ArabidopsisPhosphatidylinositol-4-Monophosphate 5-Kinase 4 Regulates Pollen Tube Growth and Polarity by Modulating Membrane Recycling

Abstract

Phosphatidylinositol-4-monophosphate 5-kinases produce phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P 2 ] and have been implicated in vesicle trafficking and cytoskeletal rearrangements. Here, we adopted a reverse genetics approach to investigate the function of the Arabidopsis thaliana pollen-expressed gene encoding phosphatidylinositol-4-monophosphate 5-kinase 4 (PIP5K4). Pollen germination, tube growth, and polarity were significantly impaired in homozygous mutant plants lacking PIP5K4 transcript. In vitro, supplementation with PtdIns(4,5)P 2 rescued these phenotypes. In vivo, mutant pollen fertilized ovules, leading to normal seed set and silique length. However, fertilization took longer than in wild-type plants, and the pip5k4 null mutant allele was transmitted through the pollen at a reduced frequency. Analysis of endocytic events using FM1-43 (or FM4-64) suggested a reduction in endocytosis and membrane recycling in pip5k4 null mutant pollen tubes. Imaging of elongating tobacco (Nicotiana tabacum) pollen tubes transiently transformed with a PIP5K4-green fluorescent protein fusion construct revealed that the protein localized to the plasma membrane, particularly in the subapical region. Overexpression of PIP5K4-GFP delocalized the protein to the apical region of the plasma membrane, perturbed pollen tube growth, and caused apical cell wall thickening. Thus, PIP5K4 plays a crucial role in regulating the polarity of pollen tubes. This study supports a model for membrane secretion and recycling where the apical and subapical regions appear to contain the components required to promote and sustain growth.

Arabidopsis phosphatidylinositol monophosphate 5-kinase 2 is involved in root gravitropism through regulation of polar auxin transport by affecting the cycling of PIN proteins

“…As shown in Figure 2D (right panel), when supplemented with exogenous PtdIns(4,5)P 2 , the pip5k2 seedlings could produce as many lateral roots as WT seedlings, while treatment with PtdIns4P or carrier only (which is used to transfer the lipid into plant cells [22,54]) had no effect. This indicates that the reduced lateral root formation of pip5k2 is indeed caused by insufficient endogenous PtdIns(4,5)P 2 production.…”

“…The catalytic activities of using PtdIns4P as the preferred substrate to generate PtdIns(4,5)P 2 have all been characterized [15][16][17][18]. Studies have revealed the physiological functions of some PIP5Ks, including PIP5K1 in abscisic acid signaling [19]; PIP5K3 in root hair formation and growth [16,20]; PIP5K4 in stomata opening [21]; and PIP5K4-6 in pollen tube growth [17,22]. In addition, PIP5K9 regulates root growth and is involved in sugar signaling [23].…”

A Dibasic Amino Acid Pair Conserved in the Activation Loop Directs Plasma Membrane Localization and Is Necessary for Activity of Plant Type I/II Phosphatidylinositol Phosphate Kinase

“…However, in terms of enzymatic activity, plant type I/II PIPKs resemble animal type I PIPKs, with a lack of activity toward the PtdIns5P found in animal type II PIPKs. In contrast to the structural and biochemical characteristics, little is known about the function of plant type I/II PIPKs in physiological processes; AtPIP5K3 regulates tip growth of root hairs (Kusano et al, 2008;Stenzel et al, 2008), whereas AtPIP5K4 and AtPIP5K5 are involved in apical growth of pollen tubes Sousa et al, 2008). In P. patens PIPKs, although the functions of PpPIPKs are not clear, it is likely that PpPIPK1 and PpPIPK2 are functionally distinct because of the unique enzymatic activity of PpPIPK2 as a PtdIns kinase (Saavedra et al, 2009).…”

“…Despite the establishment of the roles of PIPKs in polarized tip growth of root hairs and pollen tubes via their localization at plasma membranes in Arabidopsis Kusano et al, 2008;Sousa et al, 2008;Stenzel et al, 2008), the regulatory mode of this plasma membrane localization remains elusive in plants. Here, we identified a dibasic amino acid pair, KR, in the activation loop of PpPIPK1, the biological relevance of which is 2-fold.…”

“…Physiological roles of several plant PIPKs have been reported. In Arabidopsis (Arabidopsis thaliana), AtPIP5K3 is an essential regulator of tip growth of root hairs (Kusano et al, 2008;Stenzel et al, 2008), while AtPIPK4 and AtPIPK5 are essential for pollen germination and pollen tube elongation Sousa et al, 2008). In addition, AtPIP5K9 was shown to interact with the cytosolic invertase CINV1 to regulate sugar-mediated root cell elongation negatively (Lou et al, 2007).…”