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.     

MADS about the evolution of orchid flowers

Orchids have unique flowers involving three types of perianth organs: outer tepals, lateral inner tepals, and a lip. Expression studies indicate that the identity of these organs is specified by the combinatorial interaction of four different DEFICIENS-like MADS-box genes. We suggest that clarifying the evolution of these genes provides a rational framework for reconstructing the enigmatic origin and unique diversification of the orchid flower. For example, two rounds of gene duplications during early orchid evolution might have generated the genes that were probably recruited to distinguish the different types of perianth organs. This hypothesis suggests intriguing, experimentally testable mechanisms by which gene duplications followed by sub- and neo-functionalization events might have contributed to the evolutionary origin of morphological novelties in orchids - and well beyond.     

Expression of AODEF,a B-functional MADS-box gene,in stamens and inner tepals of the dioecious species Asparagus officinalis L.

Garden asparagus (Asparagus officinalis L.) is a dioecious species with male and female flowers on separate unisexual individuals. Since B- and C-functional MADS-box genes specify male and female reproductive organs, it is important to characterize these genes to clarify the mechanism of sex determination in monoecious and dioecious species. In this study, we isolated and characterized AODEF gene, a B-functional gene in the development of male and female flowers of A. officinalis. Southern hybridization identified a single copy of AODEF gene in asparagus genome. Northern blot analysis showed that this gene was specifically expressed in flower buds and not in vegetative tissues. In situ hybridization showed that during early hermaphrodite stages, AODEFgene was expressed in the inner tepal and stamen whorls (whorls 2 and 3, respectively), but not in the outer tepals (whorl 1), in both male and female flowers. In late unisexual developmental stages, the expression of AODEF gene was still detected in the inner tepals and stamens of male flowers, but the expression was reduced in whorls 2 and 3 of female flowers. Our results suggest that AODEF gene is probably not involved in tepal development in asparagus and that the expression of AODEF gene is probably controlled directly or indirectly by sex determination gene in the late developmental stages.     

Interactions of B-class complex proteins involved in tepal development in Phalaenopsis orchid

In our previous studies, we identified four DEFICIENS (DEF)-like genes and one GLOBOSA (GLO)-like gene involved in floral organ development in Phalaenopsis equestris. Revealing the DNA binding properties and protein-protein interactions of these floral homeotic MADS-box protein complexes (PeMADS) in orchids is crucial for the elucidation of the unique orchid floral morphogenesis. In this study, the interactome of B-class PeMADS proteins was assayed by the yeast two-hybrid system (Y2H) and glutathione S-transferase (GST) pull-down assays. Furthermore, the DNA binding activities of these proteins were assessed by using electrophoretic mobility shift assay (EMSA). All four DEF-like PeMADS proteins interacted individually with the GLO-like PeMADS6 in Y2H assay, yet with different strengths of interaction. Generally, the PeMADS3/PeMADS4 lineage interacted more strongly with PeMADS6 than the PeMADS2/PeMADS5 lineage did. In addition, independent homodimer formation for both PeMADS4 (DEF-like) and PeMADS6 (GLO-like) was detected. The protein-protein interactions between pairs of PeMADS proteins were further confirmed by using a GST pull-down assay. Furthermore, both the PeMADS4 homodimer and the PeMADS6 homodimer/homomultimer per se were able to bind to the MADS-box protein-binding motif CArG. The heterodimeric complexes PeMADS2-PeMADS6, PeMADS4-PeMADS6 and PeMADS5-PeMADS6 showed CArG binding activity. Taken together, these results suggest that various complexes formed among different combinations of the five B-class PeMADS proteins may increase the complexity of their regulatory functions and thus specify the molecular basis of whorl morphogenesis and combinatorial interactions of floral organ identity genes in orchids.     

