Micromorfología y ultraestructura de las anteras y los granos de polen en diez genotipos élite de Theobroma cacao (Malvaceae) / Micromorphology and ultrastructure of anthers and pollen grains in ten elite genotypes of Theobroma cacao (Malvaceae)

Resumen Introducción: A pesar de que T. cacao es una especie importante a nivel mundial por la producción de cacao, es poco lo que se conoce sobre la micromorfología y estructura de las anteras y los granos de polen. Objetivos: Describir y analizar la estructura y micromorfología de las anteras y los granos de polen de 10 genotipos élite de esta importante especie tropical. Métodos: Se tomaron más de 30 anteras de flores en antesis de los 10 genotipos élite de T. cacao del banco de germoplasma ex situ del Centro de Investigaciones Suiza-Agrosavia (Rionegro, Santander-Colombia). El material se procesó de acuerdo con los protocolos estándar para embeber y seccionar en parafina. Las secciones obtenidas (3 μm) se tiñeron con azul de Safranina-Alcian para discriminar estructuras con paredes primarias y secundarias y polifenoles totales. Además, se usó la técnica PAS-Amidoblack para diferenciar entre polisacáridos estructurales y de reserva, así como proteínas. Para la determinación de esporopolenina y polifenoles se usó la tinción azul de toluidina y finalmente para descripciones adicionales se aplicó la tinción azul alcián-PAS-hematoxilina. Las observaciones se realizaron mediante microscopio fotónico y microscopio de epifluorescencia. Para la observación con microscopía electrónica de barrido (MEB), las anteras con los granos de polen se fijaron y deshidrataron en 2.2 dimetoxipropano, luego se desecaron hasta un punto crítico y finalmente se recubrieron con oro. Resultados: Las anteras son bitecas y están sostenidas por un largo filamento formado por un estrato epidérmico, tejido parenquimatoso y un haz vascular. La dehiscencia ocurre longitudinalmente a través del estomio. La pared de la antera madura está formada por una capa epidérmica monoestratificada, una capa de células endoteliales con engrosamientos fibrilares lignificados y se pueden apreciar restos celulares del tapete y abundantes orbículas recubriendo la cavidad de los microesporangios. Los tejidos epidérmicos y parenquimatosos de las anteras almacenan polifenoles. Las orbículas son generalmente esféricas, psiladas y exhiben las mismas reacciones de tinción y fluorescencia que la exina de los granos de polen. Los granos de polen son mónades, isopolares, pequeños (16-19 µm) con amb circular, esferoidales, tricolpados con colpos medianos o cortos (5-10 µm) con membrana ornamentada, semitectatos, reticulados, heterobrochados, las paredes del retículo ornamentadas o no, con microgránulos de diferente tamaño o escabrados. Los análisis estadísticos mostraron que existen diferencias significativas en el tamaño de los granos de polen (P ˂ 0.05). Se observa que los granos de polen más pequeños son los del genotipo TCS 19 (16.890 µm) y se diferencian del resto de genotipos, y entre estos no se observan diferencias significativas. Solo dos genotipos (SCC 19 y SCA 6) presentaron polenkit y solo uno tiene paredes perforadas (SCA 6). Conclusiones: La estructura y micromorfología de las anteras de T. cacao son similares a las descritas para otras Malvaceae. Así mismo, los granos de polen mostraron variaciones de tamaño, ornamentación de las paredes y del lumen del retículo y presencia de polenkit. Sin embargo, no se observó relación entre las variaciones de los caracteres micromorfológicos analizados en los granos de polen y los modelos de compatibilidad polínica reportados para estos genotipos.

Abstract Introduction: Despite the fact that T. cacao is an important species worldwide for cocoa production, little is known about the micromorphology and structure of anthers and pollen grains. Objectives: To describe and analyze the structure and micromorphology of the anthers and pollen grains of 10 elite genotypes of this important tropical species. Methods: More than 30 anthers of flowers in anthesis were taken of the 10 elite genotypes of T. cacao from the ex situ germplasm bank of the Suiza-Agrosavia Research Center (Rionegro, Santander-Colombia). The anthers with the pollen grains were fixated and processed according to the standard protocols for embedding and sectioning in paraffin. Sections obtained (3 μm thick) were stained with Safranin-Alcian blue to discriminate structures with primary and secondary walls and total polyphenols. Additionally, the samples were also stained with the PAS-Amidoblack technique was used to differentiate between structural and reserve polysaccharides as well as proteins. Toluidine blue staining was used for the determination of sporopollenin and polyphenols and finally Alcian blue-PAS-Hematoxylin staining was applied for additional descriptions. Observations were made using photonic microscopy and epifluorescence microscopy. For observation with scanning electron microscopy (SEM) the anthers with the pollen grains were fixed and dehydrated in 2.2 Dimethoxypropane, then desiccated to critical point and finally coated with gold. Results: The anthers are dithecal and supported by a long filament made up of an epidermal stratum, parenchymal tissue, and a vascular bundle. The dehiscence occurs longitudinally through the stomium. The anther wall is made up of a monostratified epidermal layer, followed by a layer of endothecial cells with lignified fibrillar thickenings, cellular remnants of tapetum and abundant orbicules can be seen covering the cavity of the microsporangia. The epidermal and parenchymal tissues of the anthers are abundant in polyphenols. Orbicules are generally spherical, psilated, and these exhibit the same staining and fluorescence reactions as exine from pollen grains. The pollen grains are monades, isopolar, small (16-19 µm) with circular amb, spheroidal, tricolpate with medium or short colpi (5-10 µm) with sculptured membrane, semitectate, reticulated, heterobrochate, sculptured or non- sculptured walls, with microgranules of different size or scabrate. The statistical analyzes showed that there are significant differences in the size of the pollen grains (P ˂ 0.05). It is observed that the smallest pollen grains are those of the TCS 19 genotype (16.890 µm) and are different from the other genotypes, and among these there are no significant differences. Only two genotypes (SCC 19 and SCA 6) showed pollenkit and only one has perforated walls (SCA 6). Conclusions: The structure and micromorphology of the anthers of T. cacao are similar to those described for other Malvaceae. Likewise, the pollen grains showed variations in size, ornamentation of the sporoderm and the lumen of the reticulum and the presence of pollenkitt. However, no relationship was observed between the micromorphological characters analyzed in the pollen grains and the pollen compatibility models reported for these genotypes.

