Why do paralogs persist? Molecular evolution of CYCLOIDEA and related floral symmetry genes in Antirrhineae (Veronicaceae)

CYCLOIDEA (CYC) and DICHOTOMA (DICH) are paralogous genes that determine adaxial (dorsal) flower identity in the bilaterally symmetric flowers of Antirrhinum majus (snapdragon). We show here that the duplication leading to the existence of both CYC and DICH in Antirrhinum occurred before the radiation of the Antirrhineae (the tribe to which snapdragon belongs). We find no additional gene duplications within Antirrhineae. Using explicit codon-based models of evolution in a likelihood framework, we show that patterns of molecular evolution after the duplication that gave rise to CYC and DICH are consistent with purifying selection acting at both loci, despite their known functional redundancy in snapdragon. However, for specific gene regions, purifying selection is significantly relaxed across DICH lineages, relative to CYC lineages. In addition, we find evidence for relaxed purifying selection along the lineage leading to snapdragon in one of two putative functional domains of DICH. A model of selection accounting for the persistence of paralogous genes in the absence of diversifying selection is presented. This model takes into account differences in the degree of purifying selection acting at the two loci and is consistent with subfunctionalization models of paralogous gene evolution.     

Flower symmetry and shape in Antirrhinum

According to their symmetry, flowers are classified as radially symmetrical or bilaterally symmetrical. Bilateral symmetry, which is thought to have evolved from radial symmetry, results from establishment of asymmetry relative to a dorsoventral axis of flowers. Here we consider developmental genetic mechanisms underlying the generation of this asymmetry and how they relate to controls of petal shape and growth in Antirrhinum. Two genes, CYC and DICH, are expressed in dorsal domains of the Antirrhinum flower and determine its overall dorsoventral asymmetry and the asymmetries and shapes of individual floral organs, by influencing regional growth. Another gene, DIV, influences regional asymmetries and shapes in ventral regions of the flower through a quantitative effect on growth. However, DIV is not involved in determining the overall dorsoventral asymmetry of the flower and its effects on regional asymmetries depend on interactions with CYC/DICH. These interactions illustrate how gene activity, symmetry, shape and growth may be related.     

Diversification and co-option of RAD-like genes in the evolution of floral asymmetry

To understand how changes in gene regulatory networks lead to novel morphologies, we have analysed the evolution of a key target gene, RAD, controlling floral asymmetry. In Antirrhinum, flower asymmetry depends on activation of RAD in dorsal regions of the floral meristem by the upstream regulators CYC and DICH. We show that Arabidopsis, a species with radially symmetric flowers, contains six RAD-like genes, reflecting at least three duplications since the divergence of Antirrhinum and Arabidopsis. Unlike the situation in Antirrhinum, none of the Arabidopsis RAD-like genes are activated in dorsal regions of the flower meristem. Rather, the RAD-like genes are expressed in distinctive domains along radial or ab-adaxial axes, consistent with a range of developmental roles. Introduction of a RAD genomic clone from Antirrhinum into Arabidopsis leads to a novel expression pattern that is distinct from the expression pattern of RAD in Antirrhinum and from the endogenous RAD-like genes of Arabidopsis. Nevertheless, RAD is able to influence developmental targets in Arabidopsis, as ectopic expression of RAD has developmental effects in this species. Taken together, our results suggest that duplication and divergence of RAD-like genes has involved a range of cis- and trans-regulatory changes. It is possible that such changes led to the coupling of RAD to CYC regulation in the Antirrhinum lineage and hence the co-option of RAD had a role in the generation of flower dorsoventral asymmetry.     

Floral zygomorphy, the recurring evolution of a successful trait

The flowers of the primitive angiosperm plants were radially symmetrical (actinomorphic). Flowers with bilateral symmetry (zygomorphic) evolved in several clades independently as an adaptation to specialized methods of pollination and played an important role in the diversification of flowering plants. In the model species Antirrhinum majus (snapdragon), the related genes CYCLOIDEA (CYC) and DICHOTOMA (DICH) are key in the development of this trait. This raises the question of whether they played a role in the evolution of floral bilateral symmetry. To address this, the evolution of CYC in relation to the evolution of zygomorphy is being investigated. Phylogenetic and functional analyses of CYC-like genes are being carried out in groups either closely related to Antirhinum or in families where zygomorphy evolved as an independent event. In addition, the origin of zygomorphy is being studied by comparing the function of CYC-like genes in species with zygomorphic flowers with their function in species with radially symmetrical flowers.     