Evolution of class B floral homeotic proteins: obligate heterodimerization originated from homodimerization

The class B floral homeotic genes from the higher eudicot model systems Arabidopsis and Antirrhinum are involved in specifying the identity of petals and stamens during flower development. These genes exist in two different types termed DEF- and GLO-like genes. The proteins encoded by the class B genes are stable and functional in the cell only as heterodimeric complexes of a DEF- and a GLO-like protein. In line with this, heterodimerization is obligate for DNA binding in vitro. The genes whose products have to heterodimerize to be stable and functional are each other's closest relatives within their genomes. This suggests that the respective genes originated by gene duplication, and that heterodimerization is of relative recent origin and evolved from homodimerization. To test this hypothesis we have investigated the dimerization behavior of putative B proteins from phylogenetic informative taxa, employing electrophoretic mobility shift assays and the yeast two-hybrid system. We find that an ancestral B protein from the gymnosperm Gnetum gnemon binds DNA in a sequence-specific manner as a homodimer. Of the two types of B proteins from the monocot Lilium regale, the GLO-like protein is still able to homodimerize, whereas the DEF-like protein binds to DNA only as a heterodimeric complex with the GLO-like protein. These data suggest that heterodimerization evolved in two steps after a gene duplication that gave rise to DEF- and GLO-like genes. Heterodimerization may have originated after the gymnosperm-angiosperm split about 300 MYA but before the monocot-eudicot split 140-200 MYA. Heterodimerization may have become obligate for both types of flowering plant B proteins in the eudicot lineage after the monocot-eudicot split.     

Evolutionary change in flowers and inflorescences: evidence from naturally occurring terata

Records of naturally occurring, heritable floral abnormalities considerably enhance our understanding of floral evolution. Peloric mutants, frequent in natural populations of orchids and mints, have radially symmetric flowers but occur in species characterized by bilaterally symmetric flowers. Three distributions of peloric flowers across an inflorescence are: (1) complete (all flowers peloric, as in the cycloidea mutant of Antirrhinum), (2) scattered (with both peloric and zygomorphic flowers, as in the epigenetic cycloidea mutant of Linaria), and (3) terminal (only the terminal flower peloric, as in the centroradialis mutant of Antirrhinum). Genetic relationships between lateral and terminal peloria, and between peloric and pseudopeloric flowers, remain ambiguous. Complete peloria probably caused occasional evolutionary reversals from zygomorphy to actinomorphy, whereas the 'terminal-flower effect' is a less likely cause of floral evolution.     

Two ancestral APETALA3 homologs from the basal angiosperm Magnolia wufengensis (Magnoliaceae) can affect flower development of Arabidopsis

APETALA3 (AP3) homologs are involved in specifying petal and stamen identities in core eudicot model organisms. In order to investigate the functional conservation of AP3 homologs between core eudicots and basal angiosperm, we isolated and identified two AP3 homologs from Magnolia wufengensis, a woody basal angiosperm belonging to the family Magnoliaceae. Sequence and phylogenetic analyses revealed that both genes are clade members of the paleoAP3 lineage. Moreover, a highly conserved motif of paleoAP3 is found in the C-terminal regions of MAwuAP3_1/2 proteins, but the PI-derived motif, usually present in AP3/DEF-like lineage members, is missing. Semi-quantitative and real time PCR analyses showed that the expression of MAwuAP3_1/2 was restricted to tepals and stamens. However, the MAwuAP3_1 expression was maintained at a high level during the rapid increased in size of tepals and stamens, while MAwuAP3_2 mRNA was only detected at the early stage of tepal and stamen development. Furthermore, the expression of MAwuAP3_1/2 in transgenic Arabidopsis causes phenotypic changes which partly resemble those caused by ectopic expressions of the endogenous AP3 gene. Moreover, the 35S::MAwuAP3_1/2 transgenic Arabidopsis can be used partially to rescue the loss-of-function ap3 mutant (ap3-3) of Arabidopsis. These findings call for a more comprehensive understanding of the B-functional evolution from basal angiosperm to core eudicot clades.     

