Genetic Analysis of ele Mutants and Comparative Mapping of ele1 Locus in the Control of Organ Internal Asymmetry in Garden Pea

Previous study has shown that during zygomorphic development in garden pea(Pisum sativum L.),the organ internal(IN) asymmetry of lateral and ventral petals was regulated by a genetic locus,SYMMETRIC PETAL 1(SYP1),while the dorsoventral(DV) asymmetry was determined by two CYC-like TCP genes or the PsCYC genes,KEELED WINGS(K) and LOBED STANDARD 1(LST1).In this study,two novel loci,ELEPHANT EAR-LIKE LEAF 1(ELE1) and ELE2 were characterized.These mutants exhibit a similar defect of IN asymmetry as syp1 in lateral and ventral petals,but also display pleiotropic effects of enlarged organ size.Genetic analysis showed that ELE1 and ELE2 were involved in same genetic pathway and the enlarged size of petals but not compound leaves in ele2 was suppressed by introducing k and lst1,indicating that the enlargement of dorsal petal in ele2 requires the activities of K and LST1.An experimental framework of comparative genomic mapping approach was set up to map and clone LjELE1 locus in Lotus japonicus.Cloning the ELE1 gene will shed light on the underlying molecular mechanism during zygomorphic development and further provide the molecular basis for genetic improvement on legume crops.     

百脉根BIO和豌豆突变位点ELE2的比较基因组定位(英文)

豆科两侧对称花的花瓣具有背腹(DV)的分化以及可变的器官内部(IN)非对称性,在大小与形状上显示出不同的发育特征;因而花瓣的发育为克隆决定植物器官的形状与大小的关键基因提供了很好的实验系统。本研究对百脉根中BIO基因进行研究。百脉根bio突变体具有多效性,既影响花器官内部的对称性也影响器官的大小和育性,豌豆ele突变体的表型与bio相似。定位结果表明BIO和ELE2位于豆科基因组的共线性区段,提示BIO和ELE2可能是同源基因突变所致。本研究利用比较基因组定位方法,将BIO和ELE2候选基因锚定在豆科模式植物百脉根和蒺藜苜蓿基因组含有11个同源基因的BAC重叠群上。BIO和ELE2基因的克隆将有助于揭示豆科花瓣形态和大小调控的分子机理,进而为豆科作物遗传改良提供分子理论基础。     

LjCYC genes constitute floral dorsoventral asymmetry in Lotus japonicus

Previous study shows that LjCYC2, a CYC-like TCP (TB1, CYC and PCFs) gene in the model legume, Lotus japonicus, is involved in dorsal petal development, which together with the other two homologous genes,LjCYC1 and LjCYC3, belongs to an LjCYC gene cluster. In this report, we modified the transformation system in L. japonicus, and constructed different RNAi transgenes to target different LjCYC genes. The expression of three endogenous LjCYC genes was specifically suppressed by different specific RNAi transgenes, and a chimerical RNAi transgene that contains the specific sequences from LjCYC1 and LjCYC2 was found to downregulate the expression of both endogenous genes simultaneously. Effects of silencing three LjCYC genes were mainly restricted on either dorsal or lateral petals, demonstrating their dorsalizing and lateralizing activities during the development of zygomorphic flower. Furthermore,abolishing the expression of three LjCYC genes could give rise to complete loss of dorsoventral (DV) differentiation in the flower whose petals all resembled the ventral one in the wild type and displayed intact organ internal (IN) asymmetry. Our data demonstrate that during zygomorphic flower development, the DV asymmetry is constituted by the LjCYC genes, while the floral organ IN asymmetry is independently determined by other genetic factors.     

Evolution of petal epidermal micromorphology in Leguminosae and its use as a marker of petal identity

Background and Aims

The legume flower is highly variable in symmetry and differentiation of petal types. Most papilionoid flowers are zygomorphic with three types of petals: one dorsal, two lateral and two ventral petals. Mimosoids have radial flowers with reduced petals while caesalpinioids display a range from strongly zygomorphic to nearly radial symmetry. The aims are to characterize the petal micromorphology relative to flower morphology and evolution within the family and assess its use as a marker of petal identity (whether dorsal, lateral or ventral) as determined by the expression of developmental genes.