Pollen resources partitioning of stingless bees (Hymenoptera: Apidae) from the southern Atlantic forest

Meliponinae eusocial bees are among the most important pollinators in the Neotropics and their beekeeping has been growing as both recreational and economic activity. Information on the pollen preferences and niche overlap among species coexisting in the same location is still scarce. This study focused on the use of pollen resources by three native species of Meliponinae in the Atlantic Forest, Melipona quadrifasciata quadrifasciata (Lepeletier 1836), Scaptotrigona depilis (Moure 1942), and Tetragonisca angustula (Latreille 1811), at two different sites. The use of pollen resources was evaluated from reserves inside hives collected monthly between February 2015 and January 2016. It was also estimated the Mean Minimal Distance (Dm) traveled by the bees to obtain pollen. The bees differed regarding the diversity of pollen types, Dm, and proportion of pollen from different plant life-form. Local factor promoted differences only for Dm. M. q. quadrifasciata foraged a low diversity of pollen species and traveled greater distances with a higher proportion of trees plus palm species. Tetragonisca angustula foraged a higher diversity of pollen and flew shorter distances with a lower proportion of trees and palms. The species factor predominates over the local factor in the clustering pattern, based on pollen profile similarity.

Nucleus- and plastid-targeted annexin 5 promotes reproductive development in Arabidopsis and is essential for pollen and embryo formation.

BACKGROUND: Pollen development is a strictly controlled post-meiotic process during which microspores differentiate into microgametophytes and profound structural and functional changes occur in organelles. Annexin 5 is a calcium- and lipid-binding protein that is highly expressed in pollen grains and regulates pollen development and physiology. To gain further insights into the role of ANN5 in Arabidopsis development, we performed detailed phenotypic characterization of Arabidopsis plants with modified ANN5 levels. In addition, interaction partners and subcellular localization of ANN5 were analyzed to investigate potential functions of ANN5 at cellular level. RESULTS: Here, we report that RNAi-mediated suppression of ANN5 results in formation of smaller pollen grains, enhanced pollen lethality, and delayed pollen tube growth. ANN5 RNAi knockdown plants also displayed aberrant development during the transition from the vegetative to generative phase and during embryogenesis, reflected by delayed bolting time and reduced embryo size, respectively. At the subcellular level, ANN5 was delivered to the nucleus, nucleolus, and cytoplasm, and was frequently localized in plastid nucleoids, suggesting a likely role in interorganellar communication. Furthermore, ANN5-YFP co-immunoprecipitated with RABE1b, a putative GTPase, and interaction in planta was confirmed in plastidial nucleoids using FLIM-FRET analysis. CONCLUSIONS: Our findings let us to propose that ANN5 influences basal cell homeostasis via modulation of plastid activity during pollen maturation. We hypothesize that the role of ANN5 is to orchestrate the plastidial and nuclear genome activities via protein-protein interactions however not only in maturing pollen but also during the transition from the vegetative to the generative growth and seed development.

Palyno-morphological characteristics of gymnosperm flora of pakistan and its taxonomic implications with LM and SEM methods.

The present study is intended to assess gymnosperms pollen flora of Pakistan using Light Microscope (LM) and Scanning Electron Microscopy (SEM) for its taxonomic significance in identification of gymnosperms. Pollens of 35 gymnosperm species (12 genera and five families) were collected from its various distributional sites of gymnosperms in Pakistan. LM and SEM were used to investigate different palyno-morphological characteristics. Five pollen types (i.e., Inaperturate, Monolete, Monoporate, Vesiculate-bisaccate and Polyplicate) were observed. Six In equatorial view seven types of pollens were observed, in which ten species were sub-angular, nine species were Traingular, six species were Perprolate, three species were Rhomboidal, three species were semi-angular, two species were rectangular and two species were prolate. While five types of pollen were observed in polar view, in which ten species were Spheroidal, nine species were Angular, eight were Interlobate, six species were Circular, two species were Elliptic. Eighteen species has rugulate and 17 species has faveolate ornamentation. Eighteen species has verrucate and 17 have gemmate type sculpturing. The data was analysed through cluster analysis. The study showed that these palyno-morphological features have significance value in classification and identification of gymnosperms. Based on these different palyno-morphological features, a taxonomic key was proposed for the accurate and fast identifications of gymnosperms from Pakistan.