Two GLOBOSA-like genes are expressed in second and third whorls of homochlamydeous flowers in Asparagus officinalis L

Garden asparagus (Asparagus officinalis L.) has homochlamydeous flowers. Like Liliaceae plants such as lily and tulip, the perianths of asparagus have two whorls of almost identical petaloid organs, called tepals. Floral structures of these homochlamydeous flowers could be explained by a modified ABC model, in which the expression of the class B genes has expanded to whorl 1, so that the organs of whorls 1 and 2 have the same petaloid structure. In this study, we isolated and characterized two GLOBOSA-like genes (AOGLOA and AOGLOB), one of class B gene, from asparagus. Southern blot showed that AOGLOA and AOGLOB genes are single copy genes. Northern blot analysis indicated that these genes were specifically expressed in male and female flowers. In situ hybridization showed that the expression of AOGLOA and AOGLOB genes is confined to whorls 2 and 3 (inner tepal and stamen) and not detected in whorl 1 (outer tepal). The other asparagus class B gene, AODEF, was also not expressed in outer tepal [Park et al. (2003) Plant Mol Biol. 51: 867]. These results indicate that the class B genes are not involved in the outer tepal development in asparagus, not supporting the modified ABC model in asparagus.     

Hormonal changes during flower development in floral tissues of Lilium

Much effort has been focussed on better understanding the key signals that modulate floral senescence. Although ethylene is one of the most important regulators of floral senescence in several species, Lilium flowers show low sensitivity to ethylene; thus their senescence may be regulated by other hormones. In this study we have examined how (1) endogenous levels of hormones in various floral tissues (outer and inner tepals, androecium and gynoecium) vary throughout flower development, (2) endogenous levels of hormones in such tissues change in cut versus intact flowers at anthesis, and (3) spray applications of abscisic acid and pyrabactin alter flower longevity. Results show that floral tissues behave differently in their hormonal changes during flower development. Cytokinin and auxin levels mostly increased in tepals prior to anthesis and decreased later during senescence. In contrast, levels of abscisic acid increased during senescence, but only in outer tepals and the gynoecium, and during the latest stages. In addition, cut flowers at anthesis differed from intact flowers in the levels of abscisic acid and auxins in outer tepals, salicylic acid in inner tepals, cytokinins, gibberellins and jasmonic acid in the androecium, and abscisic acid and salicylic acid in the gynoecium, thus showing a clear differential response between floral tissues. Furthermore, spray applications of abscisic acid and pyrabactin in combination accelerated the latest stages of tepal senescence, yet only when flower senescence was delayed with Promalin. It is concluded that (1) floral tissues differentially respond in their endogenous variations of hormones during flower development, (2) cut flowers have drastic changes in the hormonal balance not only of outer and inner tepals but also of androecium and gynoecium, and (3) abscisic acid may accelerate the progression of tepal senescence in Lilium.     

Genomic organization of the AODEF gene in Asparagus officinalis L

The perianths of Liliaceae plants, such as lily and tulip, have two whorls of almost identical petaloid organs, which are called tepals. According to the modified ABC model proposed in tulip, the class B genes are expressed in whorl 1 as well as whorls 2 and 3, so that the organs of whorls 1 and 2 have the same petaloid structure. The floral structure of asparagus (Asparagus officinalis L.) is similar to that of Liliaceae plants, however, the expression of B-class genes (AODEF, AOGLOA, AOGLOB) was not found in whorl 1, but was confined to whorls 2 and 3. This result does not support the modified ABC model in asparagus. In order to gain a better understanding of asparagus flower development, we have characterized a genomic clone of the AODEF gene. We compared the genomic organization and promoter sequence of AODEF with three well-studied DEF-like genes, DEFICIENS (Antirrhinum), APETALA3 (Arabidopsis), and OSMADS16 (rice). Exon-intron structures of these genes are well-conserved except for the large fifth intron in the AODEF gene and the OSMADS16 gene. Putative cis-elements including CArG-boxes were found in the promoter region and forty-two microsatellites were found in the AODEF genomic sequence.     

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。