草原龙胆MADS-box基因的克隆及表达分析

植物MADS-box基因家族编码高度保守的转录因子,参与了包括花发育在内的多种发育进程。为阐释双子叶植物草原龙胆（Eustoma grandiflorum）花器官发育的分子调控机制,根据MADS-box基因保守序列设计简并引物,用3＇-RACE方法从草原龙胆中克隆了4个花器官特异表达的MADS-box家族基因。序列和系统进化树分析表明,这4个基因分别与金鱼草DEF基因、矮牵牛FBP3基因和FBP6基因以及拟南芥SEP3基因具有很高的同源性,分别属DEF/GLO、AG-like和SEP-like亚家族。从而将这4个基因分别命名为EgDEF1、EgGLO1、EgPLE1和EgSEP3-1。推导的氨基酸序列显示,这些基因编码的蛋白质都包含高度保守的MADS结构域、I结构域和K结构域,每个基因均有其亚家族特异的C-末端功能域。基因特异性RT-PCR检测结果显示：EgDEF1在萼片、花瓣、雄蕊及胚珠中高丰度表达,在心皮中微量表达;而EgGLO1在花瓣和雄蕊中高丰度表达,在萼片中微量表达;在根、茎、叶等营养器官中均未检测到上述2个基因的表达。EgPLE1在雌蕊、心皮和胚珠中特异表达,但表达的丰度存在差异,在雄蕊中的表达有所减弱。SEP-like亚家族基因EgSEP3-1在四轮花器官和胚珠中均特异表达,且表达丰度相对一致。     

草原龙胆MADS-box基因的克隆及表达分析

植物MADS-box 基因家族编码高度保守的转录因子, 参与了包括花发育在内的多种发育进程。为阐释双子叶植物草原龙胆(Eustoma grandiflorum)花器官发育的分子调控机制, 根据MADS-box基因保守序列设计简并引物, 用3'-RACE方法从 草原龙胆中克隆了4个花器官特异表达的MADS-box家族基因。序列和系统进化树分析表明, 这4个基因分别与金鱼草DEF基因、矮牵牛FBP3基因和FBP6基因以及拟南芥SEP3基因具有很高的同源性, 分别属DEF/GLO、AG-like和SEP-l ike亚家族。从而将这4个基因分别命名为EgDEF1、EgGLO1、EgPLE1和EgSEP3-1。推导的氨基酸序列显示, 这些基因编码的蛋白质都包含高度保守的MADS结构域、I结构域和K结构域, 每个基因均有其亚家族特异的C-末端功能域。基因特异性RT-PCR检测结果显示: EgDEF1 在萼片、花瓣、雄蕊及胚珠中高丰度表达, 在心皮中微量表达; 而EgGLO1在花瓣和雄蕊中高丰度表达, 在萼片中微量表达; 在根、茎、叶等营养器官中均未检测到上述2个基因的表达。EgPLE1在雌蕊、心皮和胚珠中特异表达, 但表达的丰度存在差异, 在雄蕊中的表达有所减弱。SEP-like亚家族基因EgSEP3-1在四轮花器官和胚珠中均特异表达,且表达丰度相对一致。     

A B Functional Gene Cloned from Lily Encodes an Ortholog of <Emphasis Type="BoldItalic">Arabidopsis PISTILLATA</Emphasis> (<Emphasis Type="BoldItalic">PI</Emphasis>)

The classical ABC model proposed for flower development in Arabidopsis and Antirrhinum appropriately sheds light on the biological process of flower development and differentiation and serves in manipulating the floral structure of other important ornamental plants. In this study, LLGLO1, a B functional gene from Lilium longiflorum was isolated and characterized. RT-PCR analysis elucidated that temporal and spatial expression pattern of LLGLO1. This putative gene was strongly expressed in 1, 2, and 3 whorl organs, i.e., outer whorl tepals, inner whorl tepals, and stamens. Genetic effect of LLGLO1 was assayed by ectopic expression in model plant Arabidopsis. Transformed plants showed homeotic transformation of sepals into petaloid sepals in the first whorl, which is similar to the transgenic plants of 35S::PI. So LLGLO1 was one member of GLO/PI sub-family gene to function in flower development.     

APETALA3 and PISTILLATA homologs exhibit novel expression patterns in the unique perianth of Aristolochia (Aristolochiaceae)

Several lines of evidence suggest that sterile floral organs, collectively known as the perianth, have evolved multiple times during the evolution of the angiosperms. In the family Aristolochiaceae, the perianth is formed by two whorls of organs in the genus Saruma but by only one whorl in the remaining genera, including Aristolochia. Although the morphology of Saruma is similar in appearance to the core eudicot perianth, with leaf-like sepals and showy colored petals, the unipartite perianth of Aristolochia combines morphological aspects of both calyx and corolla. To investigate the organ identity program functioning in the novel perianth of Aristolochia, we identified homologs of the B-class genes APETALA3 (AP3) and PISTILLATA (PI) in both Saruma and Aristolochia. The expression patterns of these genes in Saruma indicate they are functioning in the development of the second whorl petaloid organs and third whorl stamens. In Aristolochia, however, the expression of AP3 and PI homologs in the perianth does not suggest a role in organ identity but, rather, in promoting late aspects of cell differentiation. The implications of these findings for the evolution of both petaloidy and B gene function are discussed.     