苦苣苔科大叶石上莲CYC类基因RT-PCR表达模式研究

CYC类基因的分子系统学研究已经在苦苣苔科Gesneriaceae中展开,但是还缺乏对这些基因表达和功能的研究。因此,我们选择苦苣苔科大叶石上莲Oreocharis benthamii作为实验材料,分离出了CYC类基因的两个拷贝,经过分子系统学分析这两个基因分别属于苦苣苔科GCYC1和GCYC2两个分支,故命名为ObCYC1和ObCYC2。分区的RT-PCR实验结果显示这两个基因拥有不同的时间空间表达模式。ObCYC1与模式植物金鱼草Antirrhinum majus中的CYC基因类似,集中在花冠背部区域表达,这与它们拥有保守的功能区TCP和R相一致。但是,ObCYC1与CYC表达模式仍有区别,即,和CYC相比ObCYC1在花冠背部区域的表达提前减弱。这可能和大叶石上莲花冠微弱的两侧对称性相关。另外,由于大叶石上莲的背部花瓣较两侧和腹部花瓣小,因此,在功能上ObCYC1可能起抑制背部花瓣生长作用而CYC基因则促进背部花瓣生长。与ObCYC1不同,ObCYC2的保守功能区有更多的氨基酸位点变化,而且在RT-PCR实验中也没有检测到它的表达。因此,需要开展更深入的实验研究分析ObCYC2的基本功能,这将有助于了解GCYC2类基因在苦苣苔科及其近缘科中的功能和进化途径。     

Evolution of regulatory interactions controlling floral asymmetry

A key challenge in evolutionary biology is to understand how new morphologies can arise through changes in gene regulatory networks. For example, floral asymmetry is thought to have evolved many times independently from a radially symmetrical ancestral condition, yet the molecular changes underlying this innovation are unknown. Here, we address this problem by investigating the action of a key regulator of floral asymmetry, CYCLOIDEA (CYC), in species with asymmetric and symmetric flowers. We show that CYC encodes a DNA-binding protein that recognises sites in a downstream target gene RADIALIS (RAD) in Antirrhinum. The interaction between CYC and RAD can be reconstituted in Arabidopsis, which has radially symmetrical flowers. Overexpression of CYC in Arabidopsis modifies petal and leaf development, through changes in cell proliferation and expansion at various stages of development. This indicates that developmental target processes are influenced by CYC in Arabidopsis, similar to the situation in Antirrhinum. However, endogenous RAD-like genes are not activated by CYC in Arabidopsis, suggesting that co-option of RAD may have occurred specifically in the Antirrhinum lineage. Taken together, our results indicate that floral asymmetry may have arisen through evolutionary tinkering with the strengths and pattern of connections at several points in a gene regulatory network.     

被子植物花对称性的遗传控制

简要评述了被子植物花对称性遗传控制研究的最新进展。金鱼草中控制花背腹轴不对称性基因Cy cloidea(Cyc)和Dichotama(Dich)的克隆 ,为研究单对称花的遗传控制机理和进化历程提供了可能。在唇形目(Lamialess .l.)中的研究表明 ,在与金鱼草 (Antirrhinummajus)近缘的物种中 ,Cyc基因的同源基因可以采用相似的机制控制背腹轴上花器官的不对称性发育。最新的研究结果显示 ,在同金鱼草远缘的豆科植物 (Legu minosae)中 ,不仅存在Cyc基因的同源基因 ,而且它们也参与花背腹轴上不对称性的形成。参与花对称性控制的基因属于植物中一个新发现的基因家族———TCP结构域基因     

Significance of consensus CYC-binding sites found in the promoters of both ChCYC and ChRAD genes in Chirita heterotricha (Gesneriaceae)

