首页分享科学网—[文献翻译]CYC/TB1基因家族在菊科中的进化与分化

科学网—[文献翻译]CYC/TB1基因家族在菊科中的进化与分化

来源：花匠小妙招时间：2024-11-22 05:29

文献原文：Evolution and Diversification of the CYC/TB1 Gene Family in Asteraceae—A Comparative Study in Gerbera (Mutisieae) and Sunflower (Heliantheae)

讨论部分（Discussion）：

原文：

1）Expansion of CYC/TB1 Gene Family in Gerbera and Sunflower

Our finding that gerbera has ten CYC/TB1-like TCP genes is consistent with the results of Chapman et al. (2008), who showed that the gene family has experienced a significant expansion in sunflower. As a result, both gerbera and sun- flower have more genes in this angiosperm-specific clade of TCP genes than any other plant lineages studied thus far (Howarth and Donoghue 2006; Navaud et al. 2007). Phylo- genetic analyses by Howarth and Donoghue (2005, 2006) showed that the CYC1, CYC2, and CYC3 clades of CYC/ TB1-like genes arose from a single ancestral gene by dupli- cations within the eudicot lineage. These investigators placed CYC1 sister to the clade containing CYC2 and CYC3. Despite extensive analysis of CYC/TB1-like genes (Howarth and Donoghue 2005, 2006), no other duplica- tions within the CYC1 clade similar to those demonstrated in gerbera and sunflower have been found. Taking into ac- count the low support for grouping of gerbera GhCYC1 and GhCYC10, the presence of two genes in both species sug- gests that they may originate from the shared paleopoly- ploidization event near the base of Asteraceae 40–45 Ma (Barker et al. 2008). This is supported by divergence esti- mates for HaCYC1a and HaCYC1b to the same time period (Chapman et al. 2008). Alternatively, the gerbera ortholog of HaCYC1b may have been lost, so the GhCYC1/GhCYC10 pair could represent gerbera-specific duplicates.

In contrast to the CYC1 gene clade, the CYC2 and CYC3 gene clades have expanded in plant lineages other than the Asteraceae. In diverse asterid and rosid species, thenumber of CYC2 copies has been shown to vary from one to four (reviewed by Busch and Zachgo 2009; Howarth et al. 2011). Gene isolation from the Dipsacales (asterid) revealed that they have two CYC3 gene copies instead of the one in Arabidopsis (rosid) (Howarth and Donoghue 2005). In sev- eral species, duplication patterns of CYC3 and CYC2 genes are similar (Howarth and Donoghue 2006). This seems not to be the case in gerbera and sunflower, since they show the highest number of CYC2 paralogs (six for gerbera and five for sunflower) documented so far but have only three and two CYC3 genes, respectively. The CYC3 genes in ger- bera and sunflower form orthologous groups. An addi- tional gene duplication in sunflower, HaCYC3c/HaCYC3b, is estimated to have diverged 18.5 Ma, that is, after the basal Heliantheae whole-genome duplication 26–31 Ma (Chapman et al. 2008). The presence of four orthologous groups within the CYC2 clade points to a shared origin, but species-specific gene duplications not affected by whole- genome duplications are also apparent (GhCYC4/GhCYC9 and HaCYC2d/HaCYC2e). Genome and single-gene dupli- cations in plants are known to increase genomic complex- ity and have been suggested to produce new genes that allow the evolution of functional novelty (Jiao et al. 2011). Our discovery that especially the CYC2 clade in par- ticular both expanded and maintained duplicates appears to support the idea that members of this clade may have novel and important functions contributing to the complex inflorescence structure in Asteraceae.

