Local Positive Feedback Regulation Determines Cell Shape in Root Hair Cells

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Science  29 Feb 2008:
Vol. 319, Issue 5867, pp. 1241-1244
DOI: 10.1126/science.1152505


The specification and maintenance of growth sites are tightly regulated during cell morphogenesis in all organisms. ROOT HAIR DEFECTIVE 2 reduced nicotinamide adenine dinucleotide phosphate (RHD2 NADPH) oxidase–derived reactive oxygen species (ROS) stimulate a Ca2+ influx into the cytoplasm that is required for root hair growth in Arabidopsis thaliana. We found that Ca2+, in turn, activated the RHD2 NADPH oxidase to produce ROS at the growing point in the root hair. Together, these components could establish a means of positive feedback regulation that maintains an active growth site in expanding root hair cells. Because the location and stability of growth sites predict the ultimate form of a plant cell, our findings demonstrate how a positive feedback mechanism involving RHD2, ROS, and Ca2+ can determine cell shape.

Cells develop polarized shapes by generating and maintaining localized sites of growth. Although many proteins and physiological processes that are required for tip growth have been identified, the manner by which they interact in the stable maintenance of local growth sites is unknown in plants. Important among these processes in growing root hair cells of A. thaliana is the elevation of the concentration of cytoplasmic Ca2+ and the localized production of ROS by the RHD2 NADPH oxidase (14). We hypothesized that RHD2 protein would be located at the sites of growth and ROS accumulation and therefore determined the distribution of RHD2 protein using transgenic plants expressing RHD2 fused with green fluorescent protein (GFP) under the control of the RHD2 promoter and terminator. The GFP:RHD2 (N-terminal fusion) complemented the Rhd2 phenotype (fig. S1). RHD2 was present in all epidermal cells of the elongation zone but became restricted to the trichoblasts (specialized cells that form root hairs) in the zone just before hair outgrowth occurred (fig. S2), where it accumulated at future sites of hair outgrowth (Fig. 1, A and B). RHD2 remained at the site of growth during hair elongation and disappeared when growth stopped (Fig. 1, C to F). These data suggest that the localization of RHD2 restricts ROS production to the growth points of the cell.

Fig. 1.

RHD2 is located at the growing tip of root hair cells. GFP:RHD2 accumulation in initiating hairs (A and B) and growing hairs (C to F). (G and H) GFP:RHD2 is located at the plasma membrane (arrows), which is pulled back from cell wall (arrowheads) in a root treated with 450 mM mannitol to cause plasmolysis. (I and L) FM4-64 labeling (red). (J and M) GFP:RHD2 (green). (K and N) merged channels (yellow). Before BFA treatment, [(I) to (K)]; after treatment of 25 μM BFA for 30 min, [(L) to (N)]. (O and P) GFP: RHD2 in the ectopic hair bulges of scn1-1 mutant. (Q and R) GFP:RHD2 accumulated in cytosolic clumps of der1-2 root hairs. (S and T) GFP:RHD2 localization was not affected by dimethyl sulfoxide (DMSO) control (S), whereas treatment of 10 μM cytD for 2 hours caused GFP:RHD2 to locate to clumps inside the cell (T). (U to X) GFP:RHD2 is localized at the tip of deformed hairs after treatment with 5 μM taxol [(U) and (V)] or 5 μMoryzalin [(W) and (X)]. (Y and Z) GFP:RHD2 located at the growth sites in erh3-3 trichoblast. (A), (C), (E), (G), (O), (Q), (U), (W), and (Y), bright-field images; (B), (D), (F), (H), (P), (R), (V), (X), and (Z), GFP images from charge-coupled device camera; (I) to (N), (S), and (T), fluorescent images from confocal microscopy. Scale bars, 10 μm.

Given that RHD2 carries six putative transmembrane domains (57), we predicted that RHD2 would be located in the plasma membrane. Indeed, RHD2 colocalized with the plasma membrane when separated from the cell wall after plasmolysis (Fig. 1, G and H). This localization depended on vesicle trafficking because treatment with brefeldin A (BFA) resulted in the colocalization of RHD2 and the endocytic vesicle marker FM4-64 in small aggregates within cells (Fig. 1, I to N) (8). Thus, the decrease in root hair length observed upon BFA treatment is due at least in part to RHD2 delocalization (9), and endocytotic turnover is likely to be an important process maintaining RHD2 in the plasma membrane.

ROS accumulate ectopically in supercentipede1 (scn1) mutant trichoblasts, suggesting that the Rho guanosine triphosphate dissociation inhibitor (RhoGDI)—SCN1—restricts ROS production to a single site (2). Because these foci of ectopic ROS production result from RHD2 activity, we predicted that SCN1 controls the spatial accumulation of RHD2. We found that RHD2 accumulated in several distinct foci in the scn1 mutant as compared with the wild type, where it accumulated at a single location (Fig. 1, O and P). Thus, SCN1 RhoGDI restricts the accumulation of RHD2 to a single site within the trichoblast.

