Type B Phosphatidylinositol-4-Phosphate 5-Kinases MediateArabidopsisandNicotiana tabacumPollen Tube Growth by Regulating Apical Pectin Secretion

Abstract

Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P 2 ] occurs in the apical plasma membrane of growing pollen tubes. Because enzymes responsible for PtdIns(4,5)P 2 production at that location are uncharacterized, functions of PtdIns(4,5)P 2 in pollen tube tip growth are unresolved. Two candidate genes encoding pollen-expressed Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinases (PI4P 5-kinases) of Arabidopsis subfamily B were identified (PIP5K4 and PIP5K5), and their recombinant proteins were characterized as being PI4P 5-kinases. Pollen of T-DNA insertion lines deficient in both PIP5K4 and PIP5K5 exhibited reduced pollen germination and defects in pollen tube elongation. Fluorescence-tagged PIP5K4 and PIP5K5 localized to an apical plasma membrane microdomain in Arabidopsis and tobacco (Nicotiana tabacum) pollen tubes, and overexpression of either PIP5K4 or PIP5K5 triggered multiple tip branching events. Further studies using the tobacco system revealed that overexpression caused massive apical pectin deposition accompanied by plasma membrane invaginations. By contrast, callose deposition and cytoskeletal structures were unaltered in the overexpressors. Morphological effects depended on PtdIns(4,5)P 2 production, as an inactive enzyme variant did not produce any effects. The data indicate that excessive PtdIns(4,5)P 2 production by type B PI4P 5-kinases disturbs the balance of membrane trafficking and apical pectin deposition. Polar tip growth of pollen tubes may thus be modulated by PtdIns(4,5)P 2 via regulatory effects on membrane trafficking and/or apical pectin deposition.

Exocytosis Precedes and Predicts the Increase in Growth in Oscillating Pollen Tubes

“…While the chemical basis for PI fluorescence in the pollen tube apical wall has not been established, we suspect that its positive charge allows the dye to interact with negative charges on pectin (i.e., carboxyl residues) in a manner similar to several other pectin dyes, such as ruthenium red (Ischebeck et al, 2008;Szumlanski and Nielsen, 2009), coriphosphine O (Weis et al, 1988), and cobalt and nickel (Varner and Taylor, 1989;Jauneau et al, 1997). That PI fluorescence and DIC thickness measurements provide nearly identical results strongly supports the conclusion that these two independent assays respond to the same feature.…”

“…Although the basis for staining is not known, PI carries positive charges and may interact with the negative charges that are present on the pectins, especially the carboxyl residues. The basis for PI staining may therefore be similar to other reagents better known for their ability to react with pectin, such as ruthenium red (Ischebeck et al, 2008;Szumlanski and Nielsen, 2009), coriphosphine O (Weis et al, 1988), and nickel or cobalt (Varner and Taylor, 1989;Jauneau et al, 1997), which also carry positive charges. We have applied PI to growing pollen tubes of lily and tobacco.…”

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

“…Besides 4 homologs of yeast PIP5K, 11 PIPK isoforms can be divided into two subfamilies (A and B) according to their domain organization [14]. 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].…”

PIP kinases and their role in plant tip growing cells

“…10 It is clear that it is not the N-terminal MORN domain responsible for these differences in phenotypes observed between type A and B subfamilies, since overexpression of mutated isoforms lacking the MORN domain from AtPIP5K3 or AtPIP5K5 resulted in the same phenotypes as that of full-length proteins. 9,11 In P. patens, both PIPKs are expressed in protoplasts, protonema and gametophores. 7 The disruption of both genes by gene targeting showed that both enzymes are involved in tip growth.…”

“…It has been shown that PtdIns4P is the preferred substrate in vitro for the synthesis of PtdIns(4,5)P 2 by all AtPIPKs; [8][9][10][11][12] this is also the case for PpPIPK1, but not for PpPIPK2, which in vitro prefers PI to produce PtdIns3P. 7 However, we have shown that in vivo both PpPIPK1 and PpPIPK2 catalyze the synthesis of PtdIns(4,5)P 2 , since the knockout lipid profile showed a reduction only in PtdIns(4,5)P 2 in both pipk1 and pipk2 mutants.…”