyme activity analyses, (supplementary fig. S17, Supplementary Material online). proteomic analysis produced lower values for fold changes. This supported the use of fold change 1.3 (P-value < 0.05) as the criterion for upregulation of protein abundance in the Systematic Increases in Abundance of Metabolic proteomic analysis. In addition to NR, proteomic analysis data Enzymes in NJ-7 also showed a higher abundance of nitrite reductase (NiR) in In addition to the greatly enhanced freezing tolerance, NJ-7 NJ-7 than in UTEX259 at 20 and 4 C(fig. 6c). The higher acquired the capability to grow at 4 C. How are metabolic abundance of NR and NiR in NJ-7 may not be necessarily activities maintained in NJ-7 at such a low temperature? associated with increased mRNA level (fig. 6c). In algal cells, Nitrate reductase (NR) is the most often used enzyme in NR converts NO into NO ,then NiR converts NO into 3 2 2 studies of cold adaptation of green algae (Loppes et al. NH for synthesis of glutamine. Increased abundance of NR 1996; di Rigano et al. 2006). We first compared the amino and NiR in NJ-7 could significantly enhance the metabolism acid sequences of NR (877-aa) in NJ-7 and UTEX259 but of nitrate. In other words, before NR and NiR are cold adapted found only 19 substitutions (fig. 6a). Then, we assayed the in NJ-7, their concentrations are elevated to compensate for NADH:NR activities in NJ-7 and UTEX259 at different temper- the low specific activities, so that nitrate can be utilized ac- atures. The two curves of NR activity versus temperature were tively at low temperatures. similar to each other in shape, with the optimal temperature Many enzymes involved in other aspects of cell activities slightly shifted from 25 to 30 Cin UTEX259 to 25 Cin NJ-7 are also upregulated in NJ-7 relative to UTEX259 856 . Early Stage Adaptation of a Mesophilic Green Alga to Antarctica doi:10.1093/molbev/msz273 MBE FIG.6. Differential expression of NR and NiR genes in NJ-7 and UTEX259. (a) Structure of genes indicating amino acid substitutions. (b) Comparison of abundance and activity of NR in NJ-7 and UTEX259. Cells were cultured at 20 C, and cell-free extracts were used in enzyme activity assays at different temperatures. The inset represents a typical result of Western blot analysis of NR in the two strains. (c) Differential expression of NR and NiR genes in the two strains as shown with RNA-seq and proteomic data. Asterisks indicate significantly higher expression in NJ-7 compared with UTEX259 (RNA-seq: fold change 2, P-value< 0.05; proteomic data: fold change 1.3, P-value< 0.05). Data are means6 SD of three biological replicates. (supplementary excel S3, Supplementary Material online), es- whereas the large subunit did not. Like LEA proteins, most of pecially some involved in carbon metabolism: for two critical these enzymes from the two strains differ from each other by steps in Calvin cycle, RuBisco activase/small subunit of RuBP <3% amino acid residues (supplementary table S22, carboxylase and sedoheptulose-1,7-bisphosphatase (supple- Supplementary Material online). mentary fig. S18, Supplementary Material online); for glycol- ysis, the bifunctional enzyme phosphofructokinase (PFK-2)/ Discussion fructose-2,6-bisphosphatase, phosphoglycerate kinase, phos- phoglycerate mutase, and phosphate dikinase (fig. 7); linking Typical mesophilic microorganisms do not grow at near 0 C glycolysis to TCA cycle or fatty acid synthesis, phosphoenol- temperatures, those transported by atmospheric circulation pyruvate carboxylase and pyruvate dehydrogenase E1 (a and to Antarctica need to develop the cold-growth capability. To b subunits) (fig. 7); for degradation of polysaccharides or survive the deeper freezing conditions than encountered be- oligosaccharides (including those covalently linked to pro- fore, these species also need significantly higher levels of ice- teins and lipids), a-mannosidase and seven other enzymes binding proteins or LEA proteins. Chlorella vulgaris NJ-7 is (fig. 7); for