Ectopic Expression of an <Emphasis Type="Italic">FT</Emphasis> Homolog from <Emphasis Type="Italic">Citrus</Emphasis> Confers an Early Flowering Phenotype on Trifoliate Orange (<Emphasis Type="Italic">Poncirus trifoliata</Emphasis> L. Raf.)

Citrus FT (CiFT) cDNA, which promoted the transition from the vegetative to the reproductive phase in Arabidopsis thaliana, when constitutively expressed was introduced into trifoliate orange (Poncirus trifoliata L. Raf.). The transgenic plants in which CiFT was expressed constitutively showed early flowering, fruiting, and characteristic morphological changes. They started to flower as early as 12 weeks after transfer to a greenhouse, whereas wild-type plants usually have a long juvenile period of several years. Most of the transgenic flowers developed on leafy inflorescences, apparently in place of thorns; however, wild-type adult trifoliate orange usually develops solitary flowers in the axils of leaves. All of the transgenic lines accumulated CiFT mRNA in their shoots, but there were variations in the accumulation level. The transgenic lines showed variation in phenotypes, such as time to first flowering and tree shape. In F₁ progeny obtained by crossing ‘Kiyomi’ tangor (C. unshiu × sinensis) with the pollen of one transgenic line, extremely early flowering immediately after germination was observed. The transgene segregated in F₁ progeny in a Mendelian fashion, with complete co-segregation of the transgene and the early flowering phenotype. These results showed that constitutive expression of CiFT can reduce the generation time in trifoliate orange.     

Overexpression of <Emphasis Type="Italic">AP1-</Emphasis>like genes from <Emphasis Type="Italic">Asteraceae</Emphasis> induces early-flowering in transgenic <Emphasis Type="Italic">Chrysanthemum</Emphasis> plants

Chrysanthemum is one of the most important commercial cut flowers in the world. Early-flowering cultivars are required to produce quality chrysanthemum flowers with a lower cost of production. To shorten the vegetative growth phase of chrysanthemum, three AP1-like genes from Asteraceae were constitutively overexpressed in 80 independent transgenic chrysanthemum lines. All lines were characterized by PCR and RT-PCR and demonstrated that overexpression of compositae AP1-homologs in transgenic chrysanthemum under long-day conditions had no effect on plant development compared to non-transgenic controls. Conversely, under short-day conditions, transgenic plants commenced bud initiation 2 wk earlier than non-transgenic chrysanthemum plants. Subsequently, transgenic chrysanthemum flowers showed color earlier and resulted in full opening of inflorescences 3 wk prior to non-transgenic control plants. These results open new possibilities for genetic improvement and breeding of chrysanthemum cultivars.     

<Emphasis Type="Italic">Gemini pollen 2</Emphasis>, a male and female gametophytic cytokinesis defective mutation

Gametophytic cytokinesis is essential for the development and function of the male and female gametophytes. We have previously described the isolation and characterisation of gemini pollen 1 (gem1) that acts gametophytically to disturb asymmetric division and cytokinesis at pollen mitosis I (PMI) in Arabidopsis. Here we describe the genetic and cytological analysis of an independent gametophytic mutant, gem2, with similar characteristics to gem1, but which maps to a different genetic locus. gem2 shows reduced genetic transmission through both male and female gametes and leads to the production of divided or twin-celled pollen. Developmental analysis revealed that gem2 does not affect karyokinesis at PMI, but leads to repositioning of the cell plate, and partial or complete failure of cytokinesis, resulting in symmetrical divisions or binucleate pollen grains, respectively. Symmetrical divisions lead to altered pollen cell fate with both sister cells displaying vegetative cell fate. Moreover, we demonstrate that the predominant female defect in gem2 is a lack of cellularisation of the embryo sac during megagametogenesis. GEM2 therefore defines an independent genetic locus that is involved in the correct specification of both male and female gametophytic cytokinesis.     

