RESUMEN
Cold acclimation requires substantial alteration in membrane property. In contrast to well-documented fatty acid unsaturation during cold acclimation, changes in phospholipid biosynthesis during cold acclimation are less understood. Here, we isolated and characterized two aminoalcoholphosphotransferase (AAPT) cDNAs, TaAAPT1 and TaAAPT2, from wheat. AAPTs utilize diacylglycerols and CDP-choline/ethanolamine as substrates and catalyze the final step of the CDP-choline/ethanolamine pathway for phosphatidylcholine (PC)/phosphatidylethanolamine (PE) synthesis, respectively. Functionality of TaAAPT1 and TaAAPT2 was demonstrated by heterologous expression in a yeast cpt1Delta ept1Delta double mutant that lacks both AAPT activities. Detailed characterization of AAPT activities from the transformed mutant cells indicated that TaAAPT1 is an ECPT-type enzyme with higher ethanolamine phosphotransferase (EPT) activity than choline phosphotransferase (CPT) activity, while TaAAPT2 is a CEPT-type with the opposite substrate preference. Transient expression of GFP-fused TaAAPT1 and TaAAPT2 proteins in wheat and onion cells indicated they are localized to both the endoplasmic reticulum and Golgi apparatus, suggesting that the final synthesis of PE and PC via the CDP-choline/ethanolamine pathway occurs in these organella. Quantitative PCR analyses revealed that TaAAPT1 expression is strongly induced by cold, while TaAAPT2 was constitutively expressed at lower levels. Measurement of phospholipid content in wheat leaves indicated that PE is more prominently increased in response to cold than PC and accordingly PE/PC ratio increased from 0.385 to 0.530 during 14 days of cold acclimation. Together, these data suggested that an increase in the PE/PC ratio during cold acclimation is regulated at the final step of the biosynthetic pathway.
Asunto(s)
Fosfotransferasas/biosíntesis , Fosfotransferasas/genética , Triticum/genética , Triticum/metabolismo , Aclimatación , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Clima Frío , ADN de Plantas/genética , Retículo Endoplásmico/enzimología , Genes de Plantas , Aparato de Golgi/enzimología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Lípidos de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fosfatidiletanolaminas/metabolismo , Fosfotransferasas/química , Plantas Modificadas Genéticamente , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
We have developed a new target plate for matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). This target plate enables direct electric transfer of analytes from the 1-dimensional gel electrophoresis (1-DE) gel to the target plate in one step. Incorporated with a one-step direct transfer technique, this novel 1-DE/MALDI-MS (1-DE/MS) system eliminates staining, extracting, loading, and many other time-consuming intermediate processes, thereby greatly reducing analysis time while providing high throughput proteome analysis. Furthermore, in peptidome analysis, during the 1-DE step this system separates or removes the high molecular weight plasma proteins in blood and the various low molecular weight substances in tissue extracts, which interfere with mass spectrometry. This system can therefore be used for peptide profiling of any biological sample without special pretreatment. In view of these advantages, the 1-DE/MS system will greatly improve the usefulness of current peptidomic modalities in the discovery and validation of biomarker molecules in various body fluids and tissue extracts, permitting early detection, diagnosis, and treatment of diseases.
Asunto(s)
Biomarcadores/análisis , Péptidos/análisis , Análisis por Matrices de Proteínas/métodos , Proteoma/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Biomarcadores/sangre , Electroforesis , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Suero/químicaRESUMEN
Studies of carrot embryogenesis have suggested that abscisic acid (ABA) is involved in somatic embryogenesis. A relationship between endogenous ABA and the induction of somatic embryogenesis was demonstrated using stress-induced system of somatic embryos. The embryonic-specific genes C-ABI3 and embryogenic cell proteins (ECPs) were expressed during stress treatment prior to the formation of somatic embryos. The stress-induction system for embryogenesis was clearly distinguished by two phases: the acquisition of embryogenic competence and the formation of a somatic embryo. Somatic embryo formation was inhibited by the application of fluridone (especially at 10(-4) M), a potent inhibitor of ABA biosynthesis, during stress treatment. The inhibitory effect of fluridone was nullified by the simultaneous application of fluridone and ABA. The level of endogenous ABA increased transiently during stress. However, somatic embryogenesis was not significantly induced by the application of only ABA to the endogenous level, in the absence of stress. These results suggest that the induction of somatic embryogenesis, in particular the acquisition of embryogenic competence, is caused not only by the presence of ABA but also by physiological responses that are directly controlled by stresses.