reutilization of monosaccharides in polysaccharide such an example. Based on comparative omics data, we sys- or oligosaccharide synthesis, UDP-N-acetylglucosamine pyro- tematically analyzed the genetic divergence between NJ-7 and phosphorylase and three other enzymes (fig. 7). Differences in the temperate strain UTEX259 and deduced the underlying protein and mRNA levels of these genes between NJ-7 and mechanism for the early stage cold adaptation of C. vulgaris. UTEX259 are shown in supplementary figure S19, The cold-growth capability may be developed in two steps: Supplementary Material online. No enzymes shown in figure 7 elevation of protein abundance and cold adaptation of are downregulated in NJ-7. Taking genes for the enzymes in enzymes. Adaptation of NJ-7 to the Antarctic cold environ- figure 7 as a gene set, we also performed GSEA of the differ- ment is basically at the early stage—elevation of protein abun- ential expression between two strains at the protein level and dance. Positive selection of genes (leading to cold-adapted found significant positive enrichments in NJ-7 at both tem- enzymes/proteins) should have contributed to the cold adap- peratures (supplementary fig. S16b, Supplementary Material tation of NJ-7 (supplementary excel S1, Supplementary online). Systematic increases in abundance of critical enzymes Material online) but is apparently not the dominant contrib- would significantly accelerate carbon metabolisms at low utor. Instead, most homologous proteins in NJ-7 and UTEX259 temperatures. However, the early stage cold adaptation is still are nearly identical to each other. Some of them, such as NR, underway in NJ-7. For example, RuBisco activase and the NiR, and many enzymes involved in other metabolic pathways, small subunit of RuBP carboxylase increased in abundance, showed higher abundance inNJ-7 thaninUTEX259 857 . Wang et al. doi:10.1093/molbev/msz273 MBE FIG.7. Differential expression of enzymes involved in carbohydrate metabolism in NJ-7 and UTEX259. Biochemical reactions shown in this figure include hydrolysis of glycosidic linkages, reutilization of monosaccharides and glycolysis; two reactions (phosphoenolpyruvate to oxaloacetate, pyruvate to acetyl-CoA) that link glycolysis to TCA cycle and fatty acid synthesis are also included. Most of the high-lighted enzymes showed higher abundance in NJ-7 than in UTEX259 at both 20 and 4 C(supplementary fig. S19, Supplementary Material online). MNS1, mannosyl-oligosac- charide 1,2-alpha-mannosidase; GlcNAc, N-acetyl-D-glucosamine; GlcNAc-6P, N-acetyl-D-glucosamine-6-phosphate; GlcNAc-1P, N-acetyl-D-glu- cosamine-1-phosphate. irrespective of temperature (supplementary fig. S19 and excel translation, or posttranslation (protein stability) levels rather S3, Supplementary Material online). The capability to grow at than increased gene copy numbers. near 0 C temperatures may depend on almost all aspects of In coping with seasonally changing temperatures, meso- cellular activities and involves a large number of enzymes/ philic microorganisms developed the capability to relieve the proteins. Relative to positive selection of enzymes for higher cold stress through cold acclimation (Morgan-Kiss et al. specific activities, increases in cellular concentrations would be 2006); based on “anticipation and associative learning” a quick adaptation pathway for promoting biochemical reac- (Bleuven and Landry 2016), the cold acclimation also prepares tions at low temperatures. Presumptively, the increase in cel- for the freezing condition that may follow. In cold acclimation lular concentration is only necessary for enzymes whose of C. vulgaris, sphingolipid delta(4)-desaturase, RNA helicases, activities are not sufficient to support cell proliferation and and other enzymes are upregulated for resumption of cellular preferentially occurs at rate-limiting steps in metabolic path- activities at 4 C (predominantly for relief of cold stress), LEA ways (such as fructose-2,6-bisphosphatase/phosphofructoki- proteins