RESUMEN
In Arabidopsis thaliana and Oryza sativa, two types of PI 4-kinase (PI4Ks) have been isolated and functionally characterized. The alpha-type PI4Ks (approximately 220 kDa) contain a PH domain, which is lacking in beta-type PI4Ks (approximately 120 kDa). Beta-type PI4Ks, exemplified by Arabidopsis AtPI4Kbeta and rice OsPI4K2, contain a highly charged repetitive segment designated PPC (Plant PI4K Charged) region, which is an unique domain only found in plant beta-type PI4Ks at present. The PPC region has a length of approximately 300 amino acids and harboring 11 (AtPI4Kbeta) and 14 (OsPI4K2) repeats, respectively, of a 20-aa motif. Studies employing a modified yeast-based "Sequence of Membrane-Targeting Detection" system demonstrate that the PPC(OsPI4K2) region, as well as the former 8 and latter 6 repetitive motifs within the PPC region, are able to target fusion proteins to the plasma membrane. Further detection on the transiently expressed GFP fusion proteins in onion epidermal cells showed that the PPC(OsPI4K2) region alone, as well as the region containing repetitive motifs 1-8, was able to direct GFP to the plasma membrane, while the regions containing less repetitive motifs, i.e. 6, 4, 2 or single motif(s) led to predominantly intracellular localization. Agrobacterium-mediated transient expression of PPC-GFP fusion protein further confirms the membrane-targeting capacities of PPC region. In addition, the predominant plasma membrane localization of AtPI4Kbeta was mediated by the PPC region. Recombinant PPC peptide, expressed in E. coli, strongly binds phosphatidic acid, PI and PI4P, but not phosphatidylcholine, PI5P, or PI(4,5)P2 in vitro, providing insights into potential mechanisms for regulating sub-cellular localization and lipid binding for the plant beta-type PI4Ks.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/metabolismo , Membrana Celular/metabolismo , Fosfolípidos/metabolismo , Proteínas de Plantas/metabolismo , 1-Fosfatidilinositol 4-Quinasa/química , 1-Fosfatidilinositol 4-Quinasa/genética , Secuencia de Aminoácidos , Arabidopsis/enzimología , Biolística/métodos , Transporte Biológico , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía Confocal , Datos de Secuencia Molecular , Cebollas/citología , Cebollas/genética , Cebollas/metabolismo , Oryza/enzimología , Epidermis de la Planta/citología , Epidermis de la Planta/genética , Epidermis de la Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Unión Proteica , Secuencias Repetitivas de Aminoácido/genética , Homología de Secuencia de Aminoácido , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
Phytohormone abscisic acid (ABA) was critical for many plant growth and developmental processes including seed maturation, germination and response to environmental factors. With the purpose to detect the possible ABA related signal transduction pathways, we tried to isolate ABA-regulated genes through cDNA macroarray technology using ABA-treated rice seedling as materials (under treatment for 2, 4, 8 and 12 h). Of 6144 cDNA clones tested, 37 differential clones showing induction or suppression for at least one time, were isolated. Of them 30 and 7 were up- or down-regulated respectively. Sequence analyses revealed that the putative encoded proteins were involved in different possible processes, including transcription, metabolism and resistance, photosynthesis, signal transduction, and seed maturation. 6 cDNA clones were found to encode proteins with unknown functions. Regulation by ABA of 7 selected clones relating to signal transduction or metabolism was confirmed by reverse transcription PCR. In addition, some clones were further shown to be regulated by other plant growth regulators including auxin and brassinosteroid, which, however, indicated the complicated interactions of plant hormones. Possible signal transduction pathways involved in ABA were discussed.