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.     

RABBIT EARS is a second-whorl repressor of AGAMOUS that maintains spatial boundaries in Arabidopsis flowers

The RABBIT EARS (RBE) gene has been identified as a regulator of petal development in Arabidopsis thaliana. We find that second-whorl petals in rbe mutants can be replaced with staminoid organs, stamens or filaments and that some rbe flowers have increased numbers of sepals and exhibit fusion of sepals. We show that these rbe defects are due to AGAMOUS (AG) misexpression in the second whorl. Consistent with its role in maintaining the spatial boundary of AG expression, rbe enhanced the second-whorl defects present in ap2-1, lug-1 and clf-2 mutants. In the development of second-whorl organs, RBE acts in the same pathway and downstream of UNUSUAL FLORAL ORGANS (UFO). Enhanced first-whorl organ fusion in ap2-2 rbe-3, ant-4 rbe-3 and cuc2-1 rbe-3 double mutants supports an additional role for RBE in organ separation. RBE thus acts to maintain two different types of spatial boundaries in young flowers: boundaries between organ primordia within a whorl and boundaries of homeotic gene expression between whorls.     

'Living stones' reveal alternative petal identity programs within the core eudicots

Petals, defined as the showy laminar floral organs in the second floral whorl, have been shown to be under similar genetic control in distantly related core eudicot model organisms. On the basis of these findings, it is commonly assumed that the petal identity program regulated by B-class MADS-box gene homologs is invariant across the core eudicot clade. However, the core eudicots, which comprise >70% of angiosperm species, exhibit numerous instances of petal and sepal loss, transference of petal function between floral whorls, and recurrent petal evolution. In the face of these complex patterns of perianth evolution, the concept of a core eudicot petal identity program has not been tested. We therefore examined the petal identity program in the Caryophyllales, a core eudicot clade in which perianth differentiation into sepals and petals has evolved multiple times. Specifically, we analyzed the expression patterns of B- and C-class MADS-box homologs for evidence of a conserved petal identity program between sepal-derived and stamen-derived petaloid organs in the 'living stone' family Aizoaceae. We found that neither sepal-derived nor stamen-derived petaloid organs exhibit gene expression patterns consistent with the core eudicot petal identity program. B-class gene homologs are not expressed during the development of sepal-derived petals and are not implicated in petal identity in stamen-derived petals, as their transient expression coincides with early expression of the C-class homolog. We therefore provide evidence for petal development that is independent of B-class genes and suggest that different genetic control of petal identity has evolved within this lineage of core eudicots. These findings call for a more comprehensive understanding of perianth variation and its genetic causes within the core eudicots--an endeavor that will have broader implications for the interpretation of perianth evolution across angiosperms.     

The S locus-linked Primula homeotic mutant sepaloid shows characteristics of a B-function mutant but does not result from mutation in a B-function gene

Floral homeotic and flower development mutants of Primula, including double, Hose in Hose, Jack in the Green and Split Perianth, have been cultivated since the late 1500s as ornamental plants but until recently have attracted limited scientific attention. Here we describe the characterization of a new mutant phenotype, sepaloid, that produces flowers comprising only sepals and carpels. The sepaloid mutation is recessive, and is linked to the S locus that controls floral heteromorphy. The phenotype shows developmental variability, with flowers containing three whorls of sepals surrounding fertile carpels, two whorls of sepals with a diminished third whorl of sepals surrounding a fourth whorl of carpels, or three whorls of sepals surrounding abnormal carpels. In some respects, these phenotypes resemble the Arabidopsis and Antirrhinum homeotic B-function mutants apetala3/deficiens (ap3/def) and pistillata/globosa (pi/glo). We have isolated the Primula vulgaris B-function genes PvDEFICIENS (PvDEF) and PvGLOBOSA (PvGLO), expression of both of which is affected in the sepaloid mutant. PvGLO, like sepaloid, is linked to the S locus, whereas PvDEF is not. However, our analyses reveal that sepaloid and PvGLO represent different genes. We conclude that SEPALOID is an S-linked independent regulator of floral organ identity genes including PvDEF and PvGLO.     

Cloning and Characterization of Three APETALA1/FRUITFULL-like Genes in Different Flower Types of Rosa?��?hybrida L.