Because a target of SCN1 RhoGDI regulation [ROP2 guanosine triphosphatase (GTPase), which is located to the hair tip (2, 10)] regulates actin microfilament dynamics in root hair cells (11), we predicted that microfilaments would spatially control the distribution of RHD2 within the cell. RHD2 accumulated in cytoplasmic clumps in the deformed root hairs 1 (der1) mutant, which lacks the ACTIN2 protein (12) (Fig. 1, Q and R). Furthermore, RHD2 accumulated in clumps in the cytoplasm upon treatment of wild type with the microfilament-disrupting drug cytochalasin D (cytD) (Fig. 1, S and T) at the concentration that did not change the polar localization of ROP GTPase in the trichoblast (13). To test whether microtubules were also required, RHD2 localization was determined in the ectopic root hair 3 (erh3) mutant in which microtubules are disorganized and in wild type treated with the microtubule-disrupting drugs oryzalin or taxol (14, 15). RHD2 was located at the growing tips in all cases (Fig. 1, U to Z), indicating that microfilaments but not microtubules are required for the localization of RHD2 to the growing tip.

Given that RHD2 carries two conserved EF-hand motifs near its N terminus where Ca2+ binds (Fig. 2A) (5, 7) and that ROS produced by RHD2 activate hyperpolarization-activated Ca2+ channels, causing Ca2+ influx into cells (1), we hypothesized that Ca2+ could activate RHD2, forming a positive feedback network. To test this hypothesis, we determined the role of Ca2+ in regulating ROS production by the RHD2 in human embryonic kidney (HEK) 293T cells in which NADPH oxidase activity is abolished (16, 17). HEK293T cells were transfected with a full-length RHD2 cDNA fused with FLAG tag that allowed us to confirm the production of RHD2 protein by immune blotting (Fig. 2B). To determine whether Ca2+ stimulates RHD2 NADPH oxidase activity, we treated the transfected HEK293T cells with ionomycin, an ionophore that causes Ca2+ influx into cells, thereby raising the concentrations of cytoplasmic Ca2+ (18). Ionomycin treatment transiently activated RHD2 ROS production as compared with that in the empty vector transfection control (Fig. 2C), indicating that Ca2+ stimulated RHD2 NADPH oxidase activity. Ionomycin-induced ROS production in the RHD2-transfected cells was inhibited by treatment with diphenylene iodonium (DPI), a general inhibitor of flavin-containing oxidases, in a dose-dependent manner without changing RHD2 protein levels (fig. S3A). Thus, the elevation in cytoplasmic Ca2+ concentrations stimulates ROS production in the transfected cells through the activation of RHD2.

Fig. 2.

Ca2+ activates RHD2. (A) The Ca2+-binding amino acid residues (X, Y, Z, –X, –Y, and –Z) in RHD2 EF-hand motifs. Asterisks indicate nonconserved amino acids. (B) Protein levels in HEK293T cells shown by immune blotting with actin as a loading control. (C) Ionomycin treatment induced ROS production. Arrow indicates the time point when ionomycin was added. RLU, relative luminescence units. Error bars indicate SE. (D) Subcellular localization of GFP:RHD2 carrying the E250A substitution in a trichoblast. Scale bars, 10 μm. (E) Rescue of rhd2 root hair defects by GFP:RHD2 fusion genes. Scale bars, 500 μm. (F) Elongating hairs located within 2.5 mm from initiation were measured in different backgrounds and represented by percentage of hair length.

To investigate whether Ca2+-dependent activation is mediated by EF-hand motifs in the RHD2 protein, we changed the last glutamic acid residue of the Ca2+-binding loop in the first hand motif to an alanine [Glu250→Ala250 (E250A) (19)] (Fig. 2A). This glutamic acid is conserved among EF-hand motifs and is required for Ca2+ binding (20). Ionomycin-induced ROS production in cells transfected with RHD2 harboring the E250A substitution was 50 to 60% lower than that in cells transfected with wild-type (WT) RHD2 (Fig. 2C), despite the levels of RHD2 protein in each transfected cell type being the same (Fig. 2B). Similarly, other substitutions in conserved amino acid residues within the EF-hand motifs (D239A or G244E in the first hand; D283A or G288E in the second hand; Fig. 2A) decreased ROS production as compared with WT controls, without affecting the protein levels (fig. S3B). Thus, Ca2+ binding to the EF-hand motifs is required for the activation of ROS production by RHD2.

Because Ca2+ may control microfilament dynamics (3, 21), we determined whether Ca2+ binding through the EF hand regulated the subcellular localization of RHD2. The subcellular localization of GFP:RHD2E250A was identical to that of the WT GFP:RHD2 protein (Fig. 2D), indicating that although Ca2+ activates RHD2 NADPH oxidase activity through the EF hand, it does not regulate the distribution of RHD2 protein within the cell.

