Orientador: Nelson Sabino Bittencourt Júnior / Banca: Neusa Taroda Ranga / Banca: Diana Salles Sampaio / Resumo: Caracteres embriológicos possuem valor sistemático e sua utilidade foi demonstrada por diversos autores para elucidar o posicionamento filogenético de certas famílias de angiospermas. Este estudo visa analisar a ontogenia das estruturas reprodutivas de Cybistax antisyphilitica, com o propósito de acrescentar dados embriológicos relevantes ao delineamento filogenético da família, ou de categorias taxonômicas infrafamiliares. As características embriológicas observadas demonstraram similaridade com espécies pertencentes à ―Tabebuia alliance‖ cuja embriologia já foi investigada. Observou-se durante a ontogenia do óvulo de C. antisyphilitica que apenas o ginósporo calazal se desenvolve e, durante sua diferenciação em célula-mãe do saco embrionário, a face micropilar de sua parede celular assume uma conformação côncava, na qual há grande deposição de calose. No estádio octonuclear, há acúmulo de uma substância fibrogranular entre o endotélio e a parede do ginófito fazendo com que o saco embrionário apresente um característico afunilamento mediano. Em relação a ontogenia da antera da espécie estudada, verificou-se que as camadas tapetais são dimórficas, embora ao final do estádio pré-meiótico se tornem similares; embora, após a meiose, o dimorfismo se acentua novamente nas camadas tapetais. Os amiloplastos das células-mãe dos andrósporos são herdados pelos andrósporos e grãos de pólen deles resultantes. Os amiloplastos dos grãos de pólen gradualmente aumentam em número e tamanho em um único ciclo de amilogênese/amilólise. Com base nos dados obtidos conclui-se que a configuração da parede distal do ginósporo calazal e o acúmulo de secreção na porção mediana do saco embrionário são características não relatadas para outras espécies da família e podem... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Embryological characters have systematic value and its usefulness has been demonstrated by several authors to elucidate the phylogenetic position of certain angiosperms families. This study aims to analyze the ontogeny of reproductive structures of Cybistax antisyphilitica, with the purpose of adding relevant embryological data to the phylogenetic design of the family or, infra-familiar taxonomic categories. The embryological features observed was similar to species belonging to ―Tabebuia alliance‖ whose embryology has been investigated. During the ontogey of C. antisyphilitica ovule, only the chalazal megaspore develops, and in the course of its differentiation in the embryo sac mother cell, the micropylar side of its cell wall assumes a concave conformation in which there is an expressive deposition of callose. In the octonuclear stage, there is an accumulation of a fibrogranular substance between the endothelium and megagametophyte wall causing a bottleneck in the middle portion of embryo sac. For anther ontogeny of the species studied, the tapetal layers are dimorphic, although becoming similar at the late pre-meiotic stage. After meiosis, the dimorphism is accentuated between the two tapetal layers. The microspore mother cell amyloplasts are inherited by the microspores and the resulting pollen grains. The pollen grain amyloplasts gradually increasing in number and size in a single amylogenesis/amylolyse cycle. Based on the obtained data we concluded that the configuration of the distal wall of the chalazal megaspore and the accumulation of secretion in the median portion of embryo sac are characters not reported for other species of the family, and may possibly be autapomorphic characters. Regarding the ontogeny of the anther, Cybistax antisyphilitic showed a multistratified fibrous endothecium... (Complete abstract click electronic access below) / Mestre

Using the known sequences for serine palmitoyltransferase (SPT) and ceramide kinase (CERK) from Arabidopsis, candidates for the corresponding genes in Loblolly pine were cloned and examined during embryogenesis. The cloned two cDNA sequences from Loblolly pine, which has similarity of 81% and 88% respectively to two subunits of SPT1 and SPT2 in Arabidopsis, were presumed as the Loblolly pine SPT1 and SPT2 (Pt-SPT1 and Pt-SPT2). A few different versions of Pt-SPT1 mRNAs (2223 nts, 756 nts, 822 nts, and 754 nts respectively), most likely the alternative splicing results, were found. Three of these mRNAs are capable of encoding proteins. The long version (2223 nts) encodes a protein with 484 amino acids (Pt-SPT1); two short versions (822 nts, 756 nts) encode a 90 a.a. protein. Another cDNA sequence of 2396 nts encodes a protein of 493 a.a. (Pt-SPT2). Both predicted Pt-SPT1 and Pt-SPT2 proteins possess highly conserved serine palmitoyltransferase functional domains (E value 5.7e-61). Their expression patterns are different between somatic and zygotic embryogenesis. Two different versions of mRNAs, with 2786 nts (long), and 2320 nts (short) respectively, of ceramide kinases in Loblolly pine (Pt-CERKs) have been cloned. The long version encodes a protein with 721 a.a.; the short version with 560 a.a. The expression patterns for these two CERK mRNAs are different during embryo development. The long version is constitutively expressed, while the short one is only expressed in some stages with much lower expression level. Overexpression Pt-CERKL, Pt-CERKS, and Pt-CERKF in E.coli and function analysis in vitro show that all Pt-CERKs appear to have the same catalytic functions as their homologs in human and Arabidopsis, but with different efficiency. The catalytic efficiency was dramatically lower in the short Pt-CERK protein compared with the long Pt-CERK protein and Pt-CERKF. The membrane system is not necessary for the catalytic reactions of these three Pt-CERKs in vitro and Pt-CERKs were less dependent on the Ca2+ ions. Thus, these studies have provided the first information about SPT- and CERK- like proteins in loblolly pine, and open new avenues of investigation for the roles of sphingolipids in embryonic development.

Indiana University-Purdue University Indianapolis (IUPUI) / Dehydrins are type II LEA proteins induced in many plants during drought, low temperature, and high salinity to confer stress tolerance. AtERD14 is an Arabidopsis thaliana dehydrin that functions in part of the cold stress pathway. AtERD14 has chaperone-like capabilities that allow it to bind and protect various proteins from dehydration stresses. In order to determine the necessary components for cold induction of AtERD14, AtERD14prom::GFP/GUS and AtERD14prom::AtERD14 in AtERD14 KO constructs were created and stably transformed into A. thaliana. Analysis of the constructs showed the AtERD14 promoter alone was insufficient to respond to cold, and it was necessary to attach the AtERD14 coding region to the promoter to induce a cold response in ERD14. On the other hand, the RD29aprom::GFP/GUS promoter did respond to cold stress, indicating that RD29a does not require its coding region to support an increased amount of reporter activity after cold stress. The protoplast transformation system, while capable of transient expression of introduced constructs in protoplasts, was difficult for use for cold-inducible expression.