Developmental Gene Expression in Amphioxus: New Insights into the Evolutionary Origin of Vertebrate Brain Regions, Neural Crest, and Rostrocaudal Segmentation

SYNOPSIS. Amphioxus is widely held to be the closest invertebraterelative of the vertebrates and the best available stand-infor the proximate ancestor of the vertebrates. The spatiotemporalexpression patterns of developmental genes can help suggestbody part homologies between vertebrates and amphioxus. Thisapproach is illustrated using five homeobox genes (AmphiHoxl,AmphiHox2, AmphiOtx, AmphiDll, and AmphiEri) to provide insightsinto the evolutionary origins of three important vertebratefeatures: the major brain regions, the neural crest, and rostrocaudalsegmentation. During amphioxus development, the neural expressionpatterns of these genes are consistent with the presence ofa forebrain (detailed neuroanatomy indicates that the forebrainis all diencephalon without any telencephalon) and an extensivehindbrain; the possible presence of a midbrain requires additionalstudy. Further, during neurulation, the expression pattern ofAmphiDll as well as migratory cell behavior suggest that theepidermal cells bordering the neural plate may represent a phylogeneticprecursor of the vertebrate neural crest. Finally, when theparaxial mesoderm begins to segment, the earliest expressionof AmphiEn is detected in the posterior part of each nascentand newly formed somite. This pattern recalls the expressionof the segment-polarity gene engrailed during establishmentof the segments of metameric protostomes. Thus, during animalevolution, the role of engrailed in establishing and maintainingmetameric body plans may have arisen in a common segmented ancestorof both the protostomes and deuterostomes.     

Phylogenetic and Evolutionary Analyses of the Frizzled Gene Family in Common Carp (Cyprinus carpio) Provide Insights into Gene Expansion from Whole-Genome Duplications

In humans, the frizzled (FZD) gene family encodes 10 homologous proteins that commonly localize to the plasma membrane. Besides being associated with three main signaling pathways for cell development, most FZDs have different physiological effects and are major determinants in the development process of vertebrates and. Here, we identified and annotated the FZD genes in the whole-genome of common carp (Cyprinus carpio), a teleost fish, and determined their phylogenetic relationships to FZDs in other vertebrates. Our analyses revealed extensive gene duplications in the common carp that have led to the 26 FZD genes that we detected in the common carp genome. All 26 FZD genes were assigned orthology to the 10 FZD genes of on-land vertebrates, with none of genes being specific to the fish lineage. We postulated that the expansion of the FZD gene family in common carp was the result of an additional whole genome duplication event and that the FZD gene family in other teleosts has been lost in their evolution history with the reason that the functions of genes are redundant and conservation. Through the expression profiling of FZD genes in common carp, we speculate that the ancestral gene was likely capable of performing all functions and was expressed broadly, while some descendant duplicate genes only performed partial functions and were specifically expressed at certain stages of development.     

Evolutionary Analysis for Functional Divergence of the Toll-Like Receptor Gene Family and Altered Functional Constraints

The Toll-like receptor (TLR) gene family consists of type 1 transmembrane receptors, which play essential roles in both innate immunity and adaptive immune response by ligand recognition and signal transduction. Using all available vertebrate TLR protein sequences, we inferred the phylogenetic tree and then characterized critical amino acid residues for functional divergence by detecting altered functional constraints after gene duplications. We found that the extracellular domain of TLR genes showed higher functional divergence than that of the cytoplasmic domain, particularly in the region between leucine-rich repeat (LRR) 10 and LRR 15 of TLR 4. Our finding supports the concept that sequence evolution in the extracellular domain may be responsible for the broad diversity of TLR ligand-binding affinity, providing a testable hypothesis for potential targets that could be verified by further experimentation.     