A living fossil Tetrapus fig wasp (Hymenoptera: Agaoninae) developing in extant Neotropical fig species (Moraceae: Ficus, section Pharmacosycea ) / Un fósil viviente de avispas de los higos (Hymenoptera, Agaoninae) que se desarrollan en especies de higos (Moraceae) existentes en el Neotrópico

Abstract:It has been assumed that Tetrapus female wasps (Agaonidae s.s.), the pollinators of the figs of the New World, section Pharmacosycea, are mainly characterized by the presence of one mandibular appendange only, and that it is the most ancestral clade of extant Agaonidae s.s., and the males are tetrapodous. The main objective of this work was to study five Tetrapus morphotypes, whose females have two mandibular appendages and the males are hexapodous, their fig host association and phylogenetic position to the family Agaonidae. The question of which group of fig pollinating wasps and associated figs are the sister to the rest of the pollinating agaonids, and figs respectively remain open. I report a group of New World extant Tetrapus morphotypes (Agaonidae: Agaoninae) provisionally assigned to Hexapus subg. nov. in preparation. Currently, Tetrapus appears as the sister taxon to all other fig pollinating taxa. Howeveer, morphologically, ecologically, geographically and historically Hexapus seems to be the ancestral clade of the extant Agaoninae. Hexapus morphotypes develop in fig species of subsection Petenenses (section Pharmacosycea). In the known extant Tetrapus, the females have one mandibular appendage and the males have reduced one or two short-lobe atrophied non-functional midlegs (tetrapodous). Hexapus females have two free mandibular appendages, and the males have five segmented functional mid-legs (hexapodous). Molecularly Hexapus seems to be the ancestral clade of extant Agaoninae; e.g., a Tetrapus sp. of Ficus crassivenosa was placed by other author as the ancestral clade of 101 wasp species, representing 19 worldwide Agaoninae genera, including four Tetrapus species. In Tetrapus sp. of Ficus crassivenosa, the female has two mandibular appendages and the male is hexapodous. The females of T. apopnus and T. delclosi, preserved in Early to mid-Miocene amber from the Dominican Republic, also have two mandibular appendages and the general morphology of extant Hexapus. I suggest that Hexapus stands up as a living fossil and the sister clade of Tetrapus s.s. The presence of extant Hexapus; as well as extant Tetrapus and their fig host species, especially in South America supports a Southern Gondwanaland origin for both of them, but not a trans-Pacific migrating connection with tropical America for section Pharmacosycea (the host of Tetrapus and Hexapus), a long oceanic dispersal, high levels of stem extinction of Tetrapus or Atlantic land connections, as proposed by other authors. However, lastly it has been assumed that figs and their pollinators arose simultaneously in Eurasia during early Tertiary and spread southwards from it. Most of the morphological and molecular studies of the fig biology of Agaoninae and Ficus, did not include Hexapus morphotypes and their fig hosts, and assumed that Tetrapus is the most ancestral clade of the extant fig pollinating wasps. Rev. Biol. Trop. 64 (4): 1721-1735. Epub 2016 December 01.

Resumen:Se ha asumido que las hembras de las avispas Tetrapus (Agaonidae s.s.), las polinizadoras de los higos de la sección Pharmacosycea del Nuevo Mundo, se caracterizan principalmente por la presencia de un solo apéndice mandibular, que es el clado más ancestral de los Agaonidae s.s. existentes y los machos son tetrápodos. El principal objetivo de este trabajo fue estudiar algunos morfotipos de avispas Tetrapus, cuyas hembras poseen dos apéndices mandibulares y los machos tres pares de patas funcionales (hexápodos); su asociación con sus higos hospederos y la posición filogenética en la familia Agaonidae. Cuestiono cuál grupo de avispas polinizadoras de los higos, constituye el grupo hermano de otras avispas polinizadoras de los higos (Agaonidae). Informo sobre un grupo de avispas (Tetrapus: Agaoninae) existentes, provisionalmente asignado a Hexapus subg. nov. en preparación. Morfológica, ecológica, geográfica e históricamente, Hexapus parece ser el clado ancestral de los polinizadores de higos Agaoninae existentes. Los morfotipos de Hexapus se desarrollan en especies de higos de la subsección Petenenses (sección Pharmacosycea). Las hembras de Tetrapus existentes tienen únicamente un apéndice mandibular y los machos tienen uno o dos lóbulos cortos, en lugar de las patas medias y por lo tanto no son funcionales (tetrápodos). Las hembras de Hexapus tienen dos apéndices mandibulares libres y los machos tienen patas medias funcionales con cinco segmentos (hexápodos). Molecularmente, Hexapus parece ser el clado ancestral de los Agaoninae existentes; por ejemplo, una especie de Tetrapus de Ficus crassivenosa fue colocado por otros autores como el clado ancestral de 101 especies de avispas que representan 19 géneros de Agaoninae mundiales, incluidas cuatro especies de Tetrapus. En Tetrapus sp. de Ficus crassivenosa, la hembra tiene dos apéndices mandibulares y el macho es hexápodo. Las hembras de T. apopnus y T. delclosi, descritas del Mioceno Temprano (Burdigaliense), conservadas en ámbar de la República Dominicana, también tienen dos apéndices mandibulares y la morfología general de Hexapus existente. El autor sugiere que Hexapus representa un fósil viviente y es un clado hermano de Tetrapus s.s. La presencia de Hexapus y Tetrapus actuales, y sus higos hospederos, especialmente en Suramérica, sugieren que tienen un origen en la Gondwana meridional del Cretácico. La presencia de Hexapus y Tetrapus en el Nuevo Mundo, no parece apoyar una conexión migratoria a través del Pacífico con América tropical, para los higos de la sección Pharmacosycea (los hospederos de Tetrapus y Hexapus), una amplia dispersión transoceánica, altos niveles de extinción básica de Tetrapus y conexiones terrestres a través del Atlántico, propuesto por otros autores. Sin embargo, últimamente se ha aceptado que los higos y sus polinizadores se originaron simultáneamente en Eurasia, durante el Terciario Primario y se diseminaron hacia el sur. La mayoría de estudios morfológicos y moleculares de biólogos de los Agaoninae y Ficus, no incluyeron morfotipos de Hexapus, o sus especies hospederas de Ficus y aceptadaron que Tetrapus es el clado más ancestral de las avispas polinizadoras de los higos existentes.