翻译：

1）CYC/TB1基因家族在非洲菊与向日葵之中的扩增

本研究在非洲菊中找到了10个类CYC/TB1的TCP成员，该结果与Chapman等人2008年的研究结论是一致的，该团队认为这个基因家族在向日葵之中曾经发生过显著的扩增事件。在迄今为止所研究的所有植物家系之中，向日葵与非洲菊无疑在这个专属于被子植物的TCP基因分支中拥有者最多的成员（Howarth and Donoghue 2006；Navaud et al.2007）。Howarth与Donoghue在2005，2006两年中所进行的系统发育分析显示，在核心双子叶植物家系类CYC/TB1基因中的CYC1，CYC2，CYC3分支都来源于共同的祖先——由单一的基因复制而来，这些系统发育分析证明CYC1分支与CYC2和CYC3的大支互为姐妹关系。类CYC/TB1基因有着广泛的研究基础（Howarth and Donoghue 2005，2006），但在非洲菊与向日葵之中发生的复制事件确实是一个新的发现。GhCYC1与GhCYC10基因在系统发育分析中聚在一起（虽然支持率不高），说明该两基因可能来源于同一祖先，并且该祖先在40-45Ma的时候就经历了古多倍体化事件（paleo-polyploidization event），此时的菊科正处在进化历程的初始状态（Barker et al. 2008）。对于HaCYC1a与HaCYC1b在同一时段的分歧评估分析也支持以上假说，并与非洲菊中的GhCYC1和GhCYC10是对应的（Chapman et al. 2008）。除以上外，还有另一种可能性：非洲菊中与HaCYC1b直系同源的基因已经丢失，GhCYC1与GhCYC2的出现可能只是来自于一次仅发生于非洲菊之中的复制事件。

与CYC1分支相比较，CYC2与CYC3分支除在菊科植物中也在其他的植物家系之中发生过扩展的现象。在多样性丰富的菊类与蔷薇类类群之中，不同种类所包含的CYC2成员从1至4个不等（Busch and Zachgo 2009；Howarth et al. 2011）。相比在拟南芥（蔷薇类）中CYC3只拥有一个成员，菊类分支川断绿目则为两个拷贝。在不同物种之间，CYC2与CYC3的复制模式是相似的，但在非洲菊与向日葵之中则例外。这两者在CYC2分支中拥有大量的旁系基因（非洲菊6，向日葵5），CYC3中则较少(非洲菊3，向日葵2)，该两者的的CYC3基因则汇聚为单源的一支。而在向日葵之中的HaCYC3c/HaCYC3b，则估算是来源于18.5Ma之前发生的复制事件，且在基部Heliantheae植物的全基因组复制事件之后（发生在26-31Ma）。在CYC2分支中，有四个同源组来源于同一祖先（GhCYC4/GhCYC9, HaCYC2d/HaCYC2e），但这些单个物种所特异的基因复制事件看似并非得益于全基因组复制（WGD）。众所周知，植物中发生的基因组以及单基因复制事件能够提升基因组的复杂度，并且能够产生一些新的基因，使得出现一些有趣的功能进化。对于我们的发现（特别是CYC2），这些基因不论是保留了还是复制了，都足以支持这样一个说法：该分支成员对于菊科植物复杂的花序结构来说有着新奇且重要的作用。

原文：

2）Expression Analysis Indicates Both Early and LateFunctions for the Conserved CYC2 Clade Genesduring Flower Type Differentiation

The CYC2 clade genes that showed the greatest numberof secondary duplications in each species also shared strik-ingly similar expression domains. In both species, all CYC2 genes were expressed in developing ray flower primordia. Ingerbera, however, following its more gradual transitionfrom ray to disc flower structure, most CYC2 clade genesseem to be also involved in defining the development ofbilaterally symmetrical trans flowers and are absent onlyfrom the centermost, most strongly actinomorphic discflowers.

