Analysing the changes of amino acid usage frequencies during viridiplantae evolution.doc

Analysing the changes of amino acid usage frequencies during viridiplantae evolutionBian Chunxiang1, 2Email: Ruan Qiping1*Corresponding authorZhao Hong2Liao Min21Key laboratory for Molecular Biology and Biopharmaceuticals, Mianyang Normal University, Sichuan, 621000, China2 College of Life Sciences and Biotechnology, Mianyang Normal University, Sichuan, 621000, ChinaAbstractViridiplantae are known to originate（来源） from one phylum（门） of green algae（海藻）, streptophyte algae, and form a single evolutionary lineage. The evolution of viridiplantae mainly follows the course from the earliest green algae to bryophyte（苔藓）, pteridophytes and spermatophytes. As the result analysis of their proteomes（蛋白质组） has the potential of revealing（透露真情的，启发人的） how amino acid usage frequencies within proteins have evolved over biological time. In the paper, 655 proteins (including 452,672aa) of six proteomes from green algae (Chlamydomonas reinhardtii; Ostreococcus lucimarinus), bryophyte (Physcomitrella patens) and spermatophytes (Cryptomeria japonica; Nicotiana tabacum; Arabidopsis thaliana) were analysed. The results showed that most amino acid usage frequencies decreasing (especially AGVE) or increasing (especially YFNIKS) follow fixed patterns from green algae to bryophyte and spermatophytes. By exploring discrepancies of amino acid usage frequencies among taxonomic（分类的） groups, it was found that the frequencies performed a bigger change when plant evolved form algae to bryophyte than those from bryophyte to spermatophytes. Meanwhile data also showed that amino acid residues（剩余物，残渣） have the similar usage frequencies in species with close evolutionary distance. In addition, 239 proteins (including 130,138aa) from red algae proteomes (Guillardia theta and Hemiselmis andersenii) were also analysed. Comparing with green algae, these amino acid frequencies have distinctly（清楚地，无疑的） differences. The frequencies of AGVR in red algae are extremely lower than those in viridiplantae we have discussed. The studies hint（暗示，提示） red algae can not follow the pattern of green algae to decrease these amino acids, which have proved indirectly（间接地） the fact that embryophyte （有胚植物）originated from green algae rather than red algae. KeywordsAmino acids, usage frequency, viridiplantae, evolution, red algaeIntroductionIncluding all green algae and embryophyte plants, viridiplantae (green plants in English) represent a monophyletic（ 单一门的;单源的）group of organisms（生物，有机体）, which display a surprising diversity with respect to their morphology（形态学，形态论）, cell architecture, life histories and reproduction（繁殖）, and biochemistry (Becker and Marin, 2009). It was suggested by sequence comparisons all living green plants belong to one of the two major phyla（类群） of green algae (Melkonian et al., 1995; Friedl, 1997): Streptophyta（链形植物） (Bremer, 1985) and Chlorophyta（绿藻门） (Sluiman, 1985). About 7251200 MY ago, the Viridiplantae split（分裂） early into two evolutionary lineages: Chlorophyta and Streptophyta, according to different estimates（判断） by molecular（分子的） clock methods (Hedges et al., 2004; Yoon et al., 2004; Zimmer et al., 2007). Then the descendents（祖传的，派生的） of streptophyte algae (ancestors of land plants) began to colonized（殖民于, 移植）terrestrial(陆地的) habitats approximate(接近) 470450 MY ago (Ordovician奥陶纪的 period) (Sanderson et al., 2004; Becker and Marin, 2009), which was undoubtedly one of the most important steps in the evolution of life on earth (Graham, 1993; Kenrick and Crane, 1997; Bateman et al., 1998). It was said their primary freshwater (淡水的)adaptation apparently（显然的） played a key role in the colonization of dry land habitats (Becker and Marin, 2009). The appearance of various groups of land plants (bryophytes, pteridophytes and spermatophytes) resulted in our current terrestrial（陆地的）ecosystems（生态系统） (Waters, 2003). The evolution of viridiplantae follows the tendency（倾向，趋向） of increasing levels of complexity, from the earliest green algae to the complex gymnosperms（裸子植物）and angiosperms （被子植物）of today. In this paper we focused on analysing the amino acid composition within proteins from green plant proteomes and seeking to determine any of the 20 amino acids occurred more or less frequently during the evolution. There are many examples to examine the relationship between amino acid occurrence and the evolution of life. To explore the