Targeted expression of cystatin restores fertility in cysteine protease induced male sterile tobacco plants.

Fertility restoration in male sterile plants is an essential requirement for their utilization in hybrid seed production. In an earlier investigation, we have demonstrated that the targeted expression of a cysteine protease in tapetal cell layer resulted in complete male sterility in tobacco transgenic plants. In the present investigation, we have used a cystatin gene, which encodes for a cysteine protease inhibitor, from a wild peanut, Arachis diogoi and developed a plant gene based restoration system for cysteine protease induced male sterile transgenic tobacco plants. We confirmed the interaction between the cysteine protease and a cystatin of the wild peanut, A. diogoi through in silico modeling and yeast two-hybrid assay. Pollen from primary transgenic tobacco plants expressing cystatin gene under the tapetum specific promoter- TA29 restored fertility on cysteine protease induced male sterile tobacco plants developed earlier. This has confirmed the in vivo interaction of cysteine protease and cystatin in the tapetal cells, and the inactivation of cysteine protease and modulation of its negative effects on pollen fertility. Both the cysteine protease and cystatin genes are of plant origin in contrast to the analogous barnase-barstar system that deploys genes of prokaryotic origin. Because of the deployment of genes of plant origin, this system might not face biosafety problems in developing hybrids in food crops.

Pollen morphology of Carapa species (Meliaceae) from the Brazilian Amazon / Morfologia polínica de Carapa (Meliaceae) espécies da Amazônia brasileira

The crabwood (Carapa) represents an important source of natural resources. The objective of this work was to study the pollen morphology of the three species of Carapa (Meliaceae) registered in the Brazilian Amazon. The pollen grains were obtained from floral buds, pistillate and staminate flowers of Carapa guianensis, C. surinamensis and C. vasquezii. In order to explore the taxonomic implications of the pollen study for these species of Carapa, lactic acetolysis method was used for light microscopy study. Furthermore, the non-acetolyzed material was used in the study of the scanning electron microscopy. It was found that the three Carapa species present little variations in pollen morphology. Compared to the other species this study, Carapa vasquezii has higher values for the length and width of endoaperture; however, it has lower values for polar and equatorial diameter, equatorial diameter in polar view and apocolpium side.

A andiroba (Carapa) representa uma importante fonte de recursos naturais. O objetivo deste trabalho foi estudar a morfologia polínica das três espécies de Carapa (Meliaceae) registradas na Amazônia brasileira. Os grãos de pólen foram obtidos de botões florais, flores pistiladas e estaminadas de Carapa guianensis, C. surinamensis e C. vasquezii. A fim de explorar as implicações taxonômicas do estudo de pólen para estas espécies de Carapa, o material foi preparado de acordo com o método de acetólise láctico, permitindo a utilização de estudos de microscopia de luz. Além disso, o material não-acetolisado foi utilizado no estudo da microscopia eletrônica de varredura. O estudo mostra que as três espécies de Carapa apresentam pequenas variações na morfologia polínica. Comparada com as outras espécies deste estudo, Carapa vasquezii têm valores mais elevados para o comprimento e largura da endoabertura, entretanto, tem valores inferiores para diâmetro polar, equatorial, diâmetro equatorial em vista polar e lado do apocolpo.

Structural and developmental variability in the female gametophyte of Griffithella hookeriana, Polypleurum stylosum, and Zeylanidium lichenoides and its bearing on the occurrence of single fertilization in Podostemaceae.

