蝴蝶兰PhalPI基因的克隆及在花器官突变体中的表达分析

为深入研究兰科植物花器官发育的调控机理,从蝴蝶兰花瓣中克隆了一个B类MADS-box转录因子PhalPI（GenBank登录号为KY020416）。序列分析表明,该基因的cDNA全长为944 bp,含完整的开放阅读框,可编码210个氨基酸,属于BGLO/PI蛋白家族,与蝴蝶兰属的PhPI10和PeMADS6基因关系最近;表达模式分析表明,PhalPI基因在生殖器官中表达,在营养器官中不表达,在授粉后的子房中,该基因的表达水平降低。在5种花器官突变体中,PhalPI基因在萼片唇瓣化突变体的萼片和蕊柱中表达水平明显升高;在雄蕊花瓣化突变体的萼片和侧瓣中表达水平降低,在其唇瓣和蕊柱中显著升高;在侧瓣合柱化突变体的蕊柱中,PhalPI基因的表达也发生了显著升高;PhalPI基因表达的改变与花器官形态的突变相关;而在侧瓣唇瓣化和侧瓣花药化突变体中,PhalPI基因的表达水平没有变化。推测该基因在决定蝴蝶兰侧瓣和唇瓣的发育中起重要的调控作用。     

Four DEF-like MADS box genes displayed distinct floral morphogenetic roles in Phalaenopsis orchid

The complex flower organization of orchids offers an opportunity to discover new variant genes and different levels of complexity in the morphogenesis of flowers. In this study, four B-class Phalaenopsis DEF-like MADS-box genes were identified and characterized, including PeMADS2, PeMADS3, PeMADS4 and PeMADS5. Differential expression profiles of these genes were detected in the floral organs of P. equestris, suggesting distinctive roles in the floral morphogenesis of orchids. Furthermore, expressions of these genes were varied to different extents in the peloric mutants with lip-like petals. Expression of PeMADS4 was in lips and columns of wild type, and it extended to the lip-like petals in the peloric mutant. Expression of PeMADS5 was mainly in petals and to a lesser extent in columns in the wild type, whereas it was completely eliminated in the peloric mutant. Disruption of the PeMADS5 promoter region of the peloric mutant was detected at nucleotide +312 relative to the upstream of translational start codon, suggesting that a DNA rearrangement has occurred in the peloric mutant. Genomic structure analysis of the PeMADS5 showed that the exon length was conserved in exons 1-6, similar to DEF-like genes of other plants. Collectively, this is the first report that four DEF-like MADS genes were identified in a single monocotyledonous species and that they may play distinctive morphogenetic roles in the floral development of an orchid.     

蝴蝶兰花发育的分子生物学研究进展

蝴蝶兰花非常独特且高度进化,如萼片瓣化、瓣片特化为唇瓣、雌雄蕊合生成合蕊柱及子房发育须由授粉启动等,是单子叶植物花发育研究的理想材料。近年来蝴蝶兰花发育分子生物学取得了重要进展。该文就近年来国内外有关蝴蝶兰开花转换及花器官发育相关基因研究以及B类基因与兰花花被的进化发育关系方面的研究进展进行综述。研究表明:MADS基因在蝴蝶兰开花转换及花器官发育过程中起重要作用,推测其中的DEF(DE-FICIENS)-like基因早期经过2轮复制,形成了4类不同的DEF-like基因,进而决定兰花花被属性。蝴蝶兰花发育分子生物学的深入研究,将极大地利于通过基因工程手段提高蝴蝶兰花品质如花色改良及花期调控等,推动分子育种进程。     

A comparison of venation pattern development in wild type and a homeotic sepaloid petal mutant of Clarkia tembloriensis (Onagraceae)

Vascular system development in sepals, petals, and sepaloid petals was compared in wild-type and crinkled petal mutant plants of Clarkia tembloriensis. Patterns of vascularization in cleared whole mounts were visualized and traced under both brightfield and polarizing illumination. Wild-type sepals exhibited a basipetal pattern of maturation, with tracheary elements maturing relatively rapidly. Mature sepals had three primary veins with numerous secondary veins. In contrast, wild-type petals exhibited an acropetal pattern of maturation, with tracheary elements maturing relatively slowly. The mature petals had only one primary vein with numerous secondary veins. Sepaloid (crinkled) petals combined characteristics of both wild-type sepals and wild-type petals. They exhibited a basipetal pattern of development and a relatively rapid maturation of the tracheary elements characteristic of wild-type sepals. Venation architecture in crinkled petal mutants showed a single primary vein with numerous secondary veins, similar to wild-type petals. The crinkled petal mutant fits the definition of a homeotic mutant in that the petal has assumed characteristics of the sepal. However, homeotic transformation from petal to sepal is incomplete since the crinkled petal still retains many of the characteristics of wild-type petals.     