Functional Divergence and Evolutionary Dynamics of the Putative AAAP Gene Family in Brassica rapa

The amino acid/auxin permease (AAAP) protein family is ubiquitously present in almost all eukaryotic species and functions in various aspects of growth and development. To investigate the evolution of AAAP proteins, here 83 AAAP genes in Brassica rapa were identified, and their sequence features, and evolutionary relationships were analyzed using in silico methods. According to the phylogenetic analysis, the AAAP genes of B. rapa are divided into six clades, and these clades share relatively similar sequence features, including gene structures, conserved motifs, and domain organizations. Synteny mapping strongly suggested that segmental duplications could be responsible for the expansion of this family. Adaptive evolution analysis demonstrated that most of AAAP proteins were subject to purifying selection. However, the site Tyr²⁵⁷ on eight AAAP proteins from clade 2b underwent significant positive selection. Functional divergent analysis showed that type I functional divergence coefficients (θ _I ) were significantly greater than zero in six pair-wise comparisons. However, functional divergence sites (Q _k ?>?0.95) found only in the AAAP I/II and AAAP I/III comparisons were localized mainly to the trans-membrane (TM) regions, suggesting highly divergent TM structures between these groups might be associated with group-specific functions. Our results could provide a valuable clue for further investigations of the evolutionary history and biological functions of the AAAP genes in B. rapa.     

芥蓝miR156a家族进化特性及表达分析

miRNA广泛参与植物的发育过程。芥蓝形态多样,叶片发育因品种而异。为了解miR156a家族的进化特性及其在芥蓝叶片发育中的表达模式。该研究对芥蓝miR156a成员及其前体pre miR156a成员进行生物信息学分析,比较不同品种芥蓝叶形的差异,并采用qRT PCR方法分析芥蓝不同组织部位中pre miR156a的表达水平、以及叶形相似的芥蓝品种‘翠宝’和‘改良香菇’中不同类型叶片的pre miR156a成员及其靶基因的表达水平。结果表明：(1)多序列比对和进化树分析发现芥蓝miR156a家族成员和pre miR156a 3p_1在进化过程中高度保守;二级结构预测发现pre miR156a每个成员均能形成茎环结构并包含2～3个miR156a成员序列;靶基因预测显示miR156a 5p和miR156a主要靶向SPL,而miR156a 3p_1则靶向CTPS等不同的基因。(2)‘翠宝’和‘改良香菇’芥蓝的叶形最为相似,qRT PCR分析显示,pre miR156a在‘翠宝’营养生长期的叶片中高度表达。(3)pre miR156a成员在‘翠宝’和‘改良香菇’不同类型叶片中差异表达,pre miR156a主要在成熟叶和菇叶中表达,而pre miR156a 3p_1和pre miR156a 5p在第一片真叶中表达量更高。(4)靶基因分析的结果在不同品种中呈现不同趋势,‘翠宝’成熟叶中SPL10/15高表达,SPL2表达量降低;‘改良香菇’中SPL10在成熟叶和菇叶中表达降低,SPL15在菇叶中高表达,SPL2在不同类型叶片中表达无差异。研究认为,miR156a成员及其靶基因SPL2/10/15可能参与调控芥蓝叶片发育,不同品种中作用的靶基因可能存在差异,从而导致了各品种芥蓝叶片发育的差异。     

Comprehensive Expression Analysis of Rice Armadillo Gene Family During Abiotic Stress and Development

Genes in the Armadillo (ARM)-repeat superfamily encode proteins with a range of developmental and physiological processes in unicellular and multicellular eukaryotes. These 42 amino acid, long tandem repeat-containing proteins have been abundantly recognized in many plant species. Previous studies have confirmed that Armadillo proteins constitute a multigene family in Arabidopsis. In this study, we performed a computational analysis in the rice genome (Oryza sativa L. subsp. japonica), and identified 158 genes of Armadillo superfamily. Phylogenetic study classified them into several arbitrary groups based on a varying number of non-conserved ARM repeats and accessory domain(s) associated with them. An in-depth analysis of gene expression through microarray and Q-PCR revealed a number of ARM proteins expressing differentially in abiotic stresses and developmental conditions, suggesting a potential roles of this superfamily in development and stress signalling. Comparative phylogenetic analysis between Arabidopsis and rice Armadillo genes revealed a high degree of evolutionary conservation between the orthologues in two plant species. The non-synonymous and synonymous substitutions per site ratios (Ka/Ks) of duplicated gene pairs indicate a purifying selection. This genome-wide identification and expression analysis provides a basis for further functional analysis of Armadillo genes under abiotic stress and reproductive developmental condition in the plant lineage.     

