The functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] during vegetative plant growth remain obscure. Here, we targeted two related phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) PIP5K1 and PIP5K2, which are expressed ubiquitously in Arabidopsis thaliana. A pip5k1 pip5k2 double mutant with reduced PtdIns(4,5)P₂ levels showed dwarf stature and phenotypes suggesting defects in auxin distribution. The roots of the pip5k1 pip5k2 double mutant had normal auxin levels but reduced auxin transport and altered distribution. Fluorescence-tagged auxin efflux carriers PIN-FORMED (PIN1)–green fluorescent protein (GFP) and PIN2-GFP displayed abnormal, partially apolar distribution. Furthermore, fewer brefeldin A–induced endosomal bodies decorated by PIN1-GFP or PIN2-GFP formed in pip5k1 pip5k2 mutants. Inducible overexpressor lines for PIP5K1 or PIP5K2 also exhibited phenotypes indicating misregulation of auxin-dependent processes, and immunolocalization showed reduced membrane association of PIN1 and PIN2. PIN cycling and polarization require clathrin-mediated endocytosis and labeled clathrin light chain also displayed altered localization patterns in the pip5k1 pip5k2 double mutant, consistent with a role for PtdIns(4,5)P₂ in the regulation of clathrin-mediated endocytosis. Further biochemical tests on subcellular fractions enriched for clathrin-coated vesicles (CCVs) indicated that pip5k1 and pip5k2 mutants have reduced CCV-associated PI4P 5-kinase activity. Together, the data indicate an important role for PtdIns(4,5)P₂ in the control of clathrin dynamics and in auxin distribution in Arabidopsis.

The internodal maize pulvinus responds to gravistimulation with differential cell elongation on the lower side. As the site of both graviperception and response, the pulvinus is an ideal system to study how organisms sense changes in orientation. We observed a transient 5-fold increase in inositol 1,4,5-trisphosphate (IP ₃ ) within 10 s of gravistimulation in the lower half of the pulvinus, indicating that the positional change was sensed immediately. Over the first 30 min, rapid IP ₃ fluctuations were observed between the upper and lower halves. Maize plants require a presentation time of between 2 and 4 h before the cells on the lower side of the pulvinus are committed to elongation. After 2 h of gravistimulation, the lower half consistently had higher IP _3, and IP ₃ levels on the lower side continued to increase up to ≈5-fold over basal levels before visible growth. As bending became visible after 8–10 h, IP ₃ levels returned to basal values. Additionally, phosphatidylinositol 4-phosphate 5-kinase activity in the lower pulvinus half increased transiently within 10 min of gravistimulation, suggesting that the increased IP ₃ production was accompanied by an up-regulation of phosphatidylinositol 4,5-bisphosphate biosynthesis. Neither IP ₃ levels nor phosphatidylinositol 4-phosphate 5-kinase activity changed in pulvini halves from vertical control plants. Our data indicate the involvement of IP ₃ and inositol phospholipids in both short- and long-term responses to gravistimulation. As a diffusible second messenger, IP ₃ provides a mechanism to transmit and amplify the signal from the perceiving to the responding cells in the pulvinus, coordinating a synchronized growth response.

The phosphoinositide pathway and inositol-1,4,5-trisphosphate (InsP3) are implicated in plant responses to stress. To determine the downstream consequences of altered InsP3-mediated signaling, we generated transgenic Arabidopsis thaliana plants expressing the mammalian type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), which specifically hydrolyzes soluble inositol phosphates and terminates the signal. Rapid transient Ca2+ responses to a cold or salt stimulus were reduced by ∼30% in these transgenic plants. Drought stress studies revealed, surprisingly, that the InsP 5-ptase plants lost less water and exhibited increased drought tolerance. The onset of the drought stress was delayed in the transgenic plants, and abscisic acid (ABA) levels increased less than in the wild-type plants. Stomatal bioassays showed that transgenic guard cells were less responsive to the inhibition of opening by ABA but showed an increased sensitivity to ABA-induced closure. Transcript profiling revealed that the drought-inducible ABA-independent transcription factor DREB2A and a subset of DREB2A-regulated genes were basally upregulated in the InsP 5-ptase plants, suggesting that InsP3 is a negative regulator of these DREB2A-regulated genes. These results indicate that the drought tolerance of the InsP 5-ptase plants is mediated in part via a DREB2A-dependent pathway and that constitutive dampening of the InsP3 signal reveals unanticipated interconnections between signaling pathways.

Inositol 1,4,5-trisphosphate (InsP₃) has been implicated in the early signaling events of plants linking gravity sensing to the initiation of the gravitropic response. However, at present, the contribution of the phosphoinositide signaling pathway in plant gravitropism is not well understood. To delineate the role of InsP₃ in plant gravitropism, we generated Arabidopsis (Arabidopsis thaliana) plants constitutively expressing the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme that specifically hydrolyzes InsP₃. The transgenic plants show no significant differences in growth and life cycle compared to wild-type plants, although basal InsP₃ levels are reduced by greater than 90% compared to wild-type plants. With gravistimulation, InsP₃ levels in inflorescence stems of transgenic plants show no detectable change, whereas in wild-type plant inflorescences, InsP₃ levels increase approximately 3-fold within the first 5 to 15 min of gravistimulation, preceding visible bending. Furthermore, gravitropic bending of the roots, hypocotyls, and inflorescence stems of the InsP 5-ptase transgenic plants is reduced by approximately 30% compared with the wild type. Additionally, the cold memory response of the transgenic plants is attenuated, indicating that InsP₃ contributes to gravisignaling in the cold. The transgenic roots were shown to have altered calcium sensitivity in controlling gravitropic response, a reduction in basipetal indole-3-acetic acid transport, and a delay in the asymmetric auxin-induced β-glucuronidase expression with gravistimulation as compared to the controls. The compromised gravitropic response in all the major axes of growth in the transgenic Arabidopsis plants reveals a universal role for InsP₃ in the gravity signal transduction cascade of plants.

Pleckstrin homology (PH) domains are found in many proteins involved in signal transduction, including the family of large molecular mass phosphatidylinositol (PI) 4-kinases. Although the exact function of these newly discovered domains is unknown, it is recognized that they may influence enzyme regulation by binding different ligands. In this study, the recombinant PI 4-kinase PH domain was explored for its ability to bind to different phospholipids. First, we isolated partial cDNAs of the >7-kilobase transcripts of PI 4-kinases from carrot (DcPI4Kα) andArabidopsis (AtPI4Kα). The deduced primary sequences were 41% identical and 68% similar to rat and human PI 4-kinases and contained the telltale lipid kinase unique domain, PH domain, and catalytic domain. Antibodies raised against the expressed lipid kinase unique, PH, and catalytic domains identified a polypeptide of 205 kDa in Arabidopsis microsomes and an F-actin-enriched fraction from carrot cells. The 205-kDa immunoaffinity-purified Arabidopsis protein had PI 4-kinase activity. We have used the expressed PH domain to characterize lipid binding properties. The recombinant PH domain selectively bound to phosphatidylinositol 4-monophosphate (PI-4-P), phosphatidylinositol 4,5-bisphosphate (PI-4,5-P₂), and phosphatidic acid and did not bind to the 3-phosphoinositides. The PH domain had the highest affinity for PI-4-P, the product of the reaction. Consideration is given to the potential impact that this has on cytoskeletal organization and the PI signaling pathway in cells that have a high PI-4-P/PI-4,5-P₂ ratio.