春兰AGL6-3基因的克隆及实时定量表达分析

该研究以春兰(Cymbidium goeringii)正常花及其2枚侧瓣突变成唇瓣样的花瓣(简称:蝶花)为实验材料,采用RT-PCR结合RACE技术从春兰中分离出AGL6-3基因。序列分析表明,AGL6-3基因在春兰正常花和蝶花中序列相同,该基因含有1个720bp长的开放阅读框(ORF),共编码239个氨基酸。系统进化树进行分析表明,该基因属于MADS-box基因中AP1/AGL9组的AGL6同源基因,命名为CgAGL6-3(基因登录号为KU058679)。实时荧光定量表达分析表明,CgAGL6-3在春兰正常花和蝶花各花器官中表达存在差异。在正常春兰中CgAGL6-3基因在唇瓣中强烈表达,在主萼、侧萼及蕊柱中表达量较低,在侧瓣中则微乎其微;而在蝶花中CgAGL6-3基因在唇瓣中强表达,侧瓣中的表达量次之,在主萼、侧萼和蕊柱中的表达量相近且均较低。研究说明,CgAGL6-3基因可能在春兰蝶花侧瓣唇瓣化的过程中扮演重要角色。     

The petunia AGL6 gene has a SEPALLATA-like function in floral patterning

SEPALLATA ( SEP ) MADS-box genes are required for the regulation of floral meristem determinacy and the specification of sepals, petals, stamens, carpels and ovules, specifically in angiosperms. The SEP subfamily is closely related to the AGAMOUS LIKE6 ( AGL6 ) and SQUAMOSA ( SQUA ) subfamilies. So far, of these three groups only AGL6 -like genes have been found in extant gymnosperms. AGL6 genes are more similar to SEP than to SQUA genes, both in sequence and in expression pattern. Despite the ancestry and wide distribution of AGL6 -like MADS-box genes, not a single loss-of-function mutant exhibiting a clear phenotype has yet been reported; consequently the function of AGL6 -like genes has remained elusive. Here, we characterize the Petunia hybrida AGL6 ( PhAGL6 , formerly called PETUNIA MADS BOX GENE4 / pMADS4 ) gene, and show that it functions redundantly with the SEP genes FLORAL BINDING PROTEIN2 ( FBP2 ) and FBP5 in petal and anther development. Moreover, expression analysis suggests a function for PhAGL6 in ovary and ovule development. The PhAGL6 and FBP2 proteins interact in in vitro experiments overall with the same partners, indicating that the two proteins are biochemically quite similar. It will be interesting to determine the functions of AGL6 -like genes of other species, especially those of gymnosperms.     

FRL1 is required for petal and sepal development in Arabidopsis

A novel flower mutant, frl1 (frill 1) was isolated in Arabidopsis thaliana. The frl1 mutant has serrated petals and sepals but the other floral and vegetative organs appear to be normal. To analyse the role of the FRL1 gene, morphological, cytological and double mutant analyses were carried out. The frl1 flower had broader petals and sepals as compared with the wild-type. The distal region of frl1 petals contained fewer epidermal cells but their size was variable and generally larger than that in the wild-type. However, no significant difference was found in the basal region. Observations of the early petal development revealed that the morphology of the developing frl1 petal was normal until the middle of stage 9, but the frl1 phenotype became apparent in stages later than 10. Furthermore, larger nuclei with varied sizes were observed in the distal region of frl1 petals, but not in this region in wild-type petals. This strongly suggests that abnormal endo-reduplication had occurred. These observations indicate that the frl1 mutation affects the number of cell divisions and the subsequent cell expansion during the late stage of petal lamina formation, and that FRL1 might be maintaining the mitotic state or suppressing the transition to the endo-reduplication cycle. Double mutants with the homeotic mutants apetala3-1 and agamous showed additive phenotypes. Ectopic petals in the third whorl of fr11 ag flowers were serrated, indicating that the FRL1 gene acts in petal and sepal development in an organ-specific manner.     

Negative regulation of the Arabidopsis homeotic gene AGAMOUS by the APETALA2 product.