CYC-like genes are widely conserved in controlling floral dorsoventral asymmetry (zygomorphy) through persistent expression in corresponding domains in core eudicots. To understand how CYC-like gene expression is maintained during flower development, we selected Chirita heterotricha as a material and isolated the promoter sequences of the ChCYCIC and ChCYCID genes, homologs of CYC, by inverse polymerase chain reaction. Further promoter analyses led to the identification of a putative cis-regulatory element in each promoter matching the consensus DNA binding site for Antirrhinum CYC protein: GGCCCCTC at-165 for ChCYC1C, and GGCCCCCC at-163 for ChCYCID. This indicates that both the ChCYCIC and ChCYC1D genes have probably evolved autoregulatory loops to sustain their expression in developing flowers. We also isolated the coding and promoter sequences of the ChRAD gene, a homolog of Antirrhinum RAD. Promoter analysis showed that the ChRAD gene promoter also contained a putative CYC-binding site (GGCCCAC at -134). Therefore, ChRAD is likely a direct target of the ChCYC1 genes, which is similar to Antirrhinum RAD. These results imply that the establishment of floral zygomorphy in Chirita may have been achieved by the evolution of an autoregulatory loop for CYC-like genes,which was probably accompanied by simultaneous co-option of the RAD-like gene into their regulatory network.     

百脉根LjCYC1基因克隆及其蛋白核定位

通过筛选百脉根(Lotus 7aponicus)基因组文库,克隆了LjCYC1(Lotus japonicus Cycloidea-like1)基因.L7CYC1是金鱼草(Antirrhium)CYC (Cycloidea)基因的同源基因,CYC属于TCP[TBl(teosinte branchedl),CYC,PCFs(PCFl and PCF2)]基因家族,编码转录调控子控制金鱼草花的对称性.基因组序列分析表明,LjCYC1的开放阅读框(ORF)由两个外显子和一个内含子组成,其cDNA编码的LjCYC1蛋白包含了370个氨基酸.蛋白序列分析显示,LjCYC1包含一个TCP结构域和一个R结构域,属于TCP结构域蛋白家族的CYC/TBl亚家族;LjCYCl氨基酸序列与CYC相比,一致性和相似性分别为39.0%和42.6%.不同长度的LjCYCl-cDNA与报告基因GUS融合后,通过粒子轰击(particle bombardment)方法在洋葱表皮细胞瞬时表达融合蛋白,观察到包含了TCP结构域的融合蛋白能够进行细胞核定位,提示LjCYCl可能作为转录因子行使功能;TCP结构域自身不能完成核定位过程,还需要结构域两侧旁邻氨基酸序列的协助.     

百脉根LjCYCl基因克隆及其蛋白核定位

通过筛选百脉根(Lotus japonicus)基因组文库,克隆了LjCYCl(Lotus japonicus Cvcloidea-likel基因。LjCYCl是金鱼草(Antirrhium)CYC(Cycloidea)基因的同源基因,CYC属于TCP[TBl(teosinte branched 1),CYC,PCFs(PCF1 and PCF2)]基因家族,编码转录调控子控制金鱼草花的对称性。基因组序列分析表明,LjCYCl的开放阅读框(ORF)由两个外显子和一个内含子组成,其cDNA编码的LjCYCl蛋白包含了370个氨基酸。蛋白序列分析显示,LjCYCl包含一个TCP结构域和一个R结构域,属于TCP结构域蛋白家族的CYC／TBl亚家族;LjCYCl氨基酸序列与CYC相比,一致性和相似性分别为39．0％和42．6％。不同长度的LjCYCl-cDNA与报告基因GUS融合后,通过粒子轰击(particle bombardment)方法在洋葱表皮细胞瞬时表达融合蛋白,观察到包含了TCP结构域的融合蛋白能够进行细胞核定位,提示LjCYCl可能作为转录因子行使功能;TCP结构域自身不能完成核定位过程,还需要结构域两侧旁邻氨基酸序列的协助。     

Evolution of interacting proteins in the mitochondrial electron transport system in a marine copepod