Angiosperms are characterized by the phenomenon of double fertilization with Podostemaceae as an exception that appears to extend to the entire family. Our earlier work demonstrated the cause of failure of double fertilization and ascertained the occurrence of single fertilization in Dalzellia zeylanica (Tristichoideae, Podostemaceae). In continuation with this work, three more members, i.e., Griffithella hookeriana (Tul.) Warming, Polypleurum stylosum (Wight) Hall, and Zeylanidium lichenoides (Kurz) Engl. (Podostemoideae), have been investigated in the present work. We studied the ontogenetic development of female gametophyte and tracked the path of the two sperm cells from the time of their formation in the pollen tube through their entry into the synergid and gamete fusion. We report the occurrence of a remarkably reduced 3-nucleate, 3-celled mature female gametophyte consisting of an egg cell and two synergids in all the three genera. Interestingly, the central cell is formed during female gametophyte development, but exhibits a species-specific, limited life span, and eventually degenerates prior to the entry of the pollen tube into the synergid, resulting in a failure of double fertilization. Sperm dimorphism on the basis of fluorochrome stainability has been recorded in Z. lichenoides. Further, morphogenetic constraints on the part of male (sperm selection, functional reductionism) and female gametophyte (structural reductionism, inaccessibility of central cell) presumably ensure the failure of double fertilization in these species. Thus, loss of double fertilization in this family is likely a derived condition.

Pollen morphology of Vochysiaceae tree species in the State of Santa Catarina, Southern Brazil / Morfología del polen de las especies de árboles de Vochysiaceae en el estado de Santa Catarina, sur de Brasil

Tropical Vochysiaceae includes mainly trees, and also shrubs and subshrubs. Three genera and seven species are present in the Brazilian state of Santa Catarina. The pollen morphology of six species of trees, belonging to three genera of the Vochysiaceae A. St-Hil. family, was studied. Herbaria samples were obtained, processed and treated by standard methods. The pollen grain morphology of Callisthene, Qualea and Vochysia is distinct. Medium sized pollen grains occur in Vochysia species, and small ones in Callisthene and Qualea. Specific characteristics were considered at species level [C. castellanosii H. F. Martins, C. kuhlmannii H. F.Martins, Qualea cordata Spreng var. cordata, Q. cryptantha (Spreng) Warm. var. cryptantha, Vochysia magnifica Warm. and V. tucanorum Mart.]. The presence of a fastigium (vestibulum) and a thin space devoid of nexine fixing the boundary of the apertural area is characteristic of Qualea and Vochysia species only. Rev. Biol. Trop. 62 (3): 1209-1215. Epub 2014 September 01.

Las Vochysiaceae tropicales incluyen principalmente árboles, arbustos y subarbustos. Tres géneros y siete especies están presentes en el estado brasileño de Santa Catarina. La morfología del polen de las seis especies de árboles, pertenecientes a tres géneros de la Vochysiaceae A. St.- Hil . familia, fue considerado en el presente trabajo. La morfología de los granos de polen de Callisthene Mart., Qualea Aubl. y Vochysia (Aubl.) Juss. es distinta. Los granos de polen de tamaño medio se producen en las especies de Vochysia y pequeños en Callisthene y Qualea. Características particulares fueron considerados a nivel de especie [C. castellanosii H.F. Martins, C. kuhlmannii H.F. Martins, Qualea cordata Spreng var. cordata, Q. cryptantha (Spreng) Warm. var. cryptantha, Vochysia magnifica Warm. and V. tucanorum Mart.]. La presencia de un fastigium (vestibulum) y de un espacio delgado que carece de nexina limita la zona apertural y es característica solo de las especies de Qualea y Vochysia.

Aberrant Classopollis pollen reveals evidence for unreduced (2n) pollen in the conifer family Cheirolepidiaceae during the Triassic-Jurassic transition.

Polyploidy (or whole-genome doubling) is a key mechanism for plant speciation leading to new evolutionary lineages. Several lines of evidence show that most species among flowering plants had polyploidy ancestry, but it is virtually unknown for conifers. Here, we study variability in pollen tetrad morphology and the size of the conifer pollen type Classopollis extracted from sediments of the Triassic-Jurassic transition, 200 Ma. Classopollis producing Cheirolepidiaceae were one of the most dominant and diverse groups of conifers during the Mesozoic. We show that aberrant pollen Classopollis tetrads, triads and dyads, and the large variation in pollen size indicates the presence of unreduced (2n) pollen, which is one of the main mechanisms in modern polyploid formation. Polyploid speciation may explain the high variability of growth forms and adaptation of these conifers to different environments and their resistance to extreme growth conditions. We suggest that polyploidy may have also reduced the extinction risk of these conifers during the End-Triassic biotic crisis.

A multivariate analysis of variation in genome size and endoreduplication in angiosperms reveals strong phylogenetic signal and association with phenotypic traits.

Genome size (C-value) and endopolyploidy (endoreduplication index, EI) are known to correlate with various morphological and ecological traits, in addition to phylogenetic placement. A phylogenetically controlled multivariate analysis was used to explore the relationships between DNA content and phenotype in angiosperms. Seeds from 41 angiosperm species (17 families) were grown in a common glasshouse experiment. Genome size (2C-value and 1Cx-value) and EI (in four tissues: leaf, stem, root, petal) were determined using flow cytometry. The phylogenetic signal was calculated for each measure of DNA content, and phylogenetic canonical correlation analysis (PCCA) explored how the variation in genome size and EI was correlated with 18 morphological and ecological traits. Phylogenetic signal (λ) was strongest for EI in all tissues, and λ was stronger for the 2C-value than the 1Cx-value. PCCA revealed that EI was correlated with pollen length, stem height, seed mass, dispersal mechanism, arbuscular mycorrhizal association, life history and flowering time, and EI and genome size were both correlated with stem height and life history. PCCA provided an effective way to explore multiple factors of DNA content variation and phenotypic traits in a phylogenetic context. Traits that were correlated significantly with DNA content were linked to plant competitive ability.