We characterized the distribution of AGAMOUS (AG) RNA during early flower development in Arabidopsis. Mutations in this homeotic gene cause the transformation of stamens to petals in floral whorl 3 and of carpels to another ag flower in floral whorl 4. We found that AG RNA is present in the stamen and carpel primordia but is undetectable in sepal and petal primordia throughout early wild-type flower development, consistent with the mutant phenotype. We also analyzed the distribution of AG RNA in apetela2 (ap2) mutant flowers. AP2 is a floral homeotic gene that is necessary for the normal development of sepals and petals in floral whorls 1 and 2. In ap2 mutant flowers, AG RNA is present in the organ primordia of all floral whorls. These observations show that the expression patterns of the Arabidopsis floral homeotic genes are in part established by regulatory interactions between these genes.     

Floral development of petaloid Alismatales as an insight into the origin of the trimerous Bauplan in monocot flowers

Monocots are remarkably homogeneous in sharing a common trimerous pentacyclic floral Bauplan. A major factor affecting monocot evolution is the unique origin of the clade from basal angiosperms. The origin of the floral Bauplan of monocots remains controversial, as no immediate sister groups with similar structure can be identified among basal angiosperms, and there are several possibilities for an ancestral floral structure, including more complex flowers with higher stamen and carpel numbers, or strongly reduced flowers. Additionally, a stable Bauplan is only established beyond the divergence of Alismatales. Here, we observed the floral development of five members of the three ‘petaloid’ Alismatales families Butomaceae, Hydrocharitaceae, and Alismataceae. Outer stamen pairs can be recognized in mature flowers of Alismataceae and Butomaceae. Paired stamens always arise independently, and are either shifted opposite the sepals or close to the petals. The position of stamen pairs is related to the early development of the petals. In Butomaceae, the perianth is not differentiated and the development of the inner tepals is not delayed; the larger inner tepals (petals) only permit the initiation of stamens in antesepalous pairs. Alismataceae has delayed petals and the stamens are shifted close to the petals, leading to an association of stamen pairs with petals in so-called stamen–petal complexes. In the studied Hydrocharitaceae species, which have the monocot floral Bauplan, paired stamens are replaced by larger single stamens and the petals are not delayed. These results indicate that the origin of the floral Bauplan, at least in petaloid Alismatales, is closely linked to the position of stamen pairs and the rate of petal development. Although the petaloid Alismatales are not immediately at the base of monocot divergence, the floral evolution inferred from the results should be a key to elucidate the origin of the floral Bauplan of monocots.     

Are Petals Sterile Stamens or Bracts? The Origin and Evolution of Petals in the Core Eudicots

BACKGROUND: The aim of this paper is to discuss the controversial origins of petals from tepals or stamens and the links between the morphological expression of petals and floral organ identity genes in the core eudicots. SCOPE: I challenge the widely held classical view that petals are morphologically derived from stamens in the core eudicots, and sepals from tepals or bracts. Morphological data suggest that tepal-derived petals have evolved independently in the major lineages of the core eudicots (i.e. asterids, Santalales and rosids) from Berberidopsis-like prototypes, and that staminodial petals have arisen only in few isolated cases where petals had been previously lost (Caryophyllales, Rosales). The clear correlation between continuous changes in petal morphology, and a scenario that indicates numerous duplications to have taken place in genes controlling floral organ development, can only be fully understood within a phylogenetic context. B-gene expression plays a fundamental role in the evolution of the petals by controlling petaloidy, but it does not clarify petal homology. CONCLUSIONS: An increased synorganization of the flower in the core eudicots linked with the establishment of floral whorls restricts the petaloid gene expression to the second whorl, reducing the similarities of petals with tepals from which they were originally derived. An increased flower size linked with secondary polyandry or polycarpelly may lead to a breakdown of the restricted gene expression and a reversal to ancestral characteristics of perianth development. An altered 'sliding boundary' hypothesis is proposed for the core eudicots to explain shifts in petaloidy of the perianth and the event of staminodial petals. The repetitive changes of function in the perianth of the core eudicots are linked with shifts in petaloidy to the outer perianth whorl, or losses of petal or sepal whorls that can be secondarily compensated for by the inclusion of bracts in the flower. The origin and evolution of petals appears to be as complex on a molecular basis as it is from a morphological point of view.     

Optimal pollinator attraction strategies in Trollius ranunculoides Hemsl. (Ranunculaceae) at different altitudes: increased floral display or promotion of nectar output?

