Distinct ROPGEFs successively drive polarization and outgrowth of root hairs

Abstract

35Root hairs are tubular protrusions of the root epidermis that significantly enlarge the exploitable soil 36 volume in the rhizosphere. Trichoblasts, the cell type responsible for root hair formation, switch 37 from cell elongation to tip growth through polarization of the growth machinery to a pre-defined root 38 hair initiation domain (RHID) at the plasma membrane. The emergence of this polar domain 39 resembles the establishment of cell polarity in other eukaryotic systems [1][2][3]. Rho-type GTPases 40 of plants (ROPs) are among the first molecular determinants of the RHID [4, 5] and later play a 41 central role in polar growth [6]. Numerous studies have elucidated mechanisms that position the 42 RHID in the cell [7][8][9] or regulate ROP activity [10][11][12][13][14][15][16][17][18]. The molecular players that target ROPs to 43 the RHID and initiate outgrowth, however, have not been identified. We dissected the timing of the 44 growth machinery assembly in polarizing hair cells and found that positioning of molecular players 45 and outgrowth are temporally separate processes that are each controlled by specific ROP guanine 46 nucleotide exchange factor (GEFs). A functional analysis of trichoblast-specific GEFs revealed 47 GEF3 to be required for normal ROP polarization and thus efficient root hair emergence, while 48 GEF4 predominantly regulates subsequent tip growth. Ectopic expression of GEF3 induced the 49 formation of spatially confined, ROP-recruiting domains in other cell types, demonstrating the role 50 of GEF3 to serve as a membrane landmark during cell polarization. 51 3 RESULTS AND DISCUSSION 52 53 Temporal analysis of hair cell polarization reveals phased deployment of the tip-growth 54 machinery 55To dissect the process of breaking cellular symmetry and initiating polar growth in plants, we 56 analyzed the gradual assembly of the tip growth machinery in trichoblasts using specific markers 57 for cytoskeletal rearrangement, plasma membrane specialization, cell wall modification, vesicle 58 trafficking, and Rho-GTPase signaling. Using stable transgenic Arabidopsis lines expressing 59 fluorescently labeled versions of these protein markers, we determined the timing of their 60 polarization at the RHID during trichoblast differentiation. As the Arabidopsis root represents a time-61 axis of development from stem cells to mature cells, we numbered the developmental stages (-7 62 to +3) within a cell file, with the last cell before bulging labeled -1 and the first cell after the onset 63 of bulging labeled +1 ( Figure 1A). We used the integral plasma membrane marker GFP-LTI6B [19] 64 as a reference and calculated the polarity index of fluorescently labeled marker proteins to quantify 65 protein accumulation at the RHID (Figure 1B, C). Our survey unveiled a two-phase assembly of 66 the tip growth machinery: an initiation phase, during which the RHID is positioned and predefined, 67 followed by the tip growth phase. Consistent with previous reports [4, 5], mCitrine-labeled (mCit) 68GTPase ROP2 associ...

iDePP: a genetically encoded system for the inducible depletion of PI(4,5)P2inArabidopsis thaliana

“…Upon dex treatment, all three 205 MAP-mCH-dOCRL, MAP-mCH-dOCRL dead and MAP-3xmCH were expressed in root hairs, and 206 localized to the plasma membrane and intracellular compartments ( Figure 5A, B). Root hairs accumulate 207 PI(4,5)P2 on the plasma membrane at their tip (Denninger et al, 2019;Kusano et al, 2008;Stenzel et 208 al., 2008;Van Leeuwen et al, 2007;Vincent et al, 2005) and expression of MAP-3xmCH did not 209 affected plasma membrane localization of mCIT-2xPH PLC , which remained enriched at the tip of the cell 210 ( Figure 5A We next investigated if the iDePP system was suitable to deplete PI(4,5)P2 in the aerial part of the plant. 215…”

Plant Cell Biology: How to Give Root Hairs Enough ROPs?

“…How ROP proteins are precisely positioned at the RHID in the absence of the core planar polarity pathway is still unclear. A new study in this issue of Current Biology tackled this question and uncovered ROP GEF3 as a key protein driving the initial ROP polarization for the correct positioning of root hairs [3].…”

“…The Arabidopsis thaliana genome encodes for 11 ROPs, which control fundamental cellular processes including cytoskeleton dynamics and vesicular trafficking [5]. To determine the timing of ROP polarization at the RHID, Deniger, Reichelt et al, took advantage on the fact that cells along the root tip are ordered according to their developmental age, with younger cells located rootward and older cells shootward [3]. In this developmental timeline, the cell with the first visible root hair bulge is numbered as +1 ( Figure 1).…”

“…In this developmental timeline, the cell with the first visible root hair bulge is numbered as +1 ( Figure 1). Using this staging system, the authors separated root hair formation in three phases: initiation (from cells -7 to -1, in blue), bulging (cells +1 and +2, in green), and polar growth/elongation (starting at cell +3 and onward, in orange) [3]. Using confocal imaging and fluorescently-tagged ROPs, Deniger, Reichelt et al, showed that three ROPs (ROP2, ROP4 and ROP6) polarize at the RHID much before root hair initiation (i.e.…”

“…at cell -4 before root hair bulging, Figure 1). Accordingly, genetic analyses with double and triple rop mutants suggest that these three ROPs redundantly regulate root hair development [3,6].…”