Methods

Petals were analysed using the scanning electron microscope and light microscope. A total of 175 species were studied representing 26 tribes and 89 genera in all three subfamilies of the Leguminosae.

Key Results

The papilionoids have the highest degree of variation of epidermal types along the dorsiventral axis within the flower. In Loteae and genistoids, in particular, it is common for each petal type to have a different major epidermal micromorphology. Papillose conical cells are mainly found on dorsal and lateral petals. Tabular rugose cells are mainly found on lateral petals and tabular flat cells are found only in ventral petals. Caesalpinioids lack strong micromorphological variation along this axis and usually have only a single major epidermal type within a flower, although the type maybe either tabular rugose cells, papillose conical cells or papillose knobby rugose cells, depending on the species.

Conclusions

Strong micromorphological variation between different petals in the flower is exclusive to the subfamily Papilionoideae. Both major and minor epidermal types can be used as micromorphological markers of petal identity, at least in papilionoids, and they are important characters of flower evolution in the whole family. The molecular developmental pathway between specific epidermal micromorphology and the expression of petal identity genes has yet to be established.Key words: Epidermis, Fabaceae, Papilionoideae, Caesalpinioideae, Mimosoideae, petal surface, scanning electron microscopy, papillose conical cells, tabular rugose cells, tabular flat cells, organ identity     

The expression of D-cyclin genes defines distinct developmental zones in snapdragon apical meristems and is locally regulated by the Cycloidea gene

Three D-cyclin genes are expressed in the apical meristems of snapdragon (Antirrhinum majus). The cyclin D1 and D3b genes are expressed throughout meristems, whereas cyclin D3a is restricted to the peripheral region of the meristem, especially the organ primordia. During floral development, cyclin D3b expression is: (a) locally modulated in the cells immediately surrounding the base of organ primordia, defining a zone between lateral organs that may act as a developmental boundary; (b) locally modulated in the ventral petals during petal folding; and (c) is specifically repressed in the dorsal stamen by the cycloidea gene. Expression of both cyclin D3 genes is reduced prior to the cessation of cell cycle activity, as judged by histone H4 expression. Expression of all three D-cyclin genes is modulated by factors that regulate plant growth, particularly sucrose and cytokinin. These observations may provide a molecular basis for understanding the local regulation of cell proliferation during plant growth and development.     

Developmental instability in Brassica campestris (Cruciferae): fluctuating asymmetry of foliar and floral traits

The degree of fluctuating asymmetry of bilateral traits provides a measure of developmental instability, which can be influenced by genetic as well as environmental stress. We studied genetic variation between and within two populations of the mustard Brassica campestris for asymmetry of foliar (cotyledon width) and floral (petal length and width) traits as well as for phenological (germination and flowering) and performance (biomass and flowering) traits. The two populations differed in mean expression of most traits, including asymmetry. However, within-population estimates of genetic variability tended to be lower for asymmetry than other traits. Asymmetry was greater in the population that had lower biomass accumulation and flower production, which supports the idea that population-level asymmetry may be indicative of population-level performance. However, within each population, evidence that performance was negatively correlated with asymmetry was equivocal. Within populations there was little or no concordance among estimates of asymmetry based on different structures, i.e., plants that had highly asymmetrical cotyledons did not tend to have highly asymmetrical petals. The lack of a general buffering capacity at the individual level may be explained by developmental processes (e.g., action of different genes or morphogens) as well as evolutionary processes (e.g., selection on asymmetry of different traits).     

Wrinkled petals and stamens 1, is required for the morphogenesis of petals and stamens in Lotus japonicus

Although much progress has been made in understanding how floral organ identity is determined during the floral development, less is known about how floral organ is elaborated in the late floral developmental stages. Here we describe a novel floral mutant, wrinkled petals and stamens1 （wps1）, which shows defects in the development of petals and stamens. Genetic analysis indicates that wpsl mutant is corresponding to a single recessive locus at the long arm of chromosome 3. The early development of floral organs in wpsl mutant is similar to that in wild type, and the malfunction of the mutant commences in late developmental stages, displaying a defect on the appearance of petals and stamens. In the mature flower, petals and stamen filaments in the mutant are wrinkled or folded, and the cellular morphology under L1 layer of petals and stamen filaments is abnormal. It is found that the expression patterns of floral organ identity genes are not affected in wpsl mutants compared with that of wild type, consistent with the unaltered development of all floral organs. Furthermore, the identities of epidermal cells in different type of petals are maintained. The histological analysis shows that in wpsl flowers all petals are irregularly folded, and there are knotted structures in the petals, while the shape and arrangement of inner cells are malformed and unorganized. Based on these results, we propose that Wpsl acts downstream to the class B floral organ identity genes, and functions to modulate the cellular differentiation during the late flower developmental stages.     

