FRAGILE FIBER3, an Arabidopsis Gene Encoding a Type II Inositol Polyphosphate 5-Phosphatase, Is Required for Secondary Wall Synthesis and Actin Organization in Fiber Cells

Abstract

Type II inositol polyphosphate 5-phosphatases (5PTases) in yeast and animals have been known to regulate the level of phosphoinositides and thereby influence various cellular activities, such as vesicle trafficking and actin organization. In plants, little is known about the phosphatases involved in hydrolysis of phosphoinositides, and roles of type II 5PTases in plant cellular functions have not yet been characterized. In this study, we demonstrate that the FRAGILE FIBER3 (FRA3) gene of Arabidopsis thaliana, which encodes a type II 5PTase, plays an essential role in the secondary wall synthesis in fiber cells and xylem vessels. The fra3 mutations caused a dramatic reduction in secondary wall thickness and a concomitant decrease in stem strength. These phenotypes were associated with an alteration in actin organization in fiber cells. Consistent with the defective fiber and vessel phenotypes, the FRA3 gene was found to be highly expressed in fiber cells and vascular tissues in stems. The FRA3 protein is composed of two domains, an N-terminal localized WD-repeat domain and a C-terminal localized 5PTase catalytic domain. In vitro activity assay demonstrated that recombinant FRA3 exhibited phosphatase activity toward PtdIns(4,5)P 2 , PtdIns(3,4,5)P 3 , and Ins(1,4,5)P 3 , with the highest substrate affinity toward PtdIns(4,5)P 2 . The fra3 missense mutation, which caused an amino acid substitution in the conserved motif II of the 5PTase catalytic domain, completely abolished the FRA3 phosphatase activity. Moreover, the endogenous levels of PtdIns(4,5) 2 and Ins(1,4,5)P 3 were found to be elevated in fra3 stems. Together, our findings suggest that the FRA3 type II 5PTase is involved in phosphoinositide metabolism and influences secondary wall synthesis and actin organization.

TheArabidopsisStem Cell Factor POLTERGEIST Is Membrane Localized and Phospholipid Stimulated

“…Various studies in Arabidopsis have identified at least three putative PI(3)P phosphatases and no PI(3) kinases that may use or generate PI(3,4)P 2 (Gillaspy, 2009;Lee et al, 2009). Sequence analyses have also led to the identification of 16 putative PI(5)P phosphatases and four putative PI(5) kinases that may use or generate PI(4,5)P 2 ( Mueller-Roeber and Pical, 2002;Zhong et al, 2004;Gillaspy, 2009). Additionally, there are other potential ways that PI(4)P levels could be modulated, including lipid hydrolysis and lipid transfer between membranes or membrane bilayers, but again little is known about these processes .…”

TheArabidopsis thaliana Myo-Inositol 1-Phosphate Synthase1 Gene Is Required forMyo-inositol Synthesis and Suppression of Cell Death

“…For this work, we used a purified bovine Ins (1,4,5)P 3 receptor in radiolabeled competition binding assays that are well established and used by several plant laboratories (Carland and Nelson, 2004;Zhong et al, 2004;Gunesekera et al, 2007;Perera et al, 2008). We found no differences, with 19-d-old wild-type plants having 404 6 50 pg Ins(1,4,5)P 3 /g tissue and 19-d-old mips1-2 plants having 463 6 91 pg Ins(1,4,5)P 3 /g tissue.…”

Directional cell expansion — turning toward actin

“…Other examples of actin's involvement in cell wall synthesis and cell elongation come from members of the fragile fiber ( fra) family of mutants [46][47][48]. Cell walls in the fiber cells of fra4 (also known as root hair defective 3) are abnormally thin, have altered composition and reduced mechanical strength, and have reduced cell elongation in all organs [46].…”

“…FRA4 encodes a novel protein that has putative GTPbinding motifs [46]. FRA3 encodes an inositol polyphosphate phosphatase and FRA7 encodes a so-called 'suppressor of actin (SAC) domain' phosphatase, which is also active against inositol polyphosphates [47,48]. Both also appear to be necessary for normal actin organization and secondary cell wall formation, with fra7 mutant plants having reduced expansion in most, if not all, organs [48].…”