The Arabidopsis Rop2 GTPase is a positive regulator of both root hair initiation and tip growth - PubMed (original) (raw)

The Arabidopsis Rop2 GTPase is a positive regulator of both root hair initiation and tip growth

Mark A Jones et al. Plant Cell. 2002 Apr.

Abstract

Root hairs provide a model system for the study of cell polarity. We examined the possibility that one or more members of the distinct plant subfamily of RHO monomeric GTPases, termed Rop, may function as molecular switches regulating root hair growth. Specific Rops are known to control polar growth in pollen tubes. Overexpressing Rop2 (Rop2 OX) resulted in a strong root hair phenotype, whereas overexpressing Rop7 appeared to inhibit root hair tip growth. Overexpressing Rops from other phylogenetic subgroups of Rop did not give a root hair phenotype. We confirmed that Rop2 was expressed throughout hair development. Rop2 OX and constitutively active GTP-bound rop2 (CA-rop2) led to additional and misplaced hairs on the cell surface as well as longer hairs. Furthermore, CA-rop2 depolarized root hair tip growth, whereas Rop2 OX resulted in hairs with multiple tips. Dominant negative GDP-bound Rop2 reduced the number of hair-forming sites and led to shorter and wavy hairs. Green fluorescent protein-Rop2 localized to the future site of hair formation well before swelling formation and to the tip throughout hair development. We conclude that the Arabidopsis Rop2 GTPase acts as a positive regulatory switch in the earliest visible stage in hair development, swelling formation, and in tip growth.

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Figures

Figure 1.

Figure 1.

Root Hair Phenotypes of Wild-Type Arabidopsis and Rop7, Rop8, Rop11, and Rop2 OX Transgenic Plants. (A) and (B) Wild-type root hairs. (C) and (D) Overexpression of Rop8 and Rop11, respectively, did not affect hair cell development. (E) Overexpression of Rop7 led to some very short root hairs. (F) and (G) Overexpression of Rop2 led to a dramatic root hair phenotype. In (B) and (G), roots were grown through semisolid medium. In (A) and (C) to (F), roots were grown in contact with the air. (A), (B), (F), and (G) are bright-field images, and (C) to (E) are phase contrast images. Bars = 200 μm. (H) RT-PCR analysis of Rop transgene expression. The relative intensity of different transcripts was similar when either 20 or 30 cycles of PCR amplification were performed. The top band shows actin2 as an internal PCR amplification and template control. The bottom band shows Rop transcript specific for each construct shown. Lane 1, wild-type control*; lane 2, Rop2 OX*; lane 3, CA1-rop2 (CA1-1)*; lane 4, CA2-rop2 (CA2-2); lane 5, Rop7-2; lane 6, Rop7-34*; lane 7, Rop8-25*; lane 8, Rop8-32*; lane 9, Rop11-4*; lane 10, Rop11-7*. Asterisks indicate lines used for the characterization of phenotypes shown in this figure, although >20 independent lines with similar phenotypes were obtained for each construct. The CA-rop2 mutants (CA1-1 and CA2-2) were described previously (Li et al., 2001).

Figure 2.

Figure 2.

Rop2 Is Expressed in Root Hair Cells. (A) Nested RT-PCR using primers specific for the 5′ and 3′ untranslated regions of the native Rop2 transcript and mRNA from 16 root hair cells at the stage of swelling formation. (B) and (C) Whole mount in situ hybridization using Rop2-specific sense (B) and antisense (C) probes. (D) and (E) β-Glucuronidase staining of Rop2 promoter:GUS and CaMV 35S promoter:GUS transgenic lines, respectively, confirming that both were expressed in growing root hairs. Bars in (B) and (C) = 10 μm; bar in (E) = 100 μm for (D) and (E).

Figure 3.

Figure 3.

Rop2 Overexpression Affects the Number and Position of Hair-Forming Events in Root Hair Cells. (A) Single wild-type root hair. (B) and (C) Rop2 OX hair cells showing multiple swellings and hairs located away from the root tip end of the cell. Arrowheads show end cell walls, and arrows show root hairs or swellings. Bars = 50 μm.

Figure 4.

Figure 4.

Effects of Rop2 Transgene Expression on Tip Growth. (A) Tip of growing wild-type root hair. (B) and (C) Tips of growing Rop2 OX hairs showing different degrees of branching. (D) to (G) Root hair density and length on wild-type (D), Rop2 OX (E), DN-rop2 (F), and CA-rop2 (G) roots. (H) DN-rop2 expression led to an increased number of hairs that stopped growing before reaching 40 μm in length (i.e., they did not make the transition to tip growth). (I) Wavy DN-rop2 hair. (J) CA-rop2 expression led to depolarization of hairs grown in contact with the air. Bars in (A) to (C), (H), and (I) = 100 μm; bars in (D) to (G) = 500 μm; bar in (J) = 200 μm.

Figure 5.

Figure 5.

GFP::Rop2 Localizes to the Future Site of Hair Formation, Remains during Swelling Formation, and Localizes to the Tips of Growing Hairs throughout Hair Development. Confocal laser scanning microscopy of root hair cells on transgenic Arabidopsis plants expressing a fusion of Rop2 and GFP. (A) Bright-field image showing four cells in the same cell file, all at different stages of hair development. A box surrounds the youngest two cells. The youngest cell (far left) has not begun hair formation. The next youngest (center left) has just begun to form a swelling. The older two hairs (at right) have stopped growing. (B) GFP::Rop2 fluorescence of the cells shown in (A). (C) Enlargement of the boxed area in (B). (D) A cell that is about to make a hair. GFP::Rop2 is localized to the site of future hair formation. (E) to (K) GFP::Rop2 is localized at the hair tip during swelling formation (E), the transition to tip growth (F), and tip growth ([G] to [I] and [K]). (G) is a bright-field image of the hair shown in (H). In (J), GFP-Rop2 is absent from the tips of hairs that have stopped growing. Bars in (A) and (B) = 40 μm; bars in (C) to (K) = 10 μm.

Figure 6.

Figure 6.

Effects of CA-rop2 and DN-rop2 Expression on F-Actin Localization in Root Hairs. F-actin was visualized in live cells using transiently expressed GFP-mTalin as described in the text. Root hairs expressing GFP-mTalin were observed using confocal microscopy. Images shown are projections of scanning laser sections (1 μm) along the axes of root hairs. (A) In wild-type hairs, axial actin cables end at the subapical region (arrow), whereas the apex contains fine F-actin (arrowhead). (B) In CA-rop2 hairs, an extensive actin network was found throughout the cortex. (C) In DN-rop2 hairs, axial actin cables protruded to the extreme apex and no fine F-actin was found in the apex. For each line, more than six hairs were observed and showed identical or very similar staining patterns. Bar in (A) = 20 μm for (A) to (C).

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