Floral ontogeny of <Emphasis Type="Italic">Schisandra chinensis</Emphasis> (Schisandraceae): implications for androecial evolution within <Emphasis Type="Italic">Schisandra</Emphasis> and <Emphasis Type="Italic">Kadsura</Emphasis>

The organogenesis of staminate and carpellate flowers of Schisandra chinensis (Schisandraceae) was investigated with scanning electron microscopy, with observations on the development of tepals reported for the first time. The results showed that there is no interval between the initiation of the last tepal and that of the first stamen or carpel, and that the shapes of tepal, stamen, and carpel primordia are similar. The tepals and stamens of staminate flowers are initiated acropetally in a continuous spiral Fibonacci phyllotaxis, with no carpel structures observed; the filaments are not connate. The organogenesis of the carpellate flowers is similar to that of the staminate flowers, but with no evidence of stamen development. The carpels are ascidiate without postgenital fusion. Three androecial characters of Schisandra and Kadsura are discussed in a phylogenetic context. The subglobose or obovoid androecium of Schisandra propinqua and Schisandra plena may be homologous with that in sections Kadsura and Sarcocarpon. The plesiomorphic form of the androecium within the two genera is likely to be elongate with more than ten free stamens.     

Genetic characterization of a new cytoplasmic male sterility system (<Emphasis Type="Italic">hau</Emphasis>) in <Emphasis Type="Italic">Brassica </Emphasis><Emphasis Type="Italic">juncea</Emphasis> and its transfer to <Emphasis Type="Italic">B. napus</Emphasis>

A novel cytoplasmic male sterility (CMS) was identified in Brassica juncea, named as hau CMS (00-6-102A). Subsequently, the male sterility was transferred to B. napus by interspecific hybridization. The hau CMS has stable male sterility. Flowers on the A line are absolutely male sterile, and seeds harvested from the line following pollinations with the maintainer gave rise to 100% sterile progeny. The anthers in CMS plants are replaced by thickened petal-like structures and pollen grains were not detected. In contrast, in other CMS systems viz. pol, nap, tour, and ogu, anthers are formed but do not produce viable pollen. The sterility of hau CMS initiates at the stage of stamen primordium polarization, which is much earlier compared with the other four CMS systems. We have successfully transferred hau CMS from B. juncea to B. napus. Restorer lines for pol, ogu, nap, and tour CMS systems were found to be ineffective to restore fertility in hau CMS. Sixteen out of 40 combinations of mitochondrial probe/enzyme used for RFLP analysis distinguished the hau CMS system from the other four systems. Among these sixteen combinations, five ones alone could distinguish the five CMS systems from each other. The evidence from genetic, morphological, cytological and molecular studies confirmed that the hau CMS system is a novel CMS system. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Specialist <Emphasis Type="Italic">Osmia</Emphasis> bees forage indiscriminately among hybridizing <Emphasis Type="Italic">Balsamorhiza</Emphasis> floral hosts

Pollinators, even floral generalists (=polyleges), typically specialize during individual foraging bouts, infrequently switching between floral hosts. Such transient floral constancy restricts pollen flow, and thereby gene flow, to conspecific flowers in mixed plant communities. Where incipient flowering species meet, however, weak cross-fertility and often similar floral traits can yield mixed reproductive outcomes among pollinator-dependent species. In these cases, floral constancy by polyleges sometimes serves as an ethological mating barrier. More often, their foraging infidelities instead facilitate host introgression and hybridization. Many other bee species are oligolectic (taxonomic specialists for pollen). Oligoleges could be more discriminating connoisseurs than polyleges when foraging among their limited set of related floral hosts. If true, greater foraging constancy might ensue, contributing to positive assortative mating and disruptive selection, thereby facilitating speciation among their interfertile floral hosts. To test this Connoisseur Hypothesis, nesting females of two species of oligolectic Osmia bees were presented with randomized mixed arrays of flowers of two sympatric species of their pollen host, Balsamorhiza, a genus known for hybridization. In a closely spaced grid, the females of both species preferred the larger flowered B. macrophylla, evidence for discrimination. However, both species’ females showed no floral constancy whatsoever during their individual foraging bouts, switching randomly between species proportional to their floral preference. In a wider spaced array in which the bouquets reflected natural plant spacing, foraging oligolectic bees often transferred pollen surrogates (fluorescent powders) both between conspecific flowers (geitonogamy and xenogamy) and between the two Balsamorhiza species. The Connoisseur Hypothesis was therefore rejected. Foraging infidelity by these oligolectic Osmia bees will contribute to introgression and hybridization where interfertile species of Balsamorhiza meet and flower together. A literature review reveals that other plant genera whose species hybridize also attract numerous oligolectic bees, providing independent opportunities to test the generality of this conclusion.     

