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。     

Histone Acetylation Accompanied with Promoter Sequences Displaying Differential Expression Profiles of B-Class MADS-Box Genes for Phalaenopsis Floral Morphogenesis

Five B-class MADS-box genes, including four APETALA3 (AP3)-like PeMADS2∼5 and one PISTILLATA (PI)-like PeMADS6, specify the spectacular flower morphology in orchids. The PI-like PeMADS6 ubiquitously expresses in all floral organs. The four AP3-like genes, resulted from two duplication events, express ubiquitously at floral primordia and early floral organ stages, but show distinct expression profiles at late floral organ primordia and floral bud stages. Here, we isolated the upstream sequences of PeMADS2∼6 and studied the regulatory mechanism for their distinct gene expression. Phylogenetic footprinting analysis of the 1.3-kb upstream sequences of AP3-like PeMADS2∼5 showed that their promoter regions have sufficiently diverged and contributed to their subfunctionalization. The amplified promoter sequences of PeMADS2∼6 could drive beta-glucuronidase (GUS) gene expression in all floral organs, similar to their expression at the floral primordia stage. The promoter sequence of PeMADS4, exclusively expressed in lip and column, showed a 1.6∼3-fold higher expression in lip/column than in sepal/petal. Furthermore, we noted a 4.9-fold increase in histone acetylation (H3K9K14ac) in the translation start region of PeMADS4 in lip as compared in petal. All these results suggest that the regulation via the upstream sequences and increased H3K9K14ac level may act synergistically to display distinct expression profiles of the AP3-like genes at late floral organ primordia stage for Phalaenopsis floral morphogenesis.     

Temporal and spatial regulation of anthocyanin biosynthesis provide diverse flower colour intensities and patterning in <Emphasis Type="Italic">Cymbidium</Emphasis> orchid

Main conclusion

This study confirmed pigment profiles in different colour groups, isolated key anthocyanin biosynthetic genes and established a basis to examine the regulation of colour patterning in flowers of Cymbidium orchid. Cymbidium orchid (Cymbidium hybrida) has a range of flower colours, often classified into four colour groups; pink, white, yellow and green. In this study, the biochemical and molecular basis for the different colour types was investigated, and genes involved in flavonoid/anthocyanin synthesis were identified and characterised. Pigment analysis across selected cultivars confirmed cyanidin 3-O-rutinoside and peonidin 3-O-rutinoside as the major anthocyanins detected; the flavonols quercetin and kaempferol rutinoside and robinoside were also present in petal tissue. β-carotene was the major carotenoid in the yellow cultivars, whilst pheophytins were the major chlorophyll pigments in the green cultivars. Anthocyanin pigments were important across all eight cultivars because anthocyanin accumulated in the flower labellum, even if not in the other petals/sepals. Genes encoding the flavonoid biosynthetic pathway enzymes chalcone synthase, flavonol synthase, flavonoid 3′ hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) were isolated from petal tissue of a Cymbidium cultivar. Expression of these flavonoid genes was monitored across flower bud development in each cultivar, confirming that DFR and ANS were only expressed in tissues where anthocyanin accumulated. Phylogenetic analysis suggested a cytochrome P450 sequence as that of the Cymbidium F3′H, consistent with the accumulation of di-hydroxylated anthocyanins and flavonols in flower tissue. A separate polyketide synthase, identified as a bibenzyl synthase, was isolated from petal tissue but was not associated with pigment accumulation. Our analyses show the diversity in flower colour of Cymbidium orchid derives not from different individual pigments but from subtle variations in concentration and pattern of pigment accumulation.

     

C- and D-class MADS-box genes from Phalaenopsis equestris (Orchidaceae) display functions in gynostemium and ovule development

Gynostemium and ovule development in orchid are unique developmental processes in the plant kingdom. Characterization of C- and D-class MADS-box genes could help reveal the molecular mechanisms underlying gynostemium and ovule development in orchids. In this study, we isolated and characterized a C- and a D-class gene, PeMADS1 and PeMADS7, respectively, from Phalaenopsis equestris. These two genes showed parallel spatial and temporal expression profiles, which suggests their cooperation in gynostemium and ovule development. Furthermore, only PeMADS1 was ectopically expressed in the petals of the gylp (gynostemium-like petal) mutant, whose petals were transformed into gynostemium-like structures. Protein-protein interaction analyses revealed that neither PeMADS1 and PeMADS7 could form a homodimer or a heterodimer. An E-class protein was needed to bridge the interaction between these two proteins. A complementation test revealed that PeMADS1 could rescue the phenotype of the AG mutant. Overexpression of PeMADS7 in Arabidopsis caused typical phenotypes of the D-class gene family. Together, these results indicated that both C-class PeMADS1 and D-class PeMADS7 play important roles in orchid gynostemium and ovule development.     