Evidence of 2n microspore production in a natural diploid population of Turnera sidoides subsp. carnea and its relevance in the evolution of the T. sidoides (Turneraceae) autopolyploid complex.

Turnera sidoides is a complex of distylous perennial rhizomatous herbs that includes five subspecies. Since polyploidy has played a prominent role within this species (x = 7), ongoing studies in T. sidoides are focused on the understanding of the mechanisms involved in the origin and the establishment of polyploids. Therefore, aiming to contribute to the understanding of the mode of polyploid formation, in this study we investigated the frequency of unreduced microspores in a natural diploid population of T. sidoides subsp. carnea by analyzing the size range of pollen and the constitution of the sporads. The results showed that some of the individuals studied produced 2n and 4n microspores, both in short- and long-styled floral morphs. The analysis performed documents the production of unreduced microspores in T. sidoides subsp. carnea and provides evidence that support the hypothesis of sexual polyploidization as one of the most probable mechanisms involved in the origin of polyploids within this species complex. The role of unreduced pollen in the establishment and persistence of newly formed polyploids in diploid populations of T. sidoides is also discussed.

Characterization of monofloral honeys with multivariate analysis of their chemical profile and antioxidant activity.

UNLABELLED: Various bioactive chemical constituents were quantified for 21 honey samples obtained at Rio de Janeiro and Minas Gerais, Brazil. To evaluate their antioxidant activity, 3 different methods were used: the ferric reducing antioxidant power, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, and the 2,2'-azinobis (3-ethylbenzothiazolin)-6-sulfonate (ABTS) assays. Correlations between the parameters were statistically significant (-0.6684 ≤ r ≤-0.8410, P < 0.05). Principal component analysis showed that honey samples from the same floral origins had more similar profiles, which made it possible to group the eucalyptus, morrão de candeia, and cambara honey samples in 3 distinct areas, while cluster analysis could separate the artificial honey from the floral honeys. PRACTICAL APPLICATION: This research might aid in the discrimination of honey floral origin, by using simple analytical methods in association with multivariate analysis, which could also show a great difference among floral honeys and artificial honey, indicating a possible way to help with the identification of artificial honeys.

Equilibrative nucleoside transporter 1 (ENT1) is critical for pollen germination and vegetative growth in Arabidopsis.

ENT1 of Arabidopsis thaliana was the first member of the equilibrative nucleoside transporter (ENT) family to be identified in plants and characterized as a cellular, high-affinity nucleoside importer. Evidence is presented here for a tonoplast localization of ENT1 based on proteome data and Western blot analyses. Increased export of adenosine from reconstituted tonoplast preparations from 35S:ENT1 mutants compared with those from the wild type and ENT1-RNAi mutants support this view. Furthermore, increased vacuolar adenosine and vacuolar 2'3'-cAMP (an intermediate of RNA catabolism) contents in ENT1-RNAi mutants, but decreased contents of these metabolites in 35S:ENT1 over-expresser mutants, were observed. An up-regulation of the salvage pathway was detected in the latter mutants, leading to the conclusion that draining the vacuolar adenosine storage by ENT1 over-expression interferes with cellular nucleotide metabolism. As a consequence of the observed metabolic alterations 35S:ENT1 over-expresser mutants exhibited a smaller phenotypic appearance compared with wild-type plants. In addition, ENT1:RNAi mutants exhibited significantly lower in vitro germination of pollen and contained reduced internal and external ATP levels. This indicates that ENT1-mediated nucleosides, especially adenosine transport, is important for nucleotide metabolism, thus influencing growth and pollen germination.

The phosphomimetic mutation of an evolutionarily conserved serine residue affects the signaling properties of Rho of plants (ROPs).

Plant ROP (Rho of plants) proteins form a unique subgroup within the family of Rho-type small G-proteins of eukaryotes. In this paper we demonstrate that the phosphomimetic mutation of a serine residue conserved in all Rho proteins affects the signaling properties of plant ROPs. We found that the S74E mutation in Medicago ROP6 and Arabidopsis ROP4 prevented the binding of these proteins to their plant-specific upstream activator the plant-specific ROP nucleotide exchanger (PRONE)-domain-containing RopGEF (guanine nucleotide exchange factor) protein and abolished the PRONE-mediated nucleotide exchange reaction in vitro. Structural modeling supported the hypothesis that potential phosphorylation of the S74 residue interferes with the binding of the PRONE-domain to the adjacent plant-specific R76 residue which plays an important role in functional ROP-PRONE interaction. Moreover, we show that while the binding of constitutively active MsROP6 to the effector protein RIC (ROP-interactive CRIB-motif-containing protein) was not affected by the S74E mutation, the capability of this mutated protein to bind and activate the RRK1 kinase in vitro was reduced. These observations are in agreement with the morphology of tobacco pollen tubes expressing mutant forms of yellow fluorescent protein (YFP):MsROP6. The S74E mutation in MsROP6 had no influence on pollen tube morphology and attenuated the phenotype of a constitutively active form of MsROP6. The presented Medicago and Arabidopsis data support the notion that the phosphorylation of the serine residue in ROPs corresponding to S74 in Medicago ROP6 could be a general principle for regulating ROP activation and signaling in plants.