Expression of <Emphasis Type="Italic">Aprotinin</Emphasis> in Anther Causes Male Sterility in Tobacco var <Emphasis Type="Italic">Petit havana</Emphasis>

Expression of many proteinases has been documented during anther development. Although their roles are not completely understood, their inhibition could possibly result in impairment of anther development leading to male sterility. We proposed that such an impairment of anther development can be engineered in plants resulting in male sterile plants that can be used for hybrid seed production. Here, we report that anther-specific expression of Aprotinin gene (serine proteinase inhibitor) in tobacco has resulted in male sterility. Southern analysis and zymogram analysis confirmed the integration and expression of Aprotinin gene in the anthers of the transgenic plants. Transverse sections of anthers of transgenic male sterile plants showed damaged tapetum. The pollen germination in the transgenic plants ranged between 2% and 65% that confirmed the impairment in pollen production leading to male sterility and low seed yield. Thus, inhibition of serine proteinases that are expressed during anther development has resulted in impaired pollen production and male sterility, though the exact role of these proteinases in anther development still has to be elucidated.     

A loss-of-function mutation in <Emphasis Type="Italic">Calmodulin2</Emphasis> gene affects pollen germination in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Calmodulin (CAM) is an ubiquitous calcium binding protein whose function is to translate the signals, perceived as calcium concentration variations, into the appropriate cellular responses. In Arabidopsis thaliana there are 4 CAM isoforms which are highly similar, encoded by 7 genes, and one possible explanation proposed for the evolutionary conservation of the CAM gene family is that the different genes have acquired different functions so that they play possibly overlapping but non-identical roles. Here we report the characterization of the Arabidopsis mutant cam2-2, identified among the lines of the gene-trapping collection EXOTIC because of a distorted segregation of kanamycin resistance. Phenotypic analysis showed that in normal growth conditions cam2-2 plants were indistinguishable from the wild type while genetic analysis showed a reduced transmission of the cam2-2 allele through the male gametophyte and in vitro pollen germination revealed a reduced level of germination in comparison with the wild type. These results provide genetic evidence of the involvement of a CAM gene in pollen germination and support the theory of functional diversification of the CAM gene family.     

Developmental defects and seedling lethality in apyrase <Emphasis Type="Italic">AtAPY1</Emphasis> and <Emphasis Type="Italic">AtAPY2</Emphasis> double knockout mutants

Previously it was shown that the Arabidopsis apyrase genes AtAPY1 and AtAPY2 are crucial for male fertility because mutant pollen (apy1-1; apy2-1) with T-DNA insertions in both genes could not germinate (Steinebrunner et al. (2003) Plant Physiol. 131: 1638–1647). In this study, pollen germination was restored and apyrase T-DNA double knockouts (DKO) apy1-1/apy1-1; apy2-1/apy2-1 were generated by complementation with AtAPY2 under the control of a pollen-specific promoter. The DKO phenotype displayed developmental defects including the lack of functional root and shoot meristems. In cotyledons, morphogenetic and patterning abnormalities were apparent, e.g., unlobed pavement cells and stomatal clusters. Another set of lines was created which carried either AtAPY1 or AtAPY2 under a dexamethasone-(DEX)-inducible promoter as an additional transgene to the pollen-specific gene construct. Application of DEX did not reverse the DKO phenotype to wild-type, but some inducible lines exhibited less severe defects even in the absence of the inducer, probably due to some background expression. However, even these DKO mutants were seedling-lethal and shared other defects regarding cell division, cell expansion and stomatal patterning. Taken together, the defects in the DKO mutants demonstrate that AtAPY1 and AtAPY2 are essential for normal plant development.     