In sunflower, unlike in gerbera, the CYC1 clade gene Ha-CYC1a was the only one that showed upregulation in de-veloping disc flower primordia. In further contrast to thegerbera counterparts, the expression of the sunflower Ha-CYC3a gene suggests that it may also play a role in early rayflower development. Nevertheless, in both species, we canclearly identify genes with specific functions in ray flowersonly: GhCYC3 in gerbera and both HaCYC2d and HaCYC2cin sunflower. In both species, the restriction of expressionto ray flower primordia suggests that these genes sharea general function in defining ray flower identity, whereasthe other CYC2 clade genes are likely to have more local-ized functions at the level of single ray (and trans) flowers.These functions may include flower symmetry regulation atthe dorsoventral axis or suppression of stamen develop-ment, as previously shown for several CYC2 clade genes(Luo et al. 1996; Hileman and Baum 2003; Song et al.2009). Our previous studies showed that suppression ofGhCYC2 expression in transgenic gerbera did not cause al-terations in ray flower identity, indicating that some othergene defines ray identity and that GhCYC2 may function asa modifier gene in this process (Broholm et al. 2008). This isin accordance with classical genetic studies that have indi-cated that the absence of ray flowers (discoid flower head)in the Asteraceae is determined by one or two major genes(Gillies et al. 2002; Kloos et al. 2004). Furthermore, Bertiet al. (2005) reported on two sunflower mutants with mod-ified flower type identity. They showed that a single reces-sive gene controlled the tubular ray flower (turf) mutant trait that shows development of tubular disc-like flowers inplace of zygomorphic ray flowers, whereas in the Chrysan-themoides (Chry) mutant, all flowers were ray-like. Our datasuggest that GhCYC3 in gerbera and HaCYC2d and Ha-CYC2c in sunflower are the strongest candidates for con-ferring ray flower identity, but functional analyses arerequired to test this hypothesis.

The fact that, in both species, most CYC1 and CYC3clade genes did not show clear differential expression be-tween different flower types suggests that they, with thepossible exception of HaCYC1a and HaCYC3c, are not in-volved in the early differentiation of flower types. The rel-atively high expression levels of these genes in vegetativetissues point to their functions in other aspects of plantdevelopment, including leaf and inflorescence stem (scape)development. The CYC/TB1-like genes in Arabidopsis, inaddition to being expressed in flower and shoot primordia(Cubas et al. 2001; Aguilar-Martinez et al. 2007), are alsoexpressed in rosette leaves and in inflorescence stems(Aguilar-Martinez et al. 2007; Guo et al. 2010; Koyamaet al. 2010). The Arabidopsis CYC2 clade gene TCP1 hasbeen shown to regulate vegetative development by mod-ulating brassinosteroid biosynthesis (Guo et al. 2010),whereas BRC1/TCP18 and BRC2/TCP12 were earlier shownto regulate shoot branching (Aguilar-Martinez et al. 2007;Finlayson 2007). The possible functions of these genes inArabidopsis flower development remain to be elucidated.Our results strongly suggest that, in addition to determin-ing ray (and trans) flower specification during the early de-velopment of flower types, CYC2 genes also function inlater developmental stages of reproductive and vegetativeorgans together with CYC1 and CYC3 genes.

In conclusion, our results imply that differences, evensubtle ones, in patterns of gene expression have contrib-uted to the maintenance of CYC gene duplicates withinthe Asteraceae. Moreover, our study indicates that ofthe CYC genes, the CYC2 clade in particular has both earlyand late functions. Further studies are needed to elucidatethe functions of the different gene family members at theorgan and tissue level.

翻译：

2）保守的CYC2分支基因在花类型分化的早期与末期功能的表达分析

在大量物种中都存在二次复制事件的CYC2分支基因间有着显著相似的表达模式。在向日葵与非洲菊之中，这些基因都表达于舌状花（ray flower）原基。对于非洲菊来说，在其逐渐由舌状花向管状花（disc flower）转变的过程中，大部分的CYC2分支基因看似都与花两侧对称的定性有关——其在花序部中央区域的管状花中是不表达的，这些管状花都呈现出显著的辐射对称。