are upregulated to provide cryoprotection of cells at nase in glycolysis shown in fig. 7 and sedoheptulose-1,7- the “anticipated” subzero temperatures. Presumptively, the bisphosphatase in Calvin cycle shown in supplementary fig. expression of some of these proteins/enzymes could have S18, Supplementary Material online). been further enhanced in NJ-7 after its arrival in Antarctica. There are different mechanisms for increasing the expres- A small proportion of cold-inducible proteins in NJ-7 indeed sion of enzymes/proteins. Gene duplication is one of the rapid showed higher abundance than their orthologs in UTEX259 mechanisms (Kondrashov 2012). In an Antarctic fish, aug- (fig. 3 and supplementary table S17, Supplementary Material mented gene expression for cold adaptation is largely associ- online). However, most enzymes/proteins with higher abun- ated with gene duplication and family expansion (Chen et al. dance in NJ-7, such as those in figures 6 and 7, are not induc- 2008). In microbes, experimentally evolved phenotypes ible in response to cold stress, because increases in their (Wenger et al 2011) or development of drug resistance expression are not essential for cold stress relief but are re- (Sandegren and Andersson 2009) may also be associated quired for cell proliferation at near 0 C temperatures. The with gene amplifications. In NJ-7, however, the elevated abun- enhanced expression of “chromatin” proteins in NJ-7 (sup- dance of enzymes is apparently dependent on the upregula- plementary fig. S14e, Supplementary Material online) may tion at transcription, posttranscription (RNA stability), also contribute to the cold-growth capability. 858 . Early Stage Adaptation of a Mesophilic Green Alga to Antarctica doi:10.1093/molbev/msz273 MBE Relatively speaking, Antarctica is a permanently cold envi- starvation (48 h). For differential expression analyses, total ronment. In coastal ice-free regions, the ground surface tem- RNA and proteins were extracted from algal cells cultured at perature fluctuates at large amplitudes in December and 20 C with or without exposure to 4 C, harvested and frozen January (Guglielmin 2006). These sites could allow new- in liquid nitrogen. Considering the different rates for accumu- comer mesophilic species to grow shortly every year. Some lation of mRNA and proteins of cold-induced genes (Liu et al. species may evolve to acquire the capability to grow at near 0 2011), cells exposed to 4 C were collected at 6 h for RNA C temperatures in a way like NJ-7. Because of the energy extraction and at 24 h for protein extraction. burden for keeping higher abundance of enzymes, selective All physiological and differential expression (transcrip- pressure in permanently cold environments would continue tomic, proteomic, RT-qPCR) data were calculated from to drive the cold adaptation in enzymes in subsequent evo- results of three biological replicates. lutionary processes. Genome Assembly and Annotation High-molecular-weight genomic DNA was extracted using Materials and Methods the cetyltrimethyl ammonium bromide method (Murray A short form of “Materials and methods” is presented as and Thompson 1980) with modifications. For 454 sequencing, follows. More detailed descriptions are provided in supple- shotgun and paired-end libraries were constructed. For mentary text S1, Supplementary Material online. Illumina sequencing, two short-insert paired-end genomic DNA libraries and a long-insert mate-pair library were con- Algal Strains, Culture Conditions, and Physiological structed. Total RNA was extracted using Trizol reagent Analyses (Invitrogen) from cells cultured/treated under a variety of Chlorella vulgaris NJ-7 was isolated from rock samples col- conditions to increase the number of expressed genes, and lected (January 1999) near a transitory pond 5 km away from the pooled total RNA was used to construct the cDNA library 0 0 the Zhongshan Station (69 22 S–76 22 E) in Antarctica (Hu for 454 sequencing. et al. 2008) and deposited