The extensive interaction between mitochondrial-encoded and nuclear-encoded subunits of electron transport system (ETS) enzymes in mitochondria is expected to lead to intergenomic coadaptation. Whether this coadaptation results from adaptation to the environment or from fixation of deleterious mtDNA mutations followed by compensatory nuclear gene evolution is unknown. The intertidal copepod Tigriopus californicus shows extreme divergence in mtDNA sequence and provides an excellent model system for study of intergenomic coadaptation. Here, we examine genes encoding subunits of complex III of the ETS, including the mtDNA-encoded cytochrome b (CYTB), the nuclear-encoded rieske iron-sulfur protein (RISP), and cytochrome c(1) (CYC1). We compare levels of polymorphism within populations and divergence between populations in these genes to begin to untangle the selective forces that have shaped evolution in these genes. CYTB displays dramatic divergence between populations, but sequence analysis shows no evidence for positive selection driving this divergence. CYC1 and RISP have lower levels of sequence divergence between populations than CYTB, but, again, sequence analysis gives no evidence for positive selection acting on them. However, an examination of variation at cytochrome c (CYC), a nuclear-encoded protein that transfers electrons between complex III and complex IV provides evidence for selective divergence. Hence, it appears that rapid evolution in mitochondrial-encoded subunits is not always associated with rapid divergence in interacting subunits (CYC1 and RISP), but can be in some cases (CYC). Finally, a comparison of nuclear-encoded and mitochondrial-encoded genes from T. californicus suggests that substitution rates in the mitochondrial-encoded genes are dramatically increased relative to nuclear genes.     

非洲紫罗兰两侧与辐射对称花中两个CYC类完整基因的分离和序列分析

在已知GCYC基因部分序列基础上, 通过改进的mTAIL-PCR方法克隆非洲紫罗兰Saintpaulia ionantha两侧对称栽培种中CYC类基因的5′未知序列, 并进而从两侧与辐射对称栽培种中分离得到苦苣苔科Gesneriaceae中第一组完整基因: SiCYC1A与SiCYC1B。对以上基因的核酸和氨基酸序列比较发现, SiCYC1A与SiCYC1B序列同源性很高, 均含有完整的功能调控区域(即TCP domain和R domain)并与模式植物金鱼草Antirrhinum majus中CYC基因同源。因此, 这两个基因应具有正常功能, 是功能上互补的冗余基因。令人意外的是在辐射对称花栽培品种中的这两个基因和两侧对称花栽培品种中对应基因的序列完全相同。经过对金鱼草以及相关类群辐射对称花突变体中CYC类基因序列的比较分析, 推论在非洲紫罗兰中, SiCYC1A与SiCYC1B基因可能受上游未知的共同调控因子调控, 该调控因子的改变是导致栽培品种中花对称性发生变化的主要原因。另外, 对改进后的TAIL-PCR(mTAIL-PCR)的方法和过程进行了详细叙述, 并对其技术特征和优势开展了简单的论述。     

Low rates of silent substitution in nuclear genes of two distantly related Scrophulariaceae (Antirrhinum and Verbascum)

Low levels of genetic diversity and divergence at nuclear loci have previously been observed for cycloidea and fil1-like genes within and between several Antirrhinum species, and divergence at these loci is also low between species in genera at different levels of relatedness in the former family Scrophulariaceae (Digitalis and Verbascum). The low divergence values are surprising, because (based on the sequences of chloroplast loci) the Scrophulariaceae are thought to be polyphyletic, with two anciently diverged clades, and the species we compared belonged to the two different clades. Here, we extend our studies of sequence divergence to more nuclear genes: fil2, far, globosa, and ADH: Detailed studies revealed that in Antirrhinum these genes belong to gene families. Low levels of divergence between Antirrhinum and Verbascum were observed for four of the loci studied, fil2-1, fil2-2, far-L, and globosa, similar to our previous observations. We discuss hypotheses to explain these low synonymous divergence values. For Adh, no cases of very similar sequences were found, but, rather, our sequences from the three different genera (Antirrhinum, Digitalis, and Verbascum) were all very diverged. Repeated gene duplication and loss of elements in the Adh gene family is likely in these lineages, making it impossible to determine orthology of the Adh genes.