而在向日葵之中，CYC1分支基因HaCYC1a是唯一在发育的管状花原基中表达的成员。在与非洲菊的深入对比中，向日葵的HaCYC3a基因则只在舌状花早期的生长阶段表达。尽管如此，我们仍然能够清楚的识别出在这两个物种之中，仅在舌状花中有功能表达的基因：GhCYC3，HaCYC2d与HaCYC2c。这些基因只在舌状花原基中表达，那么说明他们很可能有着共同的功能——决定舌状花的形成，反之CYC2的其他成员在单个舌状花或两状花（trans flower）中则有着更局部的功能。像先前已经被研究的部分CYC2基因一样，这些功能可能包括有关花对称性的背腹轴的生长调控，雄蕊生长的抑制等等。我们先前的研究显示，GhCYC2基因受到抑制的转基因非洲菊的舌状花形态并未发生转变，这也预示着可能有别的基因决定这个过程，而GhCYC2在其中只起到了修饰或辅助的作用。这也与传统的基因研究的结论相一致：菊科植物中舌状花的缺失是由一到两个主要的基因控制的。Berti等人在2005年报道了在两株向日葵突变体重观察到了花类型的变异，他们认为一个单隐形基因控制了管状花（turf）向两侧对称的舌状花转变，反之在Chrysanthemoides突变株中（译者：可能是该隐形基因在所有花中表达的突变），所有的花都是舌状的。我们的数据证明了GhCYC3与HaCYC2d，HaCYC2c是最有可能控制舌状花形成的基因，但仍然缺乏功能验证。

在两物种中，CYC1与CYC3分支的基因在不同类型的花之中的表达情况并没有显著差异，而在营养器官中却有着较高的表达量。这也预示着除了HaCYC1a与HaCYC3c以外的其他成员与花类型早期的分化并不相关，而是在植物生长的其他阶段有着相应的功能，例如叶片与花序轴的生长。CYC/TB1基因在拟南芥之中不仅在花与芽原基中表达，还表达于莲座状叶片与花序轴。拟南芥的CYC2分支基因TCP1则通过修饰油菜素类固醇的生物合成来调节营养器官的生长，与此同时在早期的研究中，BRC1/TCP18与BRC2/TCP12则被证明能够调控芽的分支，但这些基因在拟南芥的花生长过程中可能起到的功能依然需要被阐明。故我们的研究强烈支持，CYC2分支基因除了决定舌状花的分化之外，还与CYC1分支、CYC3分支的成员一同在后期的生殖器官与营养器官的生长过程中起作用。

综上所述，我们的结果暗示着这些基因表达模式的不同，精确到每一点都导致了菊科中复制的CYC基因的存续。不仅如此，我们的研究还论证了CYC基因的CYC2分支在早期和晚期都有着一定的功能。然而想要验证不同基因家族成员在不同组织与器官等级上的功能情况，进一步的研究是不可少的。

3）Regulatory Network Involving CYC/TB1 Proteins

Pairwise interaction studies showed the complex networkof interactions involving CYC/TB1-like proteins. In bothspecies, the capacity of CYC/TB1-like proteins for hetero-dimerization and the overlap in their expression domainsindicate that these proteins are likely to function in com-plexes. Our data provide the first experimental proof thatthe CYC/TB1 proteins can indeed interact in vitro and mayalso form heterodimers among the CYC clades, as initiallyproposed by Howarth and Donoghue (2006). However, forsome proteins when used as baits, such as the CYC3 cladeproteins GhCYC8, HaCYC3a, and HaCYC3b, relatively largedeletions were necessary to reduce strong autoactivation.Therefore, we cannot rule out the possibility that we mayhave missed some true interactions in our analysis.

In the early stages of flower development, heterodime-rization might only occur among CYC2 clade proteins in ray and trans flowers of gerbera, whereas in sunflower,CYC2 proteins might also interact with the CYC3 proteinHaCYC3a. Although the gerbera CYC3 clade genes did notshow clear expression patterns during early primordia de-velopment, their expression in reproductive tissues duringlater stages of flower development overlapped with that ofthe CYC2 clade genes and also with the CYC1 clade geneGhCYC10. Together with the interaction data, this indicatesthat the corresponding proteins may function in the samecomplexes. Similarly, sunflower HaCYC1a interacted withboth CYC2 and CYC3 clade proteins and showed overlap-ping expression especially in ovary and leaf as shown earlierby Chapman et al. (2008). A pair of orthologous proteins inthe two species, GhCYC5 and HaCYC2c, did not interactwith any other CYC/TB1-like proteins, did not form homo-dimers, nor did they show strong transcriptional activity inyeast. Previous studies with the proliferating cell nuclearantigen gene-controlling element binding factor groupof TCP proteins have indicated that not all TCPs are ableto regulate transcription by themselves and may needother interacting proteins for transcriptional activation(Kosugi and Ohashi 2002). It remains to be elucidatedwhether this may apply to GhCYC5 and HaCYC2c as well.

