Phosphatidylinositol 4,5-Bisphosphate Influences PIN Polarization by Controlling Clathrin-Mediated Membrane Trafficking inArabidopsis   

Abstract

The functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P 2 ] 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)P 2 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 auxindependent 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)P 2 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)P 2 in the control of clathrin dynamics and in auxin distribution in Arabidopsis.

Primary root protophloem differentiation requires balanced phosphatidylinositol-4,5-biphosphate levels and systemically affects root branching

“…This interpretation also matches the previous observations that auxin efflux carrier abundance is reduced in the meristems of brx mutants (Scacchi et al, 2010) and that brx seedlings contain more free auxin per fresh weight (Sankar et al, 2011). It is also consistent with the proposed influence of the PIP to PIP 2 ratio on auxin efflux carrier polarization and plasma membrane abundance (Ischebeck et al, 2014;Tejos et al, 2014). It thus appears possible that locally disturbed polar auxin transport interferes with protophloem sieve element differentiation.…”

“…Similar to 5-phosphatases, the PIP5K genes are a family for which developmental roles are only about to emerge. For example, recently it has been reported that pip5k1 pip5k2 double mutants display severely reduced root growth and interrupted foliar vasculature (Ischebeck et al, 2014;Tejos et al, 2014), similar to our transgenic CVP2 overexpression lines. It will therefore be interesting to determine whether protophloem differentiation is impaired in pip5k1 pip5k2 double mutants as well as in pip5k1 single mutants (which already display reduced primary root growth).…”

“…Although most enzymatic and regulatory proteins that have been shown to be involved in phosphoinositide turnover and signaling are conserved across the eukaryotes, their cellular and developmental roles in plants remain comparatively obscure to date (Boss and Im, 2012;Ischebeck et al, 2014). However, recent data suggest that PIP and PIP 2 are indeed signaling molecules in plants (Munnik and Nielsen, 2011).…”

“…Although a positively skewed PIP to PIP 2 ratio could be measured in older, whole pip5k1 pip5k2 seedlings when PIP 2 levels were determined by quantifying the associated fatty acids by gas chromatography and mass spectrometry (Ischebeck et al, 2014), this could not be detected in pip5k1 pip5k2 double mutant roots with the same experimental approach using labeling as in this study (Tejos et al, 2014). This suggests that the effect is too small and too localized, and could therefore be masked by the small relative abundance of the pertinent tissue.…”

Phosphoinositide signaling in plant development

“…In fact, the physiological consequences of enhanced PI formation tend to be rather limited and highly specific, raising the question of how the specificity of PI functions is achieved (Heilmann and Heilmann, 2013). A key piece of information for understanding PI function is that these lipids do not display uniform distribution within membranes (Ischebeck et al, , 2011(Ischebeck et al, , 2013Kost et al, 1999;Kusano et al, 2008;Sousa et al, 2008;Tejos et al, 2014;van Leeuwen et al, 2007;Vermeer et al, 2009;Vincent et al, 2005). Instead, PIs are enriched in certain membrane domains, and it is likely that such microdomains play a part in mediating the specific effects of PIs (Fig.…”