To clarify the molecular mechanism of flower development in Rosa × hybrida L., three different APETALA1/FRUITFULL (AP1/FUL)-like MADS-box genes were isolated and their expression analyzed in normally developed flowers and in malformed flowers of a stable phenotype. AP1/FUL-like genes were designated as RhAP1-1, RhFUL, and RhAP1-2. Alignment of amino acid sequences showed 83% identity between RhAP1-1 and TrAP1 of Taihangia rupestris and 82% identity between RhFUL and TrFUL of T. rupestris. RhAP1-1 is 97% identical to RhAP1-2 and 58% identical to RhFUL. Expression of RhAP1-1 and RhAP1-2 in whorls 1 and 2 of rose flowers exclusively is in accordance with the expression pattern of class A genes in other plant species. In contrast, RhFUL showed a unique expression pattern and was expressed only in sepals. The roles of all putative A, B, and C class genes were examined in different flower organs of normally developed flowers and in malformed flowers that are similar to a classic C function mutant from Arabidopsis (with petals in whorl 3 and sepals in whorl 4). The expression pattern of the putative class B genes was similar in both normal and malformed flowers. However, the putative class A genes were upregulated and class C genes were downregulated in all flower organs of the mutant. These data suggest that suppression of the class C genes RhC1 and RhC2 leads to altered expression of RhAP1-1, RhFUL, and RhAP1-2 in whorls 3 and 4 that leads to the mutant flower phenotype.     

Flower form alteration by genetic transformation with the class B MADS-box genes of <Emphasis Type="Italic">Agapanthus</Emphasis><Emphasis Type="Italic">praecox</Emphasis> spp. <Emphasis Type="Italic">orientalis</Emphasis> in transgenic dicot and monocot plants

The class B genes, which belong to the MADS-box gene family, play important roles in regulating petal and stamen development in flowering plants. These genes exist in two different types termed DEF- and GLO-like genes, and the B-function is provided by heterodimers of a DEF- and a GLO-like gene product. In the present study, dicot (tobacco and lettuce) and monocot (Tricyrtis hirta) plants were transformed with the GLO-like gene of Agapanthus praecox ssp. orientalis ApGLO alone or in combination with the DEF-like gene of the same plant ApDEF. In two out of 10 transgenic tobacco plants containing ApGLO, sepals partially converted into petaloid organs. For lettuce, ray florets of four out of nine transgenic plants containing ApGLO also developed additional petaloid organs. In two out of five transgenic T. hirta plants containing both ApGLO and ApDEF, organs developed in whorl 4 showed noticeable morphological alteration: they were much longer compared with carpels of non-transgenic plants, and had purple spots overall on the surface as filaments of non-transgenic plants. No morphological alterations were observed in vegetative organs between transgenic and non-transgenic plants for all the three species. The results obtained in the present study indicate a possibility of molecular breeding for flower form alteration by genetic transformation with the class B MADS-box gene(s) of heterologous plant species.     

Ectopic expression of pMADS3 in transgenic petunia phenocopies the petunia blind mutant.

We cloned a MADS-box gene, pMADS3, from Petunia hybrida, which shows high sequence homology to the Arabidopsis AGAMOUS and Antirrhinum PLENA. pMADS3 is expressed exclusively in stamens and carpels of wild-type petunia plants. In the petunia mutant blind, which shows homeotic conversions of corolla limbs into antheroid structures with pollen grains and small parts of sepals into carpelloid tissue, pMADS3 is expressed in all floral organs as well as in leaves. Ectopic expression of pMADS3 in transgenic petunia leads to phenocopies of the blind mutant, i.e., the formation of antheroid structures on limbs and carpelloid tissue on sepals. Transgenic tobacco plants that overexpress pMADS3 exhibit an even more severe phenotype, with the sepals forming a carpel-like structure encasing the interior floral organs. Our results identify BLIND as a negative regulator of pMADS3, which specifies stamens and carpels during petunia flower development.     

Pseudodiplostemony, and its implications for the evolution of the androecium in the Caryophyllaceae