at the Freshwater Algal Culture The NJ-7 genome assembly was generated from 454 shot- Collection of the Institute of Hydrobiology (FACHB2411). gun, 454 paired-end and Illumina GAIIx paired-end reads, Chlorella vulgaris UTEX259 was purchased from the Culture using Newbler (GS De Novo Assembler, Roche) and Velvet Collection of Algae at The University of Texas at Austin (Zerbino and Birney 2008). The UTEX259 genome assembly (https://utex.org; last accessed November 24, 2019). Algal was generated from 454 shotgun, 454 paired-end, Illumina strains were purified by repeated streaking of single colonies GAIIx paired-end, Illumina MiSeq paired-end and mate pair on agar plates, and the axenicity was confirmed by micro- reads, using Newbler, Velvet, ALLPATHS-LG assembler scopic examination and culture on solid Luria-Bertani me- (Gnerre et al. 2011)and the scaffolderSSPACE (Boetzer dium and BG11 (Stanier et al. 1971)supplemented with et al. 2011). glucose. De novo transcriptome assembly was performed using Chlorella strains were cultivated in BG11 under the light of Newbler with parameters -cdna -urt -tr, generating isotigs 2 1 30 mEm s at 20 C with aeration. Algal cells grown at that represent transcripts. Genome assemblies were further 20 C were rapidly cooled to 4 C and exposed to the same improved by L_RNA_scaffolder (Xue et al. 2013), which uses temperature with aeration in a refrigerator with illumination the transcripts to order and join genome sequences into 2 1 of 30 mEm s . For comparison of their growth at 20 and larger scaffolds, and GapCloser tool in SOAPdenovo (Li 4 C, cell density was monitored turbidimetrically at 730 nm. et al. 2010), which makes use of the information of paired- For evaluation of antifreeze capability, algal cells grown at end Illumina reads to fill gaps within scaffolds. Short scaffolds 20 C were exposed to 4 C for 48 h or not. Cells collected (<500 bp) and organelle sequences were excluded from the by centrifugation were then rapidly cooled to 20 Cand assemblies of nuclear genomes. frozen at 20 C for 8 days. The frozen cells were diluted in The quality of NJ-7 and UTEX259 genome assemblies was liquid BG11 and allowed to grow on BG11 plates at 20 Cfor assessed in multiple ways, including BUSCO (Benchmarking 15 days. Colony-forming unit per OD milliliter was then Universal Single-Copy Orthologs) completeness assessment calculated. For measurements of photosynthetic activities, (Simao et al. 2015), mapping of fosmid-end sequences, and algal cells cultured at 20 C were used. Rates of photosyn- assembled transcripts onto the genome sequences. thetic oxygenic evolution were measured on a Clark-type Scaffolds and contigs corresponding to organelle genomes oxygen electrode (Oxylab2, Hansatech, United Kingdom) were extracted from the genome assemblies using BlastN 2 1 with a saturating light of 2,000 mEm s at different searches against the C. vulgaris C-27 chloroplast genome temperatures. (GenBank accession number AB001684) or Prototheca wick- For extraction of genomic DNA, cells cultured at 20 Cwere erhamii mitochondrial genome (GenBank accession number used. For transcriptome analysis, RNA was extracted from a NC_001613). The gaps were filled by sequencing of PCR frag- variety of culture conditions to increase the number of ments, producing circular organelle genomes. expressed genes. These conditions included different growth Prediction of protein-coding genes was performed using temperatures (20 and 28 C), exposure to cold (4 Cfor 24 h), both ab initio gene predictions and transcript-based salt (0.3 M NaCl for 24 h), or oxidation (0.2 mM H O for 5 h) approaches, followed by integrating them using 2 2 stress, exposure to N, P, C, Fe, Ca, Mg, K, or trace element EVidenceModeler (EVM) (Haas et al. 2008). Protein motifs 859 . Wang et al. doi:10.1093/molbev/msz273 MBE and domains of predicted gene models were annotated using gymnosperms (290–320 Ma) (Goremykin et al. 1997; InterProScan search (Zdobnov and Apweiler 2001)against Doyle 1998), the