Abscisic acid (ABA) is a key plant hormone regulating agronomically important processes including seed maturation and dormancy, stomatal opening and closure, along with the transcriptional and physiological response of plants to abiotic and biotic stresses. The current study sought to functionally characterize members of an ABA-responsive gene family encoding GRAM (Glucosyltransferases, Rab-like GTPase activators and Myotubularins) domain proteins in Arabidopsis thaliana. Utilizing reverse genetics loss- and gain-of-function lines associated with GEM-RELATED 5 (GER5) were obtained, which displayed several defects in reproductive development. Gene expression profiling, RNA in situ hybridization and immunohistochemical techniques were utilized to evaluate GER5 and two closely related GRAM genes, GEM-RELATED 1 (GER1) and GLABRA2 EXPRESSION MODULATOR (GEM) in reproductive structures. Microarray profiling of seeds from ger5-2 mutants and wild-type plants revealed transcriptional changes in carbohydrate metabolism, hormone signaling and catabolic processes accompanied seed development defects of ger5-2 mutants. Seed germination assays further revealed ger5-2 mutants exhibited reduced sensitivity to ABA. In assessing GER5, GER1 and GEM as putative ABA-response genes, a second study evaluated the expression of GRAM, AuTophaGy-related (ATG), and ABA-response genes in source and sink organs exposed to abiotic stress or within mutant backgrounds deficient in sugar signaling. Monodansylcadaverine (MDC) staining was also utilized to localize autophagosomes or autophagic bodies within vegetative or reproductive organs during plant development, or in response to carbon starvation or abiotic stress. In a third study transcriptional differences in ABA metabolism, transport and homeostasis were examined within reproductive organs (cauline leaves, inflorescence meristem, developing siliques) exposed to cold and heat stress. This study revealed reproductive organs are characterized by unique patterns of ABA metabolism which differ from tissues typically associated with classical ABA responses. Together, these studies indicate GER5, an uncharacterized ABA-responsive GRAM domain gene, plays a novel role in the reproductive development of plants and that ABA metabolism and signaling are uniquely regulated in reproductive organs.

No description available.

Abscisic acid (ABA) is a key plant hormone regulating agronomically important processes including seed maturation and dormancy, stomatal opening and closure, along with the transcriptional and physiological response of plants to abiotic and biotic stresses. The current study sought to functionally characterize members of an ABA-responsive gene family encoding GRAM (Glucosyltransferases, Rab-like GTPase activators and Myotubularins) domain proteins in Arabidopsis thaliana. Utilizing reverse genetics loss- and gain-of-function lines associated with GEM-RELATED 5 (GER5) were obtained, which displayed several defects in reproductive development. Gene expression profiling, RNA in situ hybridization and immunohistochemical techniques were utilized to evaluate GER5 and two closely related GRAM genes, GEM-RELATED 1 (GER1) and GLABRA2 EXPRESSION MODULATOR (GEM) in reproductive structures. Microarray profiling of seeds from ger5-2 mutants and wild-type plants revealed transcriptional changes in carbohydrate metabolism, hormone signaling and catabolic processes accompanied seed development defects of ger5-2 mutants. Seed germination assays further revealed ger5-2 mutants exhibited reduced sensitivity to ABA. In assessing GER5, GER1 and GEM as putative ABA-response genes, a second study evaluated the expression of GRAM, AuTophaGy-related (ATG), and ABA-response genes in source and sink organs exposed to abiotic stress or within mutant backgrounds deficient in sugar signaling. Monodansylcadaverine (MDC) staining was also utilized to localize autophagosomes or autophagic bodies within vegetative or reproductive organs during plant development, or in response to carbon starvation or abiotic stress. In a third study transcriptional differences in ABA metabolism, transport and homeostasis were examined within reproductive organs (cauline leaves, inflorescence meristem, developing siliques) exposed to cold and heat stress. This study revealed reproductive organs are characterized by unique patterns of ABA metabolism which differ from tissues typically associated with classical ABA responses. Together, these studies indicate GER5, an uncharacterized ABA-responsive GRAM domain gene, plays a novel role in the reproductive development of plants and that ABA metabolism and signaling are uniquely regulated in reproductive organs.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 139-148).

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references (leaves 238-266). Also available in electronic version. Access restricted to campus users.