Patterns and development of floral asymmetry in Senna (Leguminosae, Cassiinae)

The buzz-pollinated genus Senna (Leguminosae) is outstanding for including species with monosymmetric flowers and species with diverse asymmetric, enantiomorphic (enantiostylous) flowers. To recognize patterns of homology, we dissected the floral symmetry character complex and explored corolla morphology in 60 Senna species and studied floral development of four enantiomorphic species. The asymmetry morph of a flower is correlated with the direction of spiral calyx aestivation. We recognized five patterns of floral asymmetry, resulting from different combinations of six structural elements: deflection of the carpel, deflection of the median abaxial stamen, deflection or modification in size of one lateral abaxial stamen, and modification in shape and size of one or both lower petals. Prominent corolla asymmetry begins in the earl-stage bud (unequal development of lower petals). Androecium asymmetry begins either in the midstage bud (unequal development of thecae in median abaxial stamen; twisting of androecium) or at anthesis (stamen deflection). Gynoecium asymmetry begins in early bud (primordium off the median plane, ventral slit laterally oriented) or midstage to late bud (carpel deflection). In enantiostylous flowers, pronouncedly concave and robust petals of both monosymmetric and asymmetric corollas likely function to ricochet and direct pollen flow during buzz pollination. Occurrence of particular combinations of structural elements of floral symmetry in the subclades is shown.     

Selection for floral integration and trait variation in zygomorphic flowers of Aconitum japonicum ssp. subcuneatum (Ranunculaceae)

Pollinator-mediated selection might lead to among-trait differences in the degree and pattern of floral integration and intra-flower variation. To examine the patterns of intra-flower variation in floral traits, including nectar volume, we performed a field study using the zygomorphic flowers of Aconitum japonicum ssp. subcuneatum. We investigated (1) correlations between the sizes of the left and right sepals and petals, (2) variation in floral traits among plants, within plants and within flowers, (3) effects of sexual phases on floral integration variation in floral and nectar traits, and (4) the effect of size and intra-flower variation in traits of the left and right sepals and petals on pollen removal by pollinators. Lateral sepal area, but not lower sepal area, was highly correlated between the left and right sepals. Floral traits were more integrated during the male phase than during the female phase. Nectar standing crop in male-phase flowers correlated with helmet height and lateral and lower sepal area, but in female-phase flowers it only correlated with spur length. While intra-flower variance in lateral sepal area accounted for approximately 10% of the overall variance in these traits, the variance in lower sepal area accounted for 70% of the overall variance. Lateral sepal area had a negative effect on the number of pollen grains remaining after pollinator visits. Low variance in lateral sepals within flowers and measurements of pollen removal suggest that lateral sepals play a more important role in pollen export than the other traits. Left and right sepals may be the targets of selection for symmetry in zygomorphic flowers.

     

PETAL LOSS gene regulates initiation and orientation of second whorl organs in the Arabidopsis flower

PETAL LOSS is a new class of flower development gene whose mutant phenotype is confined mostly to the second whorl. Two properties are disrupted, organ initiation and organ orientation. Initiation is frequently blocked, especially in later-formed flowers, or variably delayed. The few petals that arise occupy a wider zone of the flower primordium than normal. Also, a minority of petals are trumpet-shaped, thread-like or stamenoid. Studies of ptl combined with homeotic mutants have revealed that the mutant effect is specific to the second whorl, not to organs with a petal identity. We propose that the PTL gene normally promotes the induction of organ primordia in specific regions of the second floral whorl. In ptl mutants, these regions are enlarged and organ induction is variably reduced, often falling below a threshold. A dominant genetic modifier of the ptl mutant phenotype was found in the Landsberg erecta strain that significantly boosts the mean number of petals per flower, perhaps by reinforcing induction so that the threshold is now more often reached. The second major disruption in ptl mutants relates to the orientation adopted by second whorl organs from early in their development. In single mutants the full range of orientations is seen, but when B function (controlling organ identity) is also removed, most second whorl organs now face outwards rather than inwards. Orientation is unaffected in B function single mutants. Thus petals apparently perceive their orientation within the flower primordium by a mechanism requiring PTL function supported redundantly by that of B class genes.     