divergence between Nymphaeales and InterPro databases (Hunter et al. 2009). Gene functions eudicots (115 Ma minimum) (Friis et al. 2001), and the were assigned based on BLASTP searches (E-value 1e-5). divergence between monocots and dicots (90–130 Ma) tRNA and rRNA genes were identified using tRNAscan-SE (Crane et al. 1995). The maximum clade credibility tree v1.3 (Lowe and Eddy 1997) and RNAmmer 1.2 (http:// was generated using TreeAnnotator v1.7.5 (Drummond www.cbs.dtu.dk/services/RNAmmer/; last accessed et al. 2012) and visualized in FigTree v1.4.0 (http://tree.bio. November 24, 2019). Organelle genomes were annotated us- ed.ac.uk/software/figtree/; last accessed November 24, ing DOGMA (http://dogma.ccbb.utexas.edu/; last accessed 2019). The estimated divergence time between C. varia- November 24, 2019), ORF-Finder (https://www.ncbi.nlm.nih. bilis and C. vulgaris was further used to calibrate the fine- gov/orffinder/; last accessed November 24, 2019), and scale analysis. BLASTX searches based on sequence similarity to genes in iii. Evolutionary rate estimates. The number of nonsynony- other annotated organelle genomes. mous substitutions per nonsynonymous site (d )and the number of synonymous substitutions per synonymous Synteny and Comparative Analyses site (d ) were estimated based on the 1,080 nuclear genes, ABLASTP alignment (E-value 1e-5) was performed be- using codeml program in the PAML package v4.6 (Yang tween all protein sequences of the two strains. OrthoMCL 2007). (Li et al. 2003) was applied to identify orthologous protein Detection of Genes with Positive Selection pairs based on the BLASTP results, with the criteria E-value The codeml program in the PAML package was applied to all 1e-5, identity 80%, andlengthcoverage 70%. the 5,899 core genes shared by NJ-7, UTEX259, and NC64A. Synteny between genomes was identified by aligning ge- Codeml analysis with branch-site model was conducted on nomic regions using orthologous gene pairs as anchors. Using either NJ-7 or UTEX259 branch. The null hypothesis (assum- MCScanX (Wang et al. 2012), pairwise syntenic segments ing x¼ 1or x< 1) and the alternative hypothesis (assuming were identified, grouped into blocks, and displayed by a dot x> 1) were compared to identify significantly higher likeli- plot graph. A dual synteny plot was generated with the lon- hood values for the alternative hypothesis. Those genes with gest 12 scaffolds of NJ-7 and the corresponding scaffolds of significance (v P-value < 0.01) were deemed to be under UTEX259. Nine chlorophyte chloroplast genomes were com- positive selection. pared using the Progressive Mauve algorithm implemented in Mauve 2.4.0 (Darling et al. 2010). Analyses of Alternative Splicing Events, Repetitive Phylogenetic and Evolutionary Analyses Elements, Gene Duplication, and Gene Families i. Phylogenetic analysis. Phylogenetic trees were constructed Alternative splicing events were detected by comparing the based on 1,080 single-copy genes shared by NJ-7, predictedgenes on genome andthe assembledisotigs and UTEX259, and other nine chlorophytes, with the land processing the clusters of aligned isotigs with at least one plant Arabidopsis thaliana as the outgroup. Using the splice site. MUSCLE program (Edgar 2004), 1,080 genes from these Repetitive elements were identified with both homology- species/strains were aligned. Poorly aligned regions were based methods and de novo repeat finding programs. The removed from multiple-alignments using Gblocks imple- library of identified repeats was used to estimate repeat con- mented in TranslatorX (Abascal et al. 2010). The cleaned tents of each genome. multiple alignments were concatenated and subjected to Duplicated genes were identified by performing self- ML (maximum likelihood) tree reconstruction using versus-self BLASTP on protein sequences. The copy number PhyML (Guindon et al. 2010). of recent duplicated genes was deduced based on highly sim- ii. Divergence time estimates. Divergence times between lin- ilar genes