For alpine plant species, patterns of resource allocation to functional floral traits for pollinator attraction can be highly significant in adaptation to low pollinator abundance and consequent pollen limitation. Increased pollination can be achieved either through a larger floral display or production of more pollen rewards. In this study, variation in resource allocation to different components for pollinator attraction was studied along an altitudinal gradient in Trollius ranunculoides, an obligate self‐incompatible out‐crosser of the Qinghai–Tibet Plateau. We compared resource allocation to conspicuous yellow sepals (which mainly provide visual attraction) and degenerate petals (which provide the major nectar reward) between populations at four altitudes. Furthermore, we investigated the contribution of sepals and petals to pollinator attraction and female reproductive success in an experiment with sepal or petal removal at sites at different altitudes. At the level of single flowers, resource allocation increased to sepals but decreased to petals with increasing altitude. Consistent with these results, sepals contributed much more to visitation rate and seed set than petals, as confirmed in the sepal or petal removal experiment. Sepals and petals contributed to female reproductive success by ensuring visitation rate rather than visitation duration. To alleviate increasing pollen limitation with increasing altitude, resource allocation patterns of T. ranunculoides altered to favour development of sepals rather than petals. This strategy may improve pollination and reproductive success through visual attraction (sepal) rather than nectar reward (petal) over a gradient of decreasing pollinator abundance.     

The seirena B Class Floral Homeotic Mutant of California Poppy (Eschscholzia californica) Reveals a Function of the Enigmatic PI Motif in the Formation of Specific Multimeric MADS Domain Protein Complexes

The products of B class floral homeotic genes specify petal and stamen identity, and loss of B function results in homeotic conversions of petals into sepals and stamens into carpels. Here, we describe the molecular characterization of seirena-1 (sei-1), a mutant from the basal eudicot California poppy (Eschscholzia californica) that shows homeotic changes characteristic of floral homeotic B class mutants. SEI has been previously described as EScaGLO, one of four B class–related MADS box genes in California poppy. The C terminus of SEI, including the highly conserved PI motif, is truncated in sei-1 proteins. Nevertheless, like the wild-type SEI protein, the sei-1 mutant protein is able to bind CArG-boxes and can form homodimers, heterodimers, and several higher order complexes with other MADS domain proteins. However, unlike the wild type, the mutant protein is not able to mediate higher order complexes consisting of specific B, C, and putative E class related proteins likely involved in specifying stamen identity. Within the PI motif, five highly conserved N-terminal amino acids are specifically required for this interaction. Several families lack this short conserved sequence, including the Brassicaceae, and we propose an evolutionary scenario to explain these functional differences.     

蓝堇草属(毛茛科)花形态发生的扫描电子显微镜观察

花的发生和发育过程研究可以发现早期进化的轨迹,为系统发育的研究提供重要线索。蓝堇草属(Leptopyrum)为毛茛科唐松草亚科一单种属,仅包含蓝堇草一种,其花的发生和发育过程仍为空白。为了深入理解唐松草亚科乃至毛茛科花发育多样性和演化规律,该文运用扫描电子显微镜(SEM)观察了蓝堇草各轮花器官的形态发生和发育过程。结果表明:该属植物所有的萼片、花瓣、雄蕊和雌蕊均为螺旋状发生,花器官排列式样也为螺旋状; 5枚萼片原基宽阔,5枚花瓣原基圆球形、位于萼片原基的间隔,且在后期表现为延迟发育现象,雄蕊原基较小、为圆球形; 花瓣原基和雄蕊原基连续发生,无明显的时空间隔,但与萼片原基有时空间隔; 心皮原基为马蹄形对折,柱头组织由单细胞乳突组成; 胚珠倒生、具单珠被。该属花器官螺旋状排列、胚珠具单珠被在唐松草亚科中是独有的性状,花发育形态学证据支持了该属的特殊性。     

Evolution of petaloid sepals independent of shifts in B-class MADS box gene expression

Attractive petals are an integral component of animal-pollinated flowers and in many flowering plant species are restricted to the second floral whorl. Interestingly, multiple times during angiosperm evolution, petaloid characteristics have expanded to adjacent floral whorls or to extra-floral organs. Here, we investigate developmental characteristics of petaloid sepals in Rhodochiton atrosanguineum, a close relative of the model species Antirrhinum majus (snapdragon). We undertook this in two ways, first using scanning electron microscopy we investigate the micromorphology of petals and sepals, followed by expression studies of genes usually responsible for the formation of petaloid structures. From our data, we conclude that R. atrosanguineum petaloid sepals lack micromorphological characteristics of petals and that petaloid sepals did not evolve through regulatory evolution of B-class MADS box genes, which have been shown to specify second whorl petal identity in a number of model flowering plant species including snapdragon. These data, in conjunction with other studies, suggests multiple convergent pathways for the evolution of showy sepals.