The capacity of CYC/TB1-like proteins to form homo-dimers appears to differ between gerbera and sunflower,whereas that of heterodimer formation was more similarin both species. Heterodimerization seemed to be most highlyconserved among those of the CYC2 clade proteins that weremost similar to each other, according to our phylogenetic andexpression analyses. In contrast, heterodimerization betweenthe proteins of the three clades show great differences. In gen-eral, heterodimerization is known to increase functional spec-ificity since heterodimers combine different DNA-bindingdomains and can therefore mediate differential gene regula-tion (Amoutzias et al. 2008). As proposed by Martin-Trillo andCubas (2010), different heteromeric combinations (as de-tected in gerbera and sunflower) may, in addition to bindingdivergent cis-regulatory elements, recognize target genes withdifferent affinity or modulate each others’ activity. This couldadd another level of complexity to the regulatory networkscontrolled by these factors.

翻译：

3）与CYC/TB1蛋白相关的调控网络

成对互作研究呈现了CYC/TB1相关蛋白互作过程中精密的调控网络。在向日葵与非洲菊中，类CYC/TB1基因产生异源二聚体的能力以及在其表达结构域上的重叠性就预示着这些蛋白可能会形成复合体。我们的数据第一次在试管中证明了CYC/TB1蛋白能够形成异质二聚体，就像Howarth与Donoghue在2006年所推断的那样。然而，一些我们用作饵剂的蛋白，例如CYC3分支的GhCYC8，HaCYC3a与HaCYC3b，减少他们强烈的自体活化则需要一些相对大的缺失部分。因此，我们很难确保我们在该分析中没有排除掉任何可能的相互作用。

在花发育的早期阶段，只有CYC2分支的蛋白在非洲菊的舌状花与管状花中都能形成异质二聚体；而在向日葵之中，CYC2的蛋白则还能与CYC3分支中的HaCYC3a相互作用。尽管在早期原基形成的阶段，非洲菊CYC3分支的基因并没有显示出清晰的表达模式，但在生殖器官生长的整个后期阶段，CYC3分支基因则与CYC2分支基因以及CYC1分支中的GhCYC10有着相同的表达模式。将以上的情形与互作的数据相结合，我们可以推论出这些蛋白可能在共同的复合体中起作用。同样的，Chapman等人在2008年时就证明了向日葵的HaCYC1a能够与CYC2与CYC3分支的蛋白相互作用，并且它们共享着很多相似的表达模式，特别是在子房和叶片之中。另外，在向日葵与非洲菊中的一对直系同源蛋白GhCYC5与HaCYC2c并不与其他类CYC/TB1蛋白相互作用，也不能形成同质二聚体，并且在酵母中也没有显示出强烈的转录活性。先前一项关于增殖细胞核抗原基因调控原件结合因子的TCP蛋白类群的研究也证明了并不是所有的TCP成员都能够自身调控转录的，它们可能需要其他的互作蛋白来激活转录过程。GhCYC5与HaCYC2c是否属于这样的情况仍然需要进一步研究。

相对于向日葵与非洲菊的类CYC/TB1蛋白形成同质二聚体的能力，其异质二聚体的形成模式是相同的。根据我们的系统发育以及表达分析来看，CYC2分支蛋白的异源二聚化过程是较为保守的。相比来看，异质二聚化在三个蛋白分支中有着诸多不同点。通常来说，异质二聚化能够提升功能的特异性，因为这些二聚体能够联接不同的DNA结合域，并且能够介导不同基因的调控。就像Martin-Trillo与Cubas在2010年认为的那样，不同的异质二聚化联接（就像在非洲菊与向日葵中发现的一样），除了结合不同的cis调控元件（顺式作用元件）外，都能够识别有着不同关系的目标基因，并能相互调节活性。这便会提升这些因子调控网络的复杂度。

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