The biosynthesis of ABA from R-[2-¹⁴C]-MVA was demonstrated in Persea americana cv. Fuerte mesocarp and in mature seeds of Hordeum vulgare cv. Dyan and cv. Himalaya. Radioactivity from R-[2-¹⁴-C]-MVA was also incorporated into the 1',4'-trans ABA diol in Persea americana mesocarp and a possible role for this metabolite as a precursor of ABA in plants is discussed. The biosynthesis of ABA from MVA could not be demonstrated in either turgid and waterstressed Hordeum vulgare cv. Dyan, Pisum sativum cv. Black-eyed Susan and Phaseolus vulgaris cv. Top-crop or in immature seeds of Pisum sativum and Phaseolus vulgaris. (R,S,)-[2-¹⁴C]-ABA was catabolised to PA, DPA and aqueous conjugates in leaves and mature seeds of Hordeum vulgare cv. Dyan, seedlings and immature seeds of Pisum sativum and Phaseolus vulgaris and in mesocarp from ripening fruits of Persea americana. PA and DPA were identified by either microchemical methods and/or capillary GC-MS. 7'-Hydroxy ABA was characterised as a novel ABA catabolite in light-grown and etiolated leaves of Hordeum vulgare by capillary GC-MS. Circular dichroism analysis revealed that it was derived predominantly from the (R)-enantiomer of ABA. This catabolite was absent in similar studies using the dicotyledons Pisum sativum and Phaseolus vulgaris. Refeeding studies with [¹⁴C]-PA, [C]-DPA and [¹⁴C]-7'-hydroxy ABA were used to confirm the metabolic interrelationship between ABA and its catabolites in both vegetative and non-vegetative tissues from monocotyledonous and dicotyledonous species. The methyl ester of (R,S,)-ABA was hydrolysed efficiently by light-grown leaves of Hordeum vulgare. Older, vegetative tissues catabolised (R,S,)-ABA more efficiently than their younger counterparts. In contrast, small, immature seeds of Pisum sativum catabolised (R,S,)-ABA more effectively than larger, immature seeds of this species. Light did not appear to influence ABA biosynthesis but markedly enhanced ABA catabolism. Light stimulated the overall rate of ABA catabolism in both vegetative and non-vegetative tissue. Water stress reduced ABA catabolism in Hordeum vulgare leaves but had little effect on this process in Phaseolus vulgaris seedlings. Pretreatment of tissues with (R,S,)-ABA retarded the catabolism of (R,S,)-[2-¹⁴C]-ABA, negating ABA-induced conversion to PA. Cycloheximide inhibited ABA biosynthesis and catabolism but did not affect ABA conjugation. Chloramphenicol and lincomycin had little or no effect on ABA metabolism suggesting that the enzymes involved were labile and cytoplasmic in origin. Ancymidol and cycocel inhibited ABA biosynthesis while AM01618 stimulated this process. The cytokinins, benzyladenine, kinetin, isopentenyl adenine and zeatin also inhibited ABA biosynthesis. These results are discussed in relation to the possible involvement of carotenoids in ABA biosynthesis. AM01618, ancymidol andcycocel did not significantly influence the conversion of ABA to PA and DPA while cytokinins appeared to enhance this process only in vegetative tissue. The information derived from these studies was then used in attempts to develop a cell-free system from higher plants capable of metabolising ABA. A cell-free system prepared from imbibed Hordeum vulgare cv. Dyan embryos biosynthesized and catabolised ABA. This is the first demonstration of a cell-free system from non-vegetative tissue capable of metabolising ABA and could prove useful for elucidating its biosynthetic route. This cell-free system generated the terpenyl pyrophosphates IPP, FPP and GGPP from MVA. ABA was produced from both MVA and IPP in the presence of 0₂ and NADPH. The biosynthesis of ABA was stimulated by the addition of the squalene 2,3-oxide cyclase and kaurene synthetase inhibitor, AM01618 and a "cold-pool trap" of (R,S,)-ABA. Ancymidol, cycocel and cytokinins reduced incorporation of label from MVA into ABA. Similar cell-free preparations, in the absence of AM01618, converted (R,S,)-[2-¹⁴-C]-ABA into PA, 7'-hydroxy ABA and water-soluble conjugates. Although the methyl ester of (R,S,)-ABA was efficiently hydrolysed in this cell-free system no DPA was generated. The possible involvement of mixed function oxidase activity and soluble oxidases is discussed in relation to ABA metabolism. While cell-free preparations from Persea americana cv. Fuerte mesocarp and immature seeds of Pisum sativum and Phaseolus vulgaris were unable to synthesize ABA from MVA, these tissue homogenates converted ABA into more polar acidic products. PA and DPA were identified as products of ABA catabolism in extracts from immature seeds of Phaseolus vulgaris and the l',4'-cis diol of ABA in extracts from Pisum sativum immature seeds