The arrest of development of abortive reproductive organs in the unisexual flower of<Emphasis Type="Italic"> Vitis vinifera</Emphasis> ssp.<Emphasis Type="Italic"> silvestris</Emphasis>

During the first stages of development, flowers of most dioecious species are hermaphroditic, with their transition to unisexual flowers being the result of the developmental arrest of one set of reproductive organs. In this work, we describe the development of male and female flowers of the dioecious wild grape species Vitis vinifera ssp. silvestris through scanning electron microscopy analysis and cytological observations, focusing our attention on the transition from bisexual to unisexual development. We divide floral development of the wild grape into eight stages. Differences between male and female flowers appear first at stage 6, when the style and stigma start to differentiate in female but not in male flowers. Cytological analysis of the slowly growing abortive pistil of male flowers shows that megagametophyte formation is, surprisingly, not inhibited. Instead of pistil abortion in the male flower, sexual determination is accomplished through programmed death of external nucellus cells and some layers of integumentary cells. Sterility of male structures in female flowers follows a different pattern, with microspore abnormalities evident from the time of their release from the tetrad. Sterile microspores and pollen grains in female flowers display an abnormal round shape, lacking colpi and possessing uniformly thickened cell walls that impede germination.     

Characterization of <Emphasis Type="Italic">SpAPETALA3</Emphasis> and <Emphasis Type="Italic">SpPISTILLATA</Emphasis>, B class floral identity genes in <Emphasis Type="Italic">Spinacia oleracea</Emphasis>, and their relationship to sexual dimorphism

Floral organ identity B class genes are generally recognized as being required for development of petals and stamens in angiosperm flowers. Spinach flowers are distinguished in their complete absence of petals in both sexes, and the absence of a developed stamen whorl in female flowers. As such, we hypothesized that differential expression of B class floral identity genes is integral to the sexual dimorphism in spinach flowers. We isolated two spinach orthologs of Arabidopsis B class genes by 3 and 5 RACE. Homology assignments were tested by comparisons of percent amino acid identities, searches for diagnostic consensus amino acid residues, conserved motifs, and phylogenetic groupings. In situ hybridization studies demonstrate that both spinach B class genes are expressed throughout the male floral meristem in early stages, and continue to be expressed in sepal primordia in reduced amounts at later stages of development. They are also highly expressed in the third whorl primordia when they arise and continue to be expressed in these tissues through the development of mature anthers. In contrast, neither gene can be detected in any stage in female flowers by in situ analyses, although northern blot experiments indicate low levels of SpAP3 within the inflorescence. The early, strong expressions of both B class floral identity genes in male floral primordia and their absence in female flowers demonstrate that B class gene expression precedes the origination of third whorl primordia (stamen) in males and is associated with the establishment of sexual floral dimorphism as it initiates in the first (sepal) whorl. These observations suggest that regulation of B class floral identity genes has a role in the development of sexual dimorphism and dioecy in spinach rather than being a secondary result of organ abortion.Electronic Supplementary Material Supplementary material is available for this article at Edited by G. Jürgens     

Specialist bees collect Asteraceae pollen by distinctive abdominal drumming (<Emphasis Type="Italic">Osmia</Emphasis>) or tapping (<Emphasis Type="Italic">Melissodes</Emphasis>, <Emphasis Type="Italic">Svastra</Emphasis>)

Four species of western US Osmia (3 Cephalosmia) that are Asteraceae specialists (mesoleges) were observed using a stereotypical means of collecting pollen—abdominal drumming—to gather pollen from 21 flowering species representing nine tribes of Asteraceae. Abdominal drumming is a rapid dorso-ventral motion of the female’s abdomen (467 pats/min) used to directly collect and place pollen in the bee’s ventral scopa. A co-occurring generalist, O. lignaria, never drummed Asteraceae flowers for pollen, but instead used its legs to harvest pollen. Observed drumming by several other osmiines is noted. A different pollen-harvesting behavior, abdominal tapping, is described for two eucerine bees (Melissodes agilis and Svastra obliqua), both oligolectic for the Asteraceae. The behavior also involves a dorso-ventral motion, but they tap their distal abdominal venter against disk flowers at a slower tempo (304 taps/min). These females’ distal sternites have distinctly dense and long hair brushes for acquiring pollen by this behavior. Brief accounts of similar abdominal pollen gathering behaviors by other megachilids are summarized.     