The Arabidopsis mutant jason produces unreduced first division restitution male gametes through a parallel/fused spindle mechanism in meiosis II.

In plants, whole-genome doubling (polyploidization) is a widely occurring process largely contributing to plant evolution and diversification. The generation and fusion of diploid gametes is now considered the major route of plant polyploidization. The parallel arrangement or fusion of meiosis II MII spindles (ps) is one of the most frequently reported mechanisms generating triploid offspring. Through a forward genetics screen of an Arabidopsis (Arabidopsis thaliana) ethyl methanesulfonate population, we identified Arabidopsis thaliana Parallel Spindles1 (AtPS1), which was recently reported as a major gene implicated in the control of the ps meiotic defect. In addition, we describe the isolation and characterization of a novel allele of JASON, involved in male gametophytic ploidy regulation in plants. Similar to atps1 mutants, jason produces more than 25% 2n pollen grains and spontaneously forms triploid offspring. By combining both cytological and genetic approaches, we demonstrate that loss of JASON causes the formation of parallel arranged and fused spindles in male MII, resulting in the production of unreduced first division restitution 2n spores. Although JASON encodes a protein of unknown function, we additionally show that the meiotic ps defect in jason is caused by a reduction in AtPS1 transcript levels, indicating that JASON positively regulates AtPS1 expression, allowing the proper organization and orientation of metaphase II spindle plates in MII.

Morfología del polen en especies de Canna (Cannaceae) y su implicancia sistemática

Pollen morphology in species of Canna (Cannaceae), and systematics implications. The morphology of pollen grains of eight taxa of Canna, C. ascendens, C. coccinea, C. compacta, C. glauca, C. indica, C. paniculata, C. variegatifolia and C. fuchsina, an unpublished new species, were studied using light and scanning electronic microscopes. We used the Wodehouse technique on samples of 20 grains per specimen to measure the intine with a light microscope; and the density of spines (in 400μm2 fields) with scanning electronic microscopy. Pollen grains are spherical, echinate, omniaperturate. The sporoderm presents a very thin exine covering a thicker intine. Corrugate micro-perforate, sub-reticulate, rugate, rugulate, striate to folded, micro-striate, micro-granulate, and smooth types of the external surface of the sporoderm were found. The spines consist of exine, partially to completely covered by tryphine. The two-layered intine is the thicker part of the wall. Echinate ornamentation is a generic character in Canna, but size, surface and color of pollen walls, and density and shape of spines, are diagnostic for species. Pollen morphology supports the view of C. indica and C. coccinea as different species. Canna fuchsina grows in wild, dense colonies, in humid riverside forests from Buenos Aires and Santa Fe Provinces, Argentina; its characters suggest relationships with a not well known group of taxa, some of them hybrids, such as C. x generalis. However, as these plants showed normal, well formed grains, close to those of C. coccinea, that germinate over the stigmatic surfaces in fresh flowers, we decided to include their pollen in this study. Rev. Biol. Trop. 58 (1): 63-79. Epub 2010 March 01.

Se estudió la morfología de los granos de polen de ocho táxones de Canna, C. ascendens, C. coccinea, C. compacta, C. glauca, C. indica, C. paniculata, C. variegatifolia y C. fuchsina, nueva especie aún no descrita, que fueron estudiadas usando microscopio de luz y microscopio electrónico de barrido. Nosotros utilizamos la técnica de Wodehouse en muestras de 20 granos por espécimen para medir la intina con el microscopio de luz; y la densidad de espinas (en campos de 400μm2) con el microscopio electrσnico de barrido. Los granos de polen son esfιricos, equinados y omniaberturados. El esporodermo presenta una exina muy delgada cubriendo una intina gruesa. La superficie del esporodermo puede ser corrugada, microperforada, sub-reticulada, rugada, rugulada, plegada-estriada, micro-estriada, microgranulada o lisa. Las espinas están formadas por exina, cubiertas total o parcialmente por trifina. La intina esta formada por dos capas, es la parte más gruesa de la pared. La ornamentación equinada es un rasgo genérico en Canna, pero el tamaño, la superficie y el color del polen, y la densidad y forma de las espinas, son rasgos diagnósticos de las especies. La morfología del polen apoya el tratamiento de C. coccinea y C. indica como especies diferentes. Canna fuchsina crece formando densas colonias silvestres en selvas ribereñas húmedas de las provincias de Buenos Aires y Santa Fe, Argentina; sus características sugieren relaciones no muy bien entendidas en el grupo de taxones, algunos son híbridos tales como C. x generalis. Sin embargo, estas plantas muestran granos normales, bien formados, cercanos a los de C. coccinea, que germinan sobre el estigma de flores frescas, nosotros decidimos incluir su polen en este estudio.