The Rice B-Box Zinc Finger Gene Family: Genomic Identification,Characterization, Expression Profiling and Diurnal Analysis

Background

The B-box (BBX) -containing proteins are a class of zinc finger proteins that contain one or two B-box domains and play important roles in plant growth and development. The Arabidopsis BBX gene family has recently been re-identified and renamed. However, there has not been a genome-wide survey of the rice BBX (OsBBX) gene family until now.

Methodology/Principal Findings

In this study, we identified 30 rice BBX genes through a comprehensive bioinformatics analysis. Each gene was assigned a uniform nomenclature. We described the chromosome localizations, gene structures, protein domains, phylogenetic relationship, whole life-cycle expression profile and diurnal expression patterns of the OsBBX family members. Based on the phylogeny and domain constitution, the OsBBX gene family was classified into five subfamilies. The gene duplication analysis revealed that only chromosomal segmental duplication contributed to the expansion of the OsBBX gene family. The expression profile of the OsBBX genes was analyzed by Affymetrix GeneChip microarrays throughout the entire life-cycle of rice cultivar Zhenshan 97 (ZS97). In addition, microarray analysis was performed to obtain the expression patterns of these genes under light/dark conditions and after three phytohormone treatments. This analysis revealed that the expression patterns of the OsBBX genes could be classified into eight groups. Eight genes were regulated under the light/dark treatments, and eleven genes showed differential expression under at least one phytohormone treatment. Moreover, we verified the diurnal expression of the OsBBX genes using the data obtained from the Diurnal Project and qPCR analysis, and the results indicated that many of these genes had a diurnal expression pattern.

Conclusions/Significance

The combination of the genome-wide identification and the expression and diurnal analysis of the OsBBX gene family should facilitate additional functional studies of the OsBBX genes.     

The Evolutionary Divergence of psbA Gene in Synechococcus and Their Myoviruses in the East China Sea

Marine Synechococcus is a principal component of the picophytoplankton and makes an important contribution to primary productivity in the ocean. Synechophages, infecting Synechococcus, are believed to have significant influences on the distribution and abundance of their hosts. Extensive previous ecological studies on cyanobacteria and viruses have been carried out in the East China Sea (ECS). Here we investigate the diversity and divergence of Synechococcus and their myoviruses (Synechomyoviruses) based on their shared photosynthesis psbA gene. Synechococcus is dominated by subclades 5.1A I, 5.1A II and 5.1A IV in the ECS, and clades I and II are the dominant groups in the Synechomyoviruses. As two phylogenetically independent clades, there is much higher diversity of the Synechomyoviruses than Synechococcus. Obvious partitioning characteristics of GC and GC3 (the GC content at the third codon position) contents are obtained among different picophytoplankton populations and their phages. The GC3 content causes the psbA gene in Synechococcus to have a higher GC content, while the opposite is true in the Synechomyoviruses. Analyzing more than one-time difference of the codon usage frequency of psbA sequences, the third position nucleotides of preferred codons for Synechococcus are all G and C, while most Synechomyoviral sequences (72.7%) have A and T at the third position of their preferred codons. This work shed light on the ecology and evolution of phage-host interactions in the environment.     

药用野生稻叶中淀粉合成酶基因家族的序列分化和特异表达

淀粉不仅是植物自身和后代生长繁殖的重要营养与能量储备,而且是人类膳食中碳水化合物的主要来源。植物中淀粉合成主要发生在两个阶段,一是在形成临时淀粉的光合作用阶段,另一个则是在成为贮藏淀粉的营养积累阶段。相对于最后的淀粉贮藏阶段,临时淀粉的形成阶段在植物整个碳水化合物代谢过程中扮演着更为重要的角色,然而却一直少有关注。为深入研究初始淀粉合成过程中相关酶在植物中的进化模式,选取了药用野生稻(Oryza officinalis)为研究对象,通过对其全叶转录组的重测序,定性、定量地调查了淀粉合成酶基因家族在稻属野生物种光合器官中的基因类型和表达变化。共有8个淀粉合成酶基因的完整编码序列在药用野生稻的叶中首次被识别。系统发育分析表明,这8个基因分别隶属SSI、SSII、SSIII、SSIV、SSV和GBSSII基因家族。序列比较和相对表达定量分析显示,药用野生稻与栽培稻的淀粉合成酶基因家族的进化模式具有高度的一致性,两个物种的同源基因在m RNA水平的序列相似度达到95%–98%。基于非同义置换和同义置换比率的统计检验表明,8个基因在两个物种间均经历了严格的纯化选择。另外,3个在栽培稻胚乳中特异表达的基因在药用野生稻的叶转录组中未筛查出来,而4个在栽培稻叶中优势表达的基因在药用野生稻叶中同样呈现相对较高水平的表达。     