Orientador: Edvaldo Aparecido Amaral da Silva / Banca: Claudio Cavariani / Banca: Juliana Pereira Bravo / Resumo: A germinação de sementes de café (Coffea arabica L.) é lenta e irregular, controlada por eventos que ocorrem, simultaneamente, no embrião e no endosperma. Embora os referidos eventos estejam determinados, ainda são necessários estudos sobre a fisiologia molecular, para auxiliar na avaliação da qualidade fisiológica das sementes durante a germinação. O objetivo do trabalho foi realizar estudos fisiológicos e moleculares durante a germinação de sementes embebidas em água e em ácido abscísico (ABA) na concentração de 1000 μM. Durante o trabalho foi determinado o teor de água, a curva de embebição, a germinação, o crescimento do embrião e a expressão dos genes associados com o crescimento do embrião e com a degradação do endosperma micropilar. Para tanto, embriões e os endospermas micropilares foram isolados para a extração de RNA total e síntese de cDNA. "Primers" específicos foram desenhados para o estudo da expressão gênica em PCR em tempo real. Foi estudada a expressão dos genes actina, ciclina e α-expansina, associados ao crescimento do embrião, e α-galactosidase, β-manosidase e endo-β-mananase, associados à degradação do endosperma micropilar. A curva de embebição apresentou um padrão trifásico. A primeira semente de café germinou com cinco dias de embebição e 50% de germinação ocorreram no décimo dia de embebição. A expressão dos genes associados com o crescimento do embrião, tais como actina, α-expansina e quinase dependente de ciclina, aumentou durante a germinação em água e inibiu parcialmente a expressão destes genes quando tratados com ABA. A expressão de β-manosidase e endo-β-mananase aumentou durante a embebição em água e ABA inibiu completamente a expressão. No entanto, α-galactosidase parece ter a expressão mais constitutiva durante a germinação em água e é menos influenciada por ABA, em comparação com outras enzimas estudadas / Abstract: Germination of coffee (Coffea arabica L.) seed is slow and uneven. The germination is a net result of events that occur simultaneously in the embryo and endosperm under the control of ABA. The aim of the study was to perform physiological and molecular studies during germination of seeds imbibed in water and 1000 μM abscisic acid (ABA). We studied the expression of the genes ciclin, α-expansin and cyclin-dependent of kinase in the embryo and α-galactosidase, β-mannosidase, endo-β-mannanase in the micropylar endosperm. The first coffee seed germinate at five days of imbibition and 50% germinate at tenth day of imbibition. Coffee embryo grew inside the seed pior radicle protrusion and ABA inhibited the embryo grow as well as radicle protrusion. The expression of the genes associated with the growth of the embryo such as ciclin, α-expansin and cyclin-dependent of kinase increased during germination and ABA partially inhibited the expression of these genes. The expression of β-mannosidase and endo-β-mannanase increased during imbibitions in water and ABA completely inhibited its expression. However, α-galactosidase seems to have a more constitutive expression during germination in water and it is less affected by ABA as compared with other enzymes studied / Mestre

No description available.

No description available.

Obesity is a disease characterized by chronic inflammation and the progressive loss in systemic insulin sensitivity. One of the more effective medications in the treatment of insulin resistance have been the thiazolidinediones (TZDs), which act through the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma ). Due to the many side-effects of TZDs, our laboratory sought out a natural phytochemical, abscisic acid (ABA), with chemical similarities to TZDs. Our first study demonstrated that ABA activates PPARgamma in vitro and significantly ameliorates white adipose tissue (WAT) inflammation and glucose tolerance in db/db mice. We next further examined the effect of ABA on the phenotype of adipose tissue macrophages (ATMs). In doing so, we discovered two separate ATM populations which differed in their expression of the macrophage surface glycoprotein and maturation marker F4/80 (F4/80hi vs F4/80lo). Dietary ABA-supplementation significantly reduced F4/80hiCCR2+ ATMs and had no effect on the F4/80lo population. Utilizing a tissue-specific knockout generated through Cre-lox recombination, we were able to determine that this effect was dependent on PPARgamma in immune cells. To further characterize the differences between the ATM subsets that were affected by ABA, we performed a multi-organ assessment (i.e., WAT, skeletal muscle and liver) of the effect of diet-induced obesity on the phenotype of infiltrating macrophages and T cells into metabolic organs. Based on our new data, we formulated a model by which F4/80hiCCR2hi ATMs infiltrate WAT and ultimately induce a CD11c+ pro-inflammatory phenotype in the resident F4/80loCCR2lo subset. Ultimately, our findings provide evidence that ABA has potential as an alternative preventive intervention, expound the role of PPARgamma in immune cells and, in general, expand our knowledge concerning the immunopathogenesis of obesity-induced insulin resistance. / Ph. D.