amino acids under primitive earth conditions, Miller (1953) made an apparatus（装置） to duplicate （复制）the primitive atmosphere of earth, and Gly and Ala were firstly identified. By analysing the hydrolyzed aqueous extract from Murchison meteorite, Gly, Ala, Val, Pro and Glu were found (Kvenvolden et al., 1970). So it was deduced these amino acids believed to have been most abundant（大量的） in the prebiotic（生命起源以前的） environment were used more frequently within proteins of the last universal ancestor of all life (LUA) than within those of modern species (Miller, 1987; Kvenvolden et al., 1970; Dayhoff et al., 1978; Brooks et al., 2002). By comparing of the LUA and modern species, the frequencies of the 20 amino acids were analysed, and the data showed that Ala, Val, Gly, Ile, Thr, Asp, Ser, Asn and His have decreased in frequency within modern species, while Trp, Gln, Leu, Lys and Glu have increased (Brooks et al., 2002). It was also suggested that at the time of the LUA, some amino acids had yet to reach equilibrium（平衡; 均衡）frequencies, so change of amino acid composition toward that predicted by neutral（中立的，中性的） evolution may be a process requiring very long time periods (Brooks and Fresco, 2002). We deduced that the equilibrium of some amino acid frequencies may also not be reached in plant, and during evolution amino acid usage frequencies within viridiplantae proteomes decreasing or increasing should also be regular and follow certain patterns. It was further inferred that 20 amino acids in two species which have a close evolutionary relationship should have similar usage frequencies. In order to test our hypotheses（假设） we examined the amino acid composition within 894 proteins including 58,2810 amino acid residues which were randomly（随便的） selected from proteomes of red algae, green algae, bryophytes and spermatophytes. All the proteins used in the paper can be identified in the appendix（附录）. Although some data of proteins in many species are rare（稀有的，罕见的） or unavailable by far, the analysis will present new understanding of the evolution of the viridiplantae, even other species.Materials and Methods Protein samplingAmino acid sequences of sampled proteins were obtained from the NCBI Protein database (/protein/) at random. All sampled proteins and their accession numbers can be identified in the appendix. Amino acid compositional analyses were carried out by ProtParam tool (/protparam/) ( Gasteiger et al., 2005).Data analysingWe compared taxonomic（分类的） group means (average（使平衡） amino acid compositional frequency per taxonomic grouping) and visualized (想象)them with bar charts（条形图） (Fig. 1). The significance of amino acid frequency difference among taxonomic groups was assessed by one-way ANOVA in SPSS statistics software (Fig. 1), and the significance between two taxonomic groups was analysed by the MannWhitney test (Table 1). In order to analyse the discrepancies（差异） of amino acid usage frequencies between algaebryophytes and bryophytesspermatophytes, two formulas, |Fi(algae)-Fi(bryophytes)| and |Fi(bryophytes)- Fi(spermatophytes)| (Fi indicating usage frequency of each of 20 amino acids), were carried out respectively（分别的，各自的） to sum up the absolute（完全的，绝对的） differences of amino acid frequencies and assess the difference between the two groups (Fig. 2). 翻译不好To detect the relationship between green and red algae in the level of amino acid usage frequencies, Line charts were drawn to visualize（想象，形象化） the amino acid fluctuations（波动，变动） for three parts, two green algae (Chlamydomonas reinhardtii and Ostreococcus lucimarinus, Fig. 3A), two red algae (Guillardia theta and Hemiselmis andersenii, Fig. 3B), and the mean frequencies of the two green algae and the two red algae (Fig. 3C). Two groups of each part were compared and the strength of their relationship was assessed by Bivariate Correlations（双变量相关/双变量相关分析） in SPSS statistics （统计）software. Results and DiscussionThe change of amino acid usage frequencies during the evolution of the viridiplantaeWe have analysed the amino acid composition within 655 proteins (including 452,672 amino acid residues) from green algae (Chlamydomonas reinhardtii, 59720aa, 77proteins; Ostreococcus lucimarinus, 94815aa, 122 proteins), bryophyte (Physcomitrella patens, 100725aa, 135 proteins) and spermatophytes (Cryptomeria japonica, 33798aa, 74 proteins; Nicotiana tabacum, 64131aa, 132 proteins; Arabidopsis thaliana, 99483aa, 115 proteins). As is shown in