The androecium of the Caryophyllaceae is varied, ranging from a two-whorled condition to a single stamen. A number of species belonging to the three subfamilies, Caryophyl-loideae, Alsinoideae and Paronychioideae have been studied ontogenetically with the SEM to understand their peculiar androecial development in the broader context of the Caryophyllales alliance. Although patterns of initiation are highly variable among species, there are three ontogenetic modes of stamen initiation: all stamens simultaneous within a whorl, the antepetalous stamens simultaneous and the antesepalous sequentially with a reversed direction, or both whorls sequentially with or without a reversed direction. The most common floral (ontogenetic) sequence of the Caryophyllaceae runs as follows: five sepals (in a 2/5 sequence), the stamens in front of the three inner sepals successively, stamens opposite the two outermost sepals, five antepetalous stamens (simultaneously or in a reversed spiral superimposed on the spiral of the antesepalous stamens), five outer sterile (petaloid) organs arising before, simultaneously or after the antesepalous stamens, often by the division of common primordia. A comparison with the floral configurations of the Phytolaccaceae and Molluginaceae indicates that the outer petaline whorl of the Caryophyllaceae corresponds positionally to the alternisepalous stamens of somePhytolacca, such asP. dodecandra. The difference withP. dodecandra lies in the fact that an extra inner or outer whorl is formed in the Caryophyl-laceae, in alternation with the sepals. A comparable arrangement exists in the Molluginaceae, though the initiation of stamens is centrifugal. A comparison of floral ontogenies and the presence of reduction series in the Caryophyllaceae support the idea that the pentamerous arrangement is derived from a trimerous prototype. Petals correspond to sterillized stamens and are comparable to two stamen pairs opposite the outer sepals and a single stamen alternating with the third and fifth sepals. Petals are often in a state of reduction; they may be confused with staminodes and they often arise from common stamenpetal primordia. The antesepalous stamen whorl represents an amalgamation of two whorls: initiation is reversed with the stamens opposite the fourth and fifth formed sepals arising before the other, while the stamens opposite the first and second formed sepals are frequently reduced or lost. Reductive trends are correlated with the mode of initiation of the androecium, as well as changes in the number of carpels, and affect the antesepalous and antepetalous whorls in different proportions. It is concluded that the androecium of the Caryophyllaceae is pseudodiplos-temonous and is not comparable to diplostemonous forms in the Dilleniidae and Rosidae. The basic floral formula of Caryophyllaceae is as follows: sepals 5—petals 5 (sterile stamens)—antesepalous stamens 3+2—antepetalous stamens 5 gynoecium 5.     

兜兰DEFICIENS(DEF)-和GLOBOSA(GLO)-like基因的克隆及表达分析

以‘同色兜兰’品种为材料,采用RT-PCR和RACE技术获得了DEFICIENS(DEF)-和GLOBOSA(GLO)-like基因的cDNA全长,命名为PcDEF和PcGLO,并用半定量RT-PCR和实时PCR研究了PcDEF和PcGLO在花芽发育过程和不同组织部位的表达特性。结果表明,PcDEF和PcGLO的全长cDNA分别为1 039bp和934bp,分别编码224和210个氨基酸;蛋白比对表明,PcDEF和PcGLO蛋白都具有典型MADS-box蛋白的MADS和K结构域;蛋白同源性分析显示,PcDEF和PcGLO与已登录的其它兰科植物的DEF/AP3和GLO/PI蛋白的相似性分别在75%～96%和87%～98%;系统进化树分析表明,PcDEF和PcGLO分别属于B类MADS-box蛋白家族的AP3和PI亚家族。表达分析显示,PcDEF和PcGLO在花芽发育中均有表达,PcDEF在成熟花、唇瓣和花瓣中的表达量高,在蕊柱、萼片、苞叶和根中次之,在花茎和叶中较低,在子房中几乎不表达;PcGLO在各组织中均有不同丰度的表达。     

Within and between Whorls: Comparative Transcriptional Profiling of Aquilegia and Arabidopsis

Background

The genus Aquilegia is an emerging model system in plant evolutionary biology predominantly because of its wide variation in floral traits and associated floral ecology. The anatomy of the Aquilegia flower is also very distinct. There are two whorls of petaloid organs, the outer whorl of sepals and the second whorl of petals that form nectar spurs, as well as a recently evolved fifth whorl of staminodia inserted between stamens and carpels.

Methodology/Principal Findings

We designed an oligonucleotide microarray based on EST sequences from a mixed tissue, normalized cDNA library of an A. formosa x A. pubescens F2 population representing 17,246 unigenes. We then used this array to analyze floral gene expression in late pre-anthesis stage floral organs from a natural A. formosa population. In particular, we tested for gene expression patterns specific to each floral whorl and to combinations of whorls that correspond to traditional and modified ABC model groupings. Similar analyses were performed on gene expression data of Arabidopsis thaliana whorls previously obtained using the Ath1 gene chips (data available through The Arabidopsis Information Resource).

Conclusions/Significance

Our comparative gene expression analyses suggest that 1) petaloid sepals and petals of A. formosa share gene expression patterns more than either have organ-specific patterns, 2) petals of A. formosa and A. thaliana may be independently derived, 3) staminodia express B and C genes similar to stamens but the staminodium genetic program has also converged on aspects of the carpel program and 4) staminodia have unique up-regulation of regulatory genes and genes that have been implicated with defense against microbial infection and herbivory. Our study also highlights the value of comparative gene expression profiling and the Aquilegia microarray in particular for the study of floral evolution and ecology.