that were detected by performing self-versus-self eages were estimated using the Bayesian MCMC ap- BlastN, or roughly estimated by aligning trimmed Illumina proach as implemented in the program BEAST v1.7.5 reads onto gene sequences to calculate the relative depth (Drummond et al. 2012) based on 36 chloroplast genes of coverage. (atpA, atpE, atpF, atpH, atpI, petA, petB, petG, psaA, psaB, Homologous protein families were constructed based on psaC, psaJ, psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, genome sequences of 11 species/strains of green algae, and all psbJ, psbL, psbN, rbcL, rpl2, rpl14, rpl16, rpl20, rpl23, rps7, predicted protein sequences were compared using all- rps8, rps11, rps12, rps14, rps18,and rps19). Two sets of against-all BLASTP. Protein families were annotated according analyses were performed: broad-scale analyses with 12–19 to Pfam domains or InterPro descriptions. The significance of species/strains of green algae (in four combinations) and family expansion or reduction was analyzed by chi-square test four species of higher plants; a fine-scale analysis with nine in R. species/strains of Chlorella. BEAST used concatenated nu- cleotide alignments as input and estimated phylogeny Identification of LEA Protein Genes and divergence times simultaneously. To calibrate nodes In addition to Ccor1/Ccor2 identified by experimental analyses in the broad-scale tree, three reliable fossil dates were (Liu et al. 2011) and genes similar to Ccor1/Ccor2,LEA protein used: the divergence between angiosperms and genes were identified by searching for LEA motifs and 860 . Early Stage Adaptation of a Mesophilic Green Alga to Antarctica doi:10.1093/molbev/msz273 MBE secondary structures (Tunnacliffe and Wise 2007; Battaglia et multiple copies were treated as one in the quantitative anal- al. 2008) using the fuzzpro program in EMBOSS package (Rice yses. Differential expression with ratio 1.3 (P-value < 0.05) et al. 2000) and the PSIPRED Server (http://bioinf.cs.ucl.ac.uk/ was defined as upregulation, that with ratio 0.77 (P-value< psipred; last accessed November 24, 2019), searching for Pfam 0.05) as downregulation. domains using hmmsearch (Eddy 2011)against LEA HMM profiles, searching for homologs using BLASTP (E-value 1e- GO Enrichment Analysis 5) against LEA proteins in the Late Embryogenesis Abundant GSEA software version 3.0 (Subramanian et al. 2005) was used Proteins DataBase (http://forge.info.univ-angers.fr/gh/ to identify significantly enriched GO gene sets. GSEA was run Leadb/index.php; last accessed November 24, 2019). with 1,000 permutations, and functional gene sets with FDR- Subcellular locations were predicted using TargetP (http:// adjusted q-value <0.05 were considered significant. www.cbs.dtu.dk/services/TargetP; last accessed November 24, 2019), MitoProt (https://ihg.gsf.de/ihg/mitoprot. html; Analyses of Regulation Networks last accessed November 24, 2019), and PSORT (http:// TFs were predicted by searching for Pfam domains using psort1.hgc.jp/form.html; last accessed November 24, 2019). hmmsearch against the HMM profiles for TF families at PlantTFDB (Jin et al. 2014). DNA-binding motifs for ortholo- Analyses of Differential Expression at mRNA Level gous TFs in C. variabilis NC64A were used to scan for recog- Differential expression at mRNA level was analyzed based on nition sites upstream of protein-coding genes in NJ-7 and RNA-seq. Processed sequencing reads were aligned to exonic UTEX259 by FIMO (Grantetal. 