Genomic Insights into the Evolutionary Origin of Xanthomonas axonopodis pv. citri and Its Ecological Relatives

Xanthomonas axonopodis pv. citri (Xac) is the causal agent of citrus bacterial canker (CBC) and is a serious problem worldwide. Like CBC, several important diseases in other fruits, such as mango, pomegranate, and grape, are also caused by Xanthomonas pathovars that display remarkable specificity toward their hosts. While citrus and mango diseases were documented more than 100 years ago, the pomegranate and grape diseases have been known only since the 1950s and 1970s, respectively. Interestingly, diseases caused by all these pathovars were noted first in India. Our genome-based phylogenetic studies suggest that these diverse pathogens belong to a single species and these pathovars may be just a group of rapidly evolving strains. Furthermore, the recently reported pathovars, such as those infecting grape and pomegranate, form independent clonal lineages, while the citrus and mango pathovars that have been known for a long time form one clonal lineage. Such an understanding of their phylogenomic relationship has further allowed us to understand major and unique variations in the lineages that give rise to these pathovars. Whole-genome sequencing studies including ecological relatives from their putative country of origin has allowed us to understand the evolutionary history of Xac and other pathovars that infect fruits.     

大白菜乙烯受体基因家族分子特征、微同线性与进化分析

采用HMMER与BLAST相结合的方法,在大白菜基因组中挖掘了10个大白菜ERT基因,被命名为BraERT1-10.基因结构分析显示,乙烯受体基因的外显子数目变异大,其范围为1-15.微同线性结果表明,白菜与拟南芥间以及白菜基因组内共有5对基因区段具有较高同线性,每对基因区段间至少共享了8个保守序列模块.蛋白保守结构域和亚细胞定位分析表明,BraERT1、BraERT2和BraERT8都具有N端疏水区域、GAF区、HisKA区、反应调节区,分别被定位于质膜、叶绿体和线粒体;BraERT3、BraERT4和BraERT7虽然有N端疏水区域、GAF区和HisKA区,但没有完整的反应调节区,分别被定位于质膜、质膜和胞质外;其余4个蛋白不具有典型区域,主要定位于核和叶绿体内.进化树结果显示,大白菜ERT基因具有3种类型,分别归属于不同3个类群.本研究为大白菜ERT基因功能研究提供线索,为进一步解析大白菜乙烯信号途径奠定基础.     

Structural,Functional, and Evolutionary Analysis of the Unusually Large Stilbene Synthase Gene Family in Grapevine

