Association of Phosphatidylinositol 3-Kinase with Nuclear Transcription Sites in Higher Plants

Abstract

The kinases responsible for phosphorylation of inositol-containing lipids are essential for many aspects of normal eukaryotic cell function. Genetic and biochemical studies have established that the phosphatidylinositol (PtdIns) 3-kinase encoded by the yeast VPS34 gene is essential for the efficient sorting and delivery of proteins to the vacuole; the kinase encoded by the human VPS34 homolog has been equally implicated in the control of intracellular vesicle traffic. The plant VPS34 homolog also is required for normal growth and development, and although a role for PtdIns 3-kinase in vesicle trafficking is likely, it has not been established. In this study, we have shown that considerable PtdIns 3-kinase activity is associated with the internal matrix of nuclei isolated from carrot suspension cells. Immunocytochemical and confocal laser scanning microscopy studies using the monoclonal antibody JIM135 (John Innes Monoclonal 135), raised against a truncated version of the soybean PtdIns 3-kinase, SPI3K-5p, revealed that this kinase appears to have a distinct and punctate distribution within the plant nucleus and nucleolus. Dual probing of root sections with JIM135 and anti-bromo-UTP antibodies, after in vitro transcription had been allowed to proceed in the presence of bromo-UTP, showed that SPI3K-5p associates with active nuclear and nucleolar transcription sites. These findings suggest a possible link between PtdIns 3-kinase activity and nuclear transcription in plants. INTRODUCTIONSince their discovery in the early to mid-1980s, the enzymes responsible for D-3 phosphorylation of phosphoinositides, that is, the phosphatidylinositol (PtdIns) 3-kinases, have been the subject of intense study (reviewed in Carpenter and Cantley, 1996). The PtdIns 3-kinases are now known to be involved in a plethora of cellular processes, ranging from mitogenesis, membrane trafficking and ruffling to glucose uptake, oxidative burst responses, chemotaxis, and apoptosis.Several distinct PtdIns 3-kinase isoforms are known to exist in eukaryotic cells. The currently identified PtdIns 3-kinases can be divided broadly into four families based on sequence homology and their preferred inositol lipid substrate or substrates. However, only one family has been found in all the eukaryotic cells studied so far, namely, the PtdIns-specific Vps34p-related 3-kinases. The other families presumably are more organism specific and have evolved later from a common primordial PtdIns 3-kinase family. Despite the high degree of sequence homology between members of this family and the other PtdIns 3-kinases, the Vps34p-related kinases all have the distinguishing biochemical feature that they phosphorylate only PtdIns, both in vivo and in vitro, and are unable to utilize PtdIns 4-monophosphate (PtdIns(4)P) or PtdIns 4,5-bisphosphate as substrates. The VPS34 gene product, Vps34p, was first identified in a screen of yeast mutants defective in vesicle sorting (Stack and Emr, 1994). Subsequently, a human homolog was identified, and Vps34p-related PtdIns 3-kinas...

Auxin‐induced reactive oxygen species production requires the activation of phosphatidylinositol 3‐kinase

“…It has been suggested that PtdIns(3)P is involved in intracellular vesicle trafficking by binding to the FYVE domain [11,21]. Bunney et al demonstrated that PtdIns 3-kinase is associated with active nuclear or nucleolar transcription sites, suggesting a role in the regulating transcription [27]. It was also reported that ABA-induced ROS production in guard cells, involved in stomatal closure, is inhibited by the PtdIns 3-kinase inhibitors wortmannin and LY294002 [28].…”

The Arabidopsis Phosphatidylinositol 3-Kinase Is Important for Pollen Development

“…Nuclear PtdIns(3)P has been reported in BHK cells and human fibroblasts by the use of FYVE domain probes (Gillooly et al, 2000), and in HL-60 cells, PtdIns(3)P level increases at G2/M phase of the cell cycle (Visnjic et al, 2003). In plants, PI3K is associated with active nuclear and nucleolar transcription sites (Bunney et al, 2000), and some of the proteins with a FYVE domain have regulator of chromosome condensation 1 tandem repeats, known to be important in nuclear processes (Drøbak and Heras, 2002). Therefore, the control of DNA replication or transcription is a possible mechanism for PI3K regulation of the cell cycle.…”

“…It is essential for normal growth, as demonstrated by the expression of AtVPS34 antisense constructs, which leads to second-generation transformed plants with very severe defects in growth and development (Welters et al, 1994). Plant PI3K is associated with active nuclear and nucleolar transcription sites (Bunney et al, 2000), which led to the suggestion that it plays a role in active transcription. Together with the fact that there is no other enzyme that can produce PtdIns(3)P, a lipid with many physiological effects in plants, these reports indicate that Vps34p-like PI3K has much broader functions in plants than in animals or budding yeast.…”

Fluorescence in situ hybridization on vibratome sections of plant tissues

“…These requirements are met by the roots and florets and other organs of many plants. We have used it for both roots and florets of wheat (Triticum aestivum L.) and related species [1][2][3] , for rye (Secale cereale L.) florets 4 , for rice (Oryza sativa L.) roots and florets 5 and maize (Zea mays L.) roots 6 among the cereals, and for roots of pea (Pisum sativum L.), bean (Vicia faba L.) 7 and soya (Glycine max L.) 8 . The only plant species with which we have been unsuccessful in vibratome sectioning is Arabidopsis thaliana, because of its small size.…”

“…The resulting vibratome sections can be used in many different types of labeling experiments. We have used such sections for DNA FISH, for RNA FISH to detect transcripts 10 , for antibody immunofluorescence labeling 8 and for labeling of nascent transcripts by BrUTP incorporation in unfixed tissue. We have also combined antibody and BrUTP labeling with FISH (e.g., ref.…”