2011). The potential role of regions of predicted genes using Bowtie (Langmead et al. a TF in gene regulation was defined based on the correlation 2009)and Tophat (Trapnell et al. 2009) allowing one mis- between its own expression and the expression of genes with match. Raw count data and RPKM (reads per kilobase of exon its recognition site. per million mapped reads) values were generated from the alignment files using bam2rpkm (http://bam2rpkm.source- NR Assays and Western Blot Analysis forge.net/; last accessed November 24, 2019). Raw count NR activity in crude extracts was assayed as described by di data were then used as input into DESeq (Anders and Rigano et al. (2006) using NADH as electron donor, with a few Huber 2010) for differential expression analyses (three biolog- modifications. Reactions (three technical repeats) were car- ical replicates). Differentially expressed genes were identified ried out for 30 min at temperatures from 5 to 45 C. with criteria: fold-change 2 (either up- or downregulated), Western blot analysis of total soluble proteins was per- FDR (false discovery rate) adjusted P-value< 0.05, and RPKM formed using the rabbit antiserum against the recombinant 10 underatleastone condition. NR. Real-Time Quantitative Polymerase Chain Reactions Data Availability DNA-free RNA was used to synthesize the first strand of Annotations of NJ-7 and UTEX259 genomes are available in cDNA using PrimeScript RT reagent Kit (Takara) and oligo the Figshare repository (DOI: 10.6084/m9.figshare.c.4678916; (dT)15 primer (Promega). SYBR Green I (Takara) was added https://figshare.com/s/66909bf96c4a06159c94). to the PCR reaction mixture according to manufacturers’ protocol, and RT-qPCR was performed on Applied Supplementary Material Biosystems ABI 7500. b-Actin was used as the internal stan- Supplementary data are available at Molecular Biology and dard. Primers were designed based on identical sequences of Evolution online. orthologous genes (supplementary table S23, Supplementary Material online). Acknowledgments The authors express great gratitude to Guoxiang Liu for his Analyses of Differential Expression at Protein Level advice on the taxonomic analysis of strains of Chlorella vul- Differential expression at the protein level was analyzed using garis based on the ribosomal RNA region. This research was the quantitative proteomics approach. Algal cells were soni- supported by the Knowledge Innovation Project of Hubei cated and centrifuged. Proteins in the supernatant were pre- Province (2017CFA021), the STS Project of Chinese cipitated and subjected to trypsin digestion. After labelling Academy of Sciences (KFJ-SW-STS-163), the national special with Tandem Mass Tags/Isobaric Tag for Relative Absolute support program “Wan-Ren-Ji-Hua” of China and the State Quantitation (TMT Kit/iTRAQ), peptides were analyzed by Key Laboratory of Freshwater Ecology and Biotechnology at liquid chromatography coupled with tandem mass spectrom- IHB, CAS (2019FBZ06). etry (MS/MS). The resulting MS/MS data were processed us- ing Maxquant search engine (v.1.5.2.8) (Cox and Mann 2008). Author Contributions Peptide and protein ratios were obtained by direct compar- ison of signals of “light” and “heavy” isotope in the same liquid X.X., J.X., and A.-Y.G. designed the project. Y.W. performed chromatography run. Cross-strain comparison of expression bioinformatic analyses. H.-M.Z. contributed to genome as- of orthologous proteins was performed using identical pep- sembly/annotation, multicopy gene analyses, and identifica- tides instead of whole protein sequences. In cases where mul- tion of LEA protein genes. X.L. performed experimental tiple copies of proteins could not be distinguished, the analyses of the expression and activity of nitrate reductase; 861 . Wang et al. doi:10.1093/molbev/msz273 MBE Drummond AJ, Suchard MA, Xie D, Rambaut A. 2012. Bayesian phylo- Y.W. and H.G. analyzed the physiological characteristics of genetics with BEAUti and the BEAST 1.7. Mol Biol Evol. algal strains; Y.W. prepared DNA and RNA samples, per- 29(8):1969–1973. formed PCR and RT-qPCR analyses. X.X., Y.W., X.L., H.G., A.- Dure L, Greenway SC, Galau GA. 1981. Developmental biochemistry of Y.G., and J.X. interpreted the results. X.X., J.X., and A.-Y.G. cotton seed embryogenesis and germination: changing messenger edited the manuscript. 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Molecular Biology and Evolution – Pubmed Central
Published: Nov 20, 2019
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