The PI/GLO-like locus in orchids: duplication and purifying selection at synonymous sites within Orchidinae (Orchidaceae)

Positive selection and relaxation of purifying constraints after duplication events have driven the functional diversification of gene families involved in development. One example of this occurred within the plant MADS-box genes. The evolution of the orchid flower was driven by duplication events followed by sub- and neo-functionalization of class B DEF-like MADS-box genes, which are present at three to four copies in the orchid genome. In contrast, the orchid PI/GLO-like class B MADS-box genes have been reported thus far as single-copy loci, with the only exception of Habenaria radiata.We isolated a novel PI/GLO-like gene (OrcPI2) in Orchis italica, which is different than the previously characterized OrcPI locus. The presence of two functional paralogs of PI/GLO-like genes in orchids is detectable only within the tribe Orchidinae. Evolutionary analyses revealed an apparent relaxation of purifying selection acting on the two PI/GLO-like paralogs of the Orchidinae when compared to the single-copy PI/GLO-like genes found in other orchid species. Furthermore, by measuring dN/dS (ω) ratios, we show that a high percentage of sites between the two PI/GLO-like paralogs have different evolutionary pressures. Interestingly, the apparent relaxation of selective constraints on the two PI/GLO-like paralogs is due to strong purifying selection at synonymous sites rather than to a high value of nonsynonymous substitution rate. This peculiar evolutionary pattern might be related to molecular processes such as mRNA folding and/or translational efficiency control. These processes could potentially be involved in or predate the functional diversification of the two PI/GLO-like paralogs within Orchidinae.     

Analysis of diversity and relationships among Chinese orchid cultivars using EST-SSR markers

Chinese orchid (Cymbidium spp.) is an important potted flower with extremely high ornamental value in China. It is important to identify the genetic diversity of its germ plasma resources for development and evaluation of its cultivars. A set of 13 expressed sequence tag (EST)-derived simple sequence repeat (SSR) was used to analyze 103 cultivars of six species of Chinese orchid, namely Cymbidium goeringii, C. faberi, Cymbidium ensifolium, C. kanran, C. sinense and C. goeringii var. longibracteatum. The 13 SSR primer pairs generated a total of 168 polymorphic bands, with an average of 12.92 bands per primer and a range of 6–24 bands which clearly revealed the difference between cultivars inter- or intra-species of Chinese orchid. Cluster analysis based on UPGMA, NJ and PCoA method showed a dendrogram with three basic clusters and splitting feature of C. ensifolium and C. goeringii which partially congruent with the current taxonomic classification.     

Why are orchid flowers so diverse? Reduction of evolutionary constraints by paralogues of class B floral homeotic genes

Background

The nearly 30 000 species of orchids produce flowers of unprecedented diversity. However, whether specific genetic mechanisms contributed to this diversity is a neglected topic and remains speculative. We recently published a theory, the ‘orchid code’, maintaining that the identity of the different perianth organs is specified by the combinatorial interaction of four DEF-like MADS-box genes with other floral homeotic genes.

Scope

Here the developmental and evolutionary implications of our theory are explored. Specifically, it is shown that all frequent floral terata, including all peloric types, can be explained by monogenic gain- or-loss-of-function mutants, changing either expression of a DEF-like or CYC-like gene. Supposed dominance or recessiveness of mutant alleles is correlated with the frequency of terata in both cultivation and nature. Our findings suggest that changes in DEF- and CYC-like genes not only underlie terata but also the natural diversity of orchid species. We argue, however, that true changes in organ identity are rare events in the evolution of orchid flowers, even though we review some likely cases.

Conclusions

The four DEF paralogues shaped floral diversity in orchids in a dramatic way by modularizing the floral perianth based on a complex series of sub- and neo-functionalization events. These genes may have eliminated constraints, so that different kinds of perianth organs could then evolve individually and thus often in dramatically different ways in response to selection by pollinators or by genetic drift. We therefore argue that floral diversity in orchids may be the result of an unprecedented developmental genetic predisposition that originated early in orchid evolution.Key words: Orchidaceae, orchid evolution, evo-devo; perianth, class B genes, DEFICIENS, subfunctionalization, neofunctionalization, gene duplication, peloria, modularization     

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。