Given the importance of the EF hand in Ca2+-regulated activation of RHD2, we predicted that the RHD2 protein with a mutated EF-hand motif would not promote root hair elongation. To test this hypothesis, we expressed a GFP:RHD2 gene carrying a E250A substitution (GFP:RHD2E250A) in the rhd2 mutant background. GFP:RHD2E250A did not complement defects of the rhd2 mutant, whereas plants transformed with the WT GFP:RHD2 construct developed WT root hairs (Fig. 2, E and F). This suggests that Ca2+ binding to the RHD2 EF-hand motif is required for ROS production and root hair tip growth.

We identified possible sites of phosphorylationon RHD2 by comparing the amino acid sequence of RHD2 with other members of the family (Fig. 3A) (22) and predicted that RHD2 would be regulated by phosphorylation. To test whether these residues were phosphorylated directly, we performed in vitro kinase assays using a recombinant peptide corresponding to amino acids 316 to 351 of RHD2 (RHD2316–351). RHD2316–351 was directly phosphorylated in a Ca2+-dependent manner (Fig. 3B). The peptide in which both serine residues were changed to alanine (RHD2316–351-S318/322A) was not phosphorylated (Fig. 3B). To determine whether the phosphorylation of RHD2 enhanced NADPH oxidase activity, we determined the effect of calyculin A (CA)—a phosphatase inhibitor that causes proteins to remain phosphorylated (23)—on ROS production by RHD2. We expressed a full-length RHD2 cDNA (RHD2-WT) in HEK293T cells (Fig. 3C). ROS production of RHD2-WT was activated upon addition of CA (Fig. 3D), indicating that phosphorylation stimulates ROS production by RHD2. CA-induced ROS production was suppressed by DPI in a dose-dependent manner (fig. S3C), indicating that the ROS production results from the stimulation of the NADPH oxidase activity of RHD2.

Fig. 3.

Ca2+-dependent phosphorylation activates RHD2. (A) Alignment of region containing putative phosphorylation residues of RHD2, AtrbohD, and AtrbohF. Arrows highlight conserved phosphorylation serine residues. (B) In vitro kinase assays of RHD2316–351. “S” indicates WT protein, and “A” indicates protein in which 318 and 322 serine residues are converted to alanines. (C) Protein levels in HEK293T cells confirmed by immune blotting. (D) CA enhanced ROS production in WT RHD2 and RHD2-S318/322A. Arrow indicatesthe time point when CA was added. Error bars indicate SE. (E) Subcellular localization of GFP:RHD2 with S318/322A substitution. Scale bars, 10 μm.

To test whether this CA-stimulated ROS production is mediated by conserved serine residues, we determined the levels of ROS produced by full-length RHD2 cDNA carrying the S318/322A substitution (RHD2-S318/322A). The mutant proteins produced less ROS than WT proteins in response to CA treatment, without changing the protein levels (Fig. 3, C and D). Furthermore, these substitutions did not change the subcellular localization of RHD2 (Fig. 3E). Taken together, these data indicate that Ca2+-dependent phosphorylation is required for the activation of RHD2.

We investigated if Ca2+ binding to the EF hand and Ca2+-dependent phosphorylation act together to control ROS production by RHD2. When cells were pretreated with CA, the transient ROS production in response to ionomycin treatment was increased as compared with that in cells treated only with CA or ionomycin (Fig. 4). This suggests that RHD2-catalyzed ROS production is synergistically activated by cytosolic Ca2+ and Ca2+-dependent phosphorylation. The amino acid substitutions in the EF-hand motifs or phosphorylation sites caused a greater than 50% reduction in ROS production as compared with that in RHD2-WT (Fig. 4 and fig. S3D), and versions of the RHD2 protein carrying amino acid substitutions in both the phosphorylation sites and EF hand produced even less ROS (Fig. 4). Thus, these different domains of the RHD2 NADPH oxidase serve as targets for Ca2+-mediated activation.

Fig. 4.

Synergistic activation of RHD2 by Ca2+ and Ca2+-dependent phosphorylation. Arrows indicate the time points when CA or ionomycin was added. Error bars indicate SE.

Our data provide evidence for a two-part mechanism that determines the shape of root hair cells (fig. S4). A prerequisite is the apical localization of RHD2 protein at the hair cell tip. Once located at the tip, a positive feedback loop involving RHD2 is initiated. ROS derived from the RHD2 NADPH oxidase may activate hyperpolarization-activated Ca2+ channels that transport Ca2+ into the cells that in turn activates RHD2 NADPH oxidase activity through its EF hand and a Ca2+-dependent protein kinase activity. Such a system of positive feedback, in concert with an independent mechanism for locating RHD2 protein to the tip of the cell, provides a robust mechanism that explains how cells such as root hairs maintain polarity during morphogenesis.

Supporting Online Material

Materials and Methods

Figs. S1 to S4

Tables S1 and S2


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