figure 1A, it was found that there are five amino acids (AGVRP) decreasing in frequency from green algae to bryophyte and spermatophytes, and AGVP are presumed（推测） to have been most abundant in the prebiotic（无生命的） environment (Miller 1953, 1987; Kvenvolden et al., 1970). Further analyse indicated the decreasing of AGVR is highly or extremely significant (G: P0.01; AVR: P0.001). That is, the usage frequencies of AGVRP, especially AGVR, are lower in the three spermatophytes than those in green algae. In contrast, nine amino acids (WCHYFNIKS) have increased in frequency during the course of viridiplantae evolution. The increasing of YFNIK shows extremely significant (P0.001) and S is highly significant (P0.01) (Fig. 1B). Most of the nine amino acids are presumed either to have been nonexistent（不存在的） or of very low abundance in the prebiotic environment. These amino acids which are thus inferred to have been late additions to the code, include several of the most biosynthetically（生物合成的） complex amino acids (for example, all three aromatic（芳香的，芳香族的） amino acids, which share a common, complex metabolic（新陈代谢的） intermediate（中间物）, are inferred to have been late additions) (Brooks et al, 2002). In addition, there are also six amino acids (LETDQM, Fig. 1C) which have no regular change in frequency among green algae, bryophyte and spermatophytes. Especially, glutamate is believed to have been available in the prebiotic environment and thought to perform the decreasing pattern, but its frequency increased significantly (6.087.01) from green algae to bryophyte and then decreased slightly (7.016.77) in spermatophytes (Fig. 1C). It may be explained that increasing charged amino acids, such as glutamate, which can maintain protein hydration（水合作用） may play an important role in colonizing terrestrial habitats for green algae (Jobson and Qiu, 2011).From the data we analysed, it was concluded that the usage frequencies of most amino acid residues increasing or decreasing follow fixed patterns during the evolution from green algae to bryophyte and spermatophytes. By comparing our results with those analysed by Brooks et al. (2002), we found the patterns of most amino acid frequency shifts（转变）are consistent. However, there are also some discrepancies. For example, our data showed that HNS are increasing, but they are decreasing in the past analysis. In addition, there are only five amino acids (AGVRP) decreasing in frequency comparing with nine ones (AVGITDSNH) analysed before decreasing in frequency. Two reasons are present to explain the results. Firstly, in the past study Brooks et al. compared the LUA with eight modern species which including two archaea（古生菌）, one eukaryote and five eubacteria. What is more, the habitats of these species vary dramatically（戏剧性的，引人注目的）, which may have a great affect on the amino acid usage. Comparing optimal（最佳的，最理想的） growth temperatures across mesophilic（嗜温细菌） versus thermophilic（适温的; 喜温的）prokaryotes（原核生物）, it was found that thermostabilizing（耐热的） polar charged amino acids, particularly the hydrophilic EKR category, are more abundant in the latter group (Haney et al., 1999; Kumar and Nussinov, 2001; McDonald, 2001; Nishio et al., 2003; Singer and Hickey, 2003; Brocchieri, 2004). While, the terrestrial environment where the discussed land plants exist is relatively similar and stable. Secondly, in the evolution of viridiplantae, some amino acids may have reached or are coming near equilibrium. For example, the mean frequencies of Met and Lys have no big change or even keep the same from bryophyte to spermatophytes.Amino acid usage frequencies reveal the endeavor made by green plants to colonize the landMore than 400 million years ago green plants strove（奋斗，努力） to colonize land from fresh water, and their morphology, cell architecture, life histories, and genetic information experienced enormous changes to cope with various unfriendly conditions. But the plants may make far more efforts than we thought. From figure 1, we have already noticed that the mean frequencies of most amino acids have relatively bigger changes between algae and bryophyte than those between bryophyte and spermatophytes, especially Ala. To analyse this point, we carried out two formulas, |Fi(algae) Fi(bryophyte)| and |Fi(bryophyte)Fi(spermatophytes)| (Fi indicating usage frequency of each of 20 amino acids), to sum up absolute value of frequency differences of 20 amino acids. As is shown in