Morfología del polen de especies neotropicales de Podostemum (Podostemaceae)

Pollen morphology of neotropical species of Podostemum (Malpighiales: Podostemaceae). Pollen morphology of neotropical species of Podostemum is described for the first time with light (LM) and scanning electronic microscopes (SEM), using pollen dehydration by critical point. Herbarium specimens under study are P. comatum, P. distichum, P. muelleri and P. rutifolium. Pollen grains are dispersed in dyads. Observed with LM, dyads range from 21 to 31μm in lengh and 12 to 18μm wide. Individual pollen grains are radially symmetrical spheroidal to subprolate, and have a tricolpate aperture. The exine is tectate, psilate, with an infratectal structure formed by simple columella under the tectum. With SEM, an abundant pollen surface coat is observed all over the pollen grains, mainly in the two grains dyad contact zone. This pollen coat would protect the grains from dehydration because the environments in which these plants grow have important water variation and pollination is not zoophilic. The shared dyad wall shows bridges that partially fusion the exines, forming calymate dyads. Some dyads bear the apertures aligned between grains and some not. The transverse condition of the aperture or aperture in "L" that occurs in the four studied species is described for the first time. It is interpreted as a trend of the genus to espiroaperture. This change in the aperture would be associated with phenology because it is a genus with very short flowering and anthesis, generally a day of anthesis in the dry season. The spiroaperture increases the chances of germination sites and would also have a harmomegata role in an environment with water changes favoring the reproductive success.Walls have a microechinate sculpture, with or without pads at the base of the microechinae. Size of ornamental processes differs, and the colpus membrane has similar ornamentation to that of the non apertural wall, but with larger processes. Individual morphology of pollen grains is similar to that described for other family genera as Apinagia and Mourera. During previous stages of anthesis, the presence of cross tetrads was observed, also a new contribution for this genus. Rev. Biol. Trop. 58 (1): 81-88. Epub 2010 March 01.

La morfología del polen de cuatro especies neotropicales del género Podostemum (Podostemaceae) se describe aquí por primera vez mediante microscopía de luz y electrónica de barrido, utilizando material deshidratado en punto crítico. Se analizaron ejemplares de P. comatum, P. distichum, P. muelleri y P. rutifolium. Los granos de polen son dispersados en díades. Se encuentra abundante cobertura polínica, principalmente en la zona de contacto entre los dos granos de la díada. En la pared compartida entre los granos se presentan puentes de ectexina que fusionan parcialmente las exinas. Los granos individuales son radialmente simétricos y tricolpados. En algunas díades las aberturas están alineadas entre los granos y en otras no. Se describe por primera vez la condición abertura transversal, que fue observada en las cuatro especies estudiadas, y que se interpreta como tendencia del género a espiroabertura. La escultura de la exina es microequinada con mamelones en la base. Los procesos ornamentales son de diferente tamaño. La membrana del colpo tiene una ornamentación similar a la pared adyacente del grano, pero los procesos tienen mayor tamaño. Se observaron tétrades decusadas en las etapas previas a la antesis, un dato hasta el momento no registrado para el género.

Monoicy in A. angustifolia (Bert. ) O. Kuntze (Araucariaceae): I. Morphological aspects of the reproductive structures

Araucaria angustifolia (Bert.) O. Kuntze is a dioecious conifer species native of Brazil. The rare occurrence of monoiceous specimens have been attributed to pathogenic infections or other injuries in adult trees. Here, the morphological characteristics of male and female cones and pollen grains of a monoiceous A. angustifolia are described. Male and female cones and pollen grains presented normal morphology, lacking any sort of injuries or infection and suggesting the existence of further grounds for the occurrence of monoicy in this conifer species.

Araucaria angustifolia (Bert.) O. Kuntze é uma conífera primariamente dióica nativa do Brasil. A ocorrência de raros exemplares monóicos tem sido creditada a infecções patogênicas ou outras injúrias em árvores adultas. Neste trabalho, as características morfológicas de cones masculinos e femininos e dos grãos de pólen de uma A. angustifolia monóica são descritas. Os cones masculinos e femininos e os grãos de pólen apresentaram morfologia normal, se a presença de qualquer tipo de injuria ou infecção, sugerindo a existência de outros mecanismos para a ocorrência de monoicia nesta conífera.

Syncyte formation in the microsporangium of Chrysanthemum (Asteraceae): a pathway to infraspecific polyploidy.

Polyploidy, which is thought to have played an important role in plant evolution and speciation, is prevalent in Chrysanthemum (x = 9). In fact, polyploid series are known in C. zawadskii (2x, 4x, 6x, 8x, and 10x) and C. indicum (2x, 4x, and 6x), but the mechanism by which polyploidization occurs is unknown. Here we show that in diploid individuals of both C. zawadskii and C. indicum, the fusion between two adjacent pollen mother cells (PMCs) occurs at a frequency of 1.1-1.3% early in the first meiotic division. While possessing the chromosomes of both PMCs, the fused cell or syncyte undertakes subsequent meiotic division processes as a single large PMC, producing four 2n pollen grains that are able to germinate. Despite their low frequency, syncyte formation may have played a major role in the production of infraspecific polyploids in Chrysanthemum.