Pleiotropic effects of the<Emphasis Type="Italic"> male sterile33</Emphasis> (<Emphasis Type="Italic">ms33</Emphasis>) mutation in<Emphasis Type="Italic"> Arabidopsis</Emphasis> are associated with modifications in endogenous gibberellins,indole-3-acetic acid and abscisic acid

Earlier, we reported that mutation in the Male Sterile33 (MS33) locus in Arabidopsis thaliana causes inhibition of stamen filament growth and a defect in the maturation of pollen grains [Fei and Sawhney (1999) Physiol Plant 105:165–170; Fei and Sawhney (2001) Can J Bot 79:118–129]. Here we report that the ms33 mutant has other pleiotropic effects, including aberrant growth of all floral organs and a delay in seed germination and in flowering time. These defects could be partially or completely restored by low temperature or by exogenous gibberellin A₄ (GA₄), which in all cases was more effective than GA₃ Analysis of endogenous GAs showed that in wild type (WT) mature flowers GA₄ was the major GA, and that relative to WT the ms33 flowers had low levels of the growth active GAs, GA₁ and GA₄, and very reduced levels of GA₉, GA₂₄ and GA₁₅, precursors of GA₄. This suggests that mutation in the MS33 gene may suppress the GA biosynthetic pathway that leads to GA₄ via GA₉ and the early 13-H C₂₀ GAs. WT flowers also possessed a much higher level of indole-3-acetic acid (IAA), and a lower level of abscisic acid (ABA), relative to ms33 flowers. Low temperature induced partial restoration of male fertility in the ms33 flowers and this was associated with partial increase in GA₄. In contrast, in WT flowers GA₁ and GA₄ were very much reduced by low temperature. Low temperature also had little effect on IAA or ABA levels of ms33 flowers, but did reduce (>2-fold) IAA levels in WT flowers. The double mutants, ms33 aba1-1 (an ABA-deficient mutant), and ms33 spy-3 (a GA signal transduction mutant) had flower phenotypes similar to ms33. Together, the data suggest that the developmental defects in the ms33 mutant are unrelated to ABA levels, but may be causally associated with reduced levels of IAA, GA₁ and GA₄, compared to WT flowers.Abbreviations ABA Abscisic acid - GA Gibberellin - GC-MS-SIM Gas chromatography-mass spectrometry-selected ion monitoring - IAA Indole-3-acetic acid - ms33 Male sterile33 mutant - PP333 Paclobutrazol - WT Wild type     

Post-pollination hybridization barriers in <Emphasis Type="Italic">Nicotiana</Emphasis> section <Emphasis Type="Italic">Alatae</Emphasis>

Nicotiana section Alatae contains eight species with variable flower sizes and morphologies. Section members readily hybridize in the glasshouse, but no hybrids have been observed in natural sympatric and parapatric populations. To investigate interspecific crossing relationships with respect to mechanisms preventing hybridization, all members of section Alatae were intercrossed in a complete diallel. We found positive correlation between the pistil length of the pollen donor and interspecific seed set relative to the conspecific cross. Pollen tube growth rate and pollen donor pistil length were positively correlated as well. Furthermore, pollen from short-pistil members of section Alatae could only grow a maximum distance proportional to, but greater than, their own pistil lengths. Our results show that pollen tube growth capacity (i.e., rate and distance), provides a hybridization barrier in long-pistil species × short-pistil species crosses. We also found another hybridization barrier not specifically related to pollen tube growth capacity in short-pistil species × long-pistil species. Taken together, these barriers can generally be described by a ‘pistil-length mismatch’ rule; in section Alatae, pollen has the most success fertilizing ovules from species with pistil lengths similar to their own. This rule could contribute to hybridization barriers in Section Alatae because the species display dramatically different pistil lengths. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">dw2</Emphasis>, a New Mutation of Beet <Emphasis Type="Italic">Beta vulgaris</Emphasis> L.

Twelve dwarf plants were found in the second hybrid generation of beet. The average height of mutant plants was 21.8 cm, their leaf blades and flowers were significantly smaller than normal, and the plants exhibited male and female sterility. This dwarfism was shown to be caused by a mutation differing from that previously described in beet, which is named dwarf2 (dw2). The experimental evidence suggests that this mutation appeared in one of the first-generation plants. Based on plant phenotype in the first hybrid generation and the number of mutant plants in the second one, this mutation is suggested to be under recessive monogenic control of the dw2 gene. The genotypic class segregation in the second hybrid generation indicates that the dw2 gene is inherited independently of genes m, a1, and ap that control choricarpousness, gene male sterility, and pollen grain aggregation into tetrads.__________Translated from Genetika, Vol. 41, No. 5, 2005, pp. 657–660.Original Russian Text Copyright © 2005 by Mglinets, Osipova.