Stilbenes are a small family of phenylpropanoids produced in a number of unrelated plant species, including grapevine (Vitis vinifera). In addition to their participation in defense mechanisms in plants, stilbenes, such as resveratrol, display important pharmacological properties and are postulated to be involved in the health benefits associated with a moderate consumption of red wine. Stilbene synthases (STSs), which catalyze the biosynthesis of the stilbene backbone, seem to have evolved from chalcone synthases (CHSs) several times independently in stilbene-producing plants. STS genes usually form small families of two to five closely related paralogs. By contrast, the sequence of grapevine reference genome (cv PN40024) has revealed an unusually large STS gene family. Here, we combine molecular evolution and structural and functional analyses to investigate further the high number of STS genes in grapevine. Our reannotation of the STS and CHS gene families yielded 48 STS genes, including at least 32 potentially functional ones. Functional characterization of nine genes representing most of the STS gene family diversity clearly indicated that these genes do encode for proteins with STS activity. Evolutionary analysis of the STS gene family revealed that both STS and CHS evolution are dominated by purifying selection, with no evidence for strong selection for new functions among STS genes. However, we found a few sites under different selection pressures in CHS and STS sequences, whose potential functional consequences are discussed using a structural model of a typical STS from grapevine that we developed.Plants produce a vast array of secondary metabolites, many of them being restricted to specific groups of plant species. This extraordinary chemical diversity is believed to have evolved from a limited number of ubiquitous biosynthetic pathways through gene duplication followed by functional divergence (Pichersky and Gang, 2000). The phenylpropanoid pathway, derived from Phe, illustrates perfectly this phenomenon, as it gives rise to a large diversity of phenolic compounds playing key roles in plants, including participation in structural polymers, defense against herbivores and pathogens, protection from abiotic stress, and important functions in plant-pollinator interactions. Stilbenes are a small family of phenylpropanoids produced in a number of unrelated plant species, including dicotyledon angiosperms such as grapevine (Vitis vinifera), peanut (Arachis hypogaea), and Japanese knotweed (Fallopia japonica, formerly Polygonum cuspidatum), monocotyledons like sorghum (Sorghum bicolor), and gymnosperms such as several Pinus and Picea species. In addition to their participation in both constitutive and inducible defense mechanisms in plants, several stilbenes display important pharmacological properties. Since resveratrol (3,5,4′-trihydroxy-trans-stilbene) was postulated to be involved in the health benefits associated with a moderate consumption of red wine (Renaud and de Lorgeril, 1992), plant stilbenes have received considerable interest. Nowadays, resveratrol ranks among the most extensively studied natural products, and hundreds of studies have shown that it can slow the progression of a wide variety of illnesses, including cancer and cardiovascular disease, as well as extend the life spans of various organisms (Baur and Sinclair, 2006). Stilbene synthases (STSs) are characteristic of stilbene-producing plants and catalyze the biosynthesis of the stilbene backbone from three malonyl-CoA and one CoA-ester of a cinnamic acid derivative. STSs are members of the type III polyketide synthases family, chalcone synthases (CHSs), which catalyze the first step of flavonoid biosynthesis, being the most ubiquitous polyketide synthase in plants. Both CHS and STS use p-coumaroyl-CoA and malonyl-CoA as substrates and synthesize the same linear tetraketide intermediate. However, STS uses a specific cyclization mechanism involving a decarboxylation to form the stilbene backbone. STS proteins share extensive amino acid sequence identity with CHS, and phylogenetic analysis of the STS and CHS gene families has shown that STS genes may have evolved from CHS genes several times independently (Tropf et al., 1994). In most stilbene-producing plants, STS genes form small families of closely related paralogs. For example, two STS cDNAs have been cloned from peanut (Schröder et al., 1988), the genome of Scots pine (Pinus sylvestris) has been shown to contain a small family of four STS genes (Preisig-Müller et al., 1999), and three STS genes have been characterized in Japanese red pine (Pinus densiflora; Kodan et al., 2002). Only one STS gene has been isolated from Japanese knotweed to date (Liu et al., 2011), and the sequencing of sorghum genome has shown that SbSTS1 was the only STS gene in this plant species (Yu et al., 2005; Paterson et al., 2009). Grapevine is a noteworthy exception among stilbene-producing plants, as its genome has been shown to contain a large family of putative STS genes. Early Southern-blot experiments suggested that the grapevine genome contained more than 20 STS genes (Sparvoli et al., 1994). Analyses of the first drafts of the grapevine genome sequence confirmed the large size of this multigene family, with an estimated number of STS genes ranging from 21 to 43 (Jaillon et al., 2007; Velasco et al., 2007). However, these relatively low-coverage sequence drafts did not allow a precise analysis of large families of highly similar genes. The more recently released 12× genome sequence of grapevine inbred Pinot Noir cultivar PN40024 offered an improved sequence quality, allowing an accurate analysis of the STS gene family. In this work, we take advantage of the improved 12× sequence of the grapevine ‘PN40024’ genome to analyze the grapevine STS gene family. Furthermore, we combine molecular evolution to structural and functional analyses to gain more insight into the significance of the remarkable amplification of the STS family in grapevine.