figure 2, whether amino acid residues are inferred decrease (AGVRP), or increase (WCHYFNIKS), or have no regular change (LETDQM) in frequency, the values of them for algaebryophyte comparison are all higher than those for bryophytespermatophytes comparison. That is, when viridiplantae evolved from green algae to bryophytes, amino acid composition in proteomes have a relative bigger fluctuation. In order to further detect the fluctuation of each amino acid residues, the change of 20 amino acid residue frequency between taxonomic groups was analysed by the MannWhitney test in SPSS software (Table 1). It was found that for algaebryophyte comparison ten amino acids, AGNKETQIRW, show significant change (P 0.05), especially ANKI which are extremely significant (P0.001). While for bryophytespermatophytes comparison, only six amino acids, ANVRIF, show significant change (P 0.05 and P 0.001). Based on the analyses, we can learn that the process of green plants colonizing land was a hard work and their amino acid composition was forced experienced a great shift. It is apparent that this change was corresponding（符合，协调） to the sharp shift in the ecological environment (freshwater versus terrestrial habitats), and it was deduced that the habitats varied more wildly, the amino acid frequencies should fluctuate more significantly. So it inspire us to think that green plants adjusted their amino acid composition, increasing or decreasing some amino acid frequencies, which may be a premeditated tactic to adapt to terrestrial habitats. However, the detail of the tactic and how it was put into effort are beyond our knowledge, which may need more discussion.Amino acids usage frequencies in red algaeChloroplast（叶绿粒） structure and genome analyses support the hypothesis（假设） that three groups of organisms originated from the primary photosynthetic endosymbiosis（骨内膜; 骨髓膜） between a cyanobacterium （蓝细菌，蓝藻）and a eukaryotic （真核的）host（长寄生虫的活物）: green plants (green algae + land plants), red algae and glaucophytes (for example, Cyanophora) (Douglas, 1998). Although phylogenies（种系发生学） based on several mitochondrial（线粒体） genes support a specific green plants/red algae relationship, the phylogenetic（系统发生的） analysis of nucleus-encoded（原子核，改为密码） genes yields（同意，生产） inconclusive（非决定性的, 不确定的） sometimes contradictory（反对的） results (Ragan and Gutell, 1995; Stiller and Hall, 1997; Burger et al., 1999), which was also supported by the analysis of amino acid usage frequencies.看不懂In the paper, the relationship between amino acid usage frequencies and evolutionary distance for different species was also illuminated（阐明）. As is shown in figure 3, amino acid usage frequencies keep the accordant（一致的） fluctuation in the same taxonomic groups, ChlamydomonasOstreococcus (belonging to green algae) comparison (Fig. 3A) or GuillardiaHemiselmis (belonging to green algae) comparison (Fig. 3B). The species in each comparison are thought to have close evolutionary relationships. The numbers of strength relationship of both comparisons which was calculated（可能的） by Bivariate Correlations in SPSS statistics software are above 0.900 (The stronger the relationship, the closer the value is to 1.0), and the P values are lower than 0.001 which indicate extremely significance. In addition, analyzing the amino acid usage frequencies among the three discussed spermatophytes in the same way yielded the similar results (data not shown). However, the fluctuations show chaos（混乱） when we compared red algae with green algae (Fig. 3C). The number of strength relationship is only 0.210, and the P value is above 0.05, which indicates no significance. Further study showed that in red algae the mean usage frequencies of AGVRP most of which are believed to have been most abundant in the prebiotic environment (Miller, 1987; Kvenvolden et al., 1970) are extremely lower than those in spermatophytes. On the contrary, the frequencies of NIKF in red algae which follow the increasing pattern in green plant are extremely higher than those in spermatophytes. For example, the mean usage frequency of Ala in red algae is 3.00%, 12.57% in green algae, 7.45% in bryophytes and 6.56% in spermatophytes. And the mean usage frequency of Lys in red algae is 11.52%, 3.99% in green algae, 6.24% in bryophytes and 6.24% in spermatophytes. It is apparent amino acid usage frequencies between red algae and green algae have vast divergences, and red algae can not follow the pattern of gr