Effect of lateral suppressor on petal initiation in tomato

Flowers developing on tomato ( Lycopersicon esculentum ) plants homozygous for the lateral suppressor ( ls ) mutation lack petals. Scanning electron micrographs revealed that in ls plants no second whorl organs were initiated. The initiation of first, third, and fourth whorl organs were unaffected by this mutation. To investigate interactions between the cells in different layers of the floral meristem during organ initiation, a periclinal chimera between wild-type and ls tomato was generated. Flowers of the chimera having ls cells in the outer meristem layer (L1) and wild-type cells in internal layers (L2 and L3) developed normally, including the initiation of organ primordia that differentiated as petals in normal positions within the second whorl. L1 of the chimera developed in a non-autonomous manner during petal development. Thus, wild-type cells occupying the internal meristem layers provided developmental cues necessary for initiation of petal primordia at appropriate positions on the floral meristem. L1 cells carrying the lateral suppressor mutation were fully capable of responding to this information and differentiated appropriately.     

非洲紫罗兰两侧与辐射对称花中两个CYC类完整基因的分离和序列分析

在已知GCYC基因部分序列基础上, 通过改进的mTAIL-PCR方法克隆非洲紫罗兰Saintpaulia ionantha两侧对称栽培种中CYC类基因的5′未知序列, 并进而从两侧与辐射对称栽培种中分离得到苦苣苔科Gesneriaceae中第一组完整基因: SiCYC1A与SiCYC1B。对以上基因的核酸和氨基酸序列比较发现, SiCYC1A与SiCYC1B序列同源性很高, 均含有完整的功能调控区域(即TCP domain和R domain)并与模式植物金鱼草Antirrhinum majus中CYC基因同源。因此, 这两个基因应具有正常功能, 是功能上互补的冗余基因。令人意外的是在辐射对称花栽培品种中的这两个基因和两侧对称花栽培品种中对应基因的序列完全相同。经过对金鱼草以及相关类群辐射对称花突变体中CYC类基因序列的比较分析, 推论在非洲紫罗兰中, SiCYC1A与SiCYC1B基因可能受上游未知的共同调控因子调控, 该调控因子的改变是导致栽培品种中花对称性发生变化的主要原因。另外, 对改进后的TAIL-PCR(mTAIL-PCR)的方法和过程进行了详细叙述, 并对其技术特征和优势开展了简单的论述。     

Organ boundary NAC‐domain transcription factors are implicated in the evolution of petal fusion

• Research rationale: Evolution of fused petals (sympetaly) is considered to be an important innovation that has repeatedly led to increased pollination efficiency, resulting in accelerated rates of plant diversification. Although little is known about the underlying regulation of sympetaly, genetic pathways ancestrally involved in organ boundary establishment (e.g. CUP SHAPED COTYLEDON [CUC] 1–3 genes) are strong candidates. In sympetalous petunia, mutations in the CUC1/2‐like orthologue NO APICAL MERISTEM (NAM) inhibit shoot apical meristem formation. Despite this, occasional ‘escape shoots’ develop flowers with extra petals and fused inter‐floral whorl organs.
• Central methods: To To determine if petunia CUC‐like genes regulate additional floral patterning, we used virus‐induced silencing (VIGS) following establishment of healthy shoot apices to re‐examine the role of NAM in petunia petal development, and uniquely characterise the CUC3 orthologue NH16.
• Key results: Confirming previous results, we found that reduced floral NAM/NH16 expression caused increased petal–stamen and stamen–carpel fusion, and often produced extra petals. However, further to previous results, all VIGS plants infected with NAM or NH16 constructs exhibited reduced fusion in the petal whorl compared to control plants.
• Main conclusions: Together with previous data, our results demonstrate conservation of petunia CUC‐like genes in establishing inter‐floral whorl organ boundaries, as well as functional evolution to affect the fusion of petunia petals.