Functional analysis of the cellulose synthase-like genes CSLD1, CSLD2, and CSLD4 in tip-growing Arabidopsis cells

Abstract

A reverse genetic approach was used to investigate the functions of three members of the cellulose synthase superfamily in Arabidopsis (Arabidopsis thaliana), CELLULOSE SYNTHASE-LIKE D1 (CSLD1), CSLD2, and CSLD4. CSLD2 is required for normal root hair growth but has a different role from that previously described for CSLD3 (KOJAK). CSLD2 is required during a later stage of hair development than CSLD3, and CSLD2 mutants produce root hairs with a range of abnormalities, with many root hairs rupturing late in development. Remarkably, though, it was often the case that in CSLD2 mutants, tip growth would resume after rupturing of root hairs. In silico, semiquantitative reverse transcription-polymerase chain reaction, and promoter-reporter construct analyses indicated that the expression of both CSLD2 and CSLD3 is elevated at reduced temperatures, and the phenotypes of mutants homozygous for insertions in these genes were partially rescued by reduced temperature growth. However, this was not the case for a double mutant homozygous for insertions in both CSLD2 and CSLD3, suggesting that there may be partial redundancy in the functions of these genes. Mutants in CSLD1 and CSLD4 had a defect in male transmission, and plants heterozygous for insertions in CSLD1 or CSLD4 were defective in their ability to produce pollen tubes, although the number and morphology of pollen grains was normal. We propose that the CSLD family of putative glycosyltransferases synthesize a polysaccharide that has a specialized structural role in the cell walls of tip-growing cells.

Figure 1.

csld2-1 has a defective root hair phenotype that is different from that of mutants with lesions in CSLD3. A to C, Images showing the root hypocotyl junction region of wild-type Col-0 (A), csld2-1 (B), and csld3-1 (C). D to F, Images of the middle portion of roots of wild-type Col-0 (D), csld2-1 (E), and csld3-1 (F). G to J, High-magnification images showing the range of aberrant root hair phenotypes observed in csld2-1. All images are of roots from 10-d-old csld2-1 seedlings. Bars = 50 μm (A–F) and 25 μm (G–J).

Figure 2.

Tip rupture in csld2-1 and csld3-2 and regrowth in csld2-1. A to F, DIC images of root hairs from csld3-2 (A and D), wild-type Col-0 (B and E), and csld2-1 (C and F). The rupturing of root hair tips and loss of cytoplasm are visible in both csld3-2 (D) and csld2-1 (F). G and H, In csld2-1, new root hair growth was apparent after rupturing of hairs both at the tip (G) and flank (H) regions. Double arrows indicate new root hair growth. Asterisks indicate the area of rupture. Bars = 50 μm (A–C), 10 μm (D–F), and 20 μm (G and H).

Figure 3.

The cellular anatomy of csld2 is normal apart from the altered root hair phenotype. A and B, Images showing resin-embedded roots from 12-d-old wild-type Col-0 (A) and csld2-1 (B) seedlings. Overall cellular patterning was normal in csld2-1 roots, and apart from root hairs (rh), all cells had normal morphologies, including atrichoblast epidermal cells (e) and cortical cells (c). C, High-magnification image of a section through a root of csld2-1 showing root hair, atrichoblast epidermal cells, and cortical cells. Double arrows indicate the region of root hair walls that are thickened or of variable thickness. D to L, Transmission electron microscopy of cell walls from wild-type Col-0 (D–F and J), csld2-1 (G–I), csld2-2 (K), and csld3-2 (L). Images were taken of equivalent flank (D, E, G, and H) and tip (F and I) regions of wild-type Col-0 and csld2-1 root hairs. In J to L, note the diffuse appearance of csld2-2 and csld3-2 walls compared with wild-type Col-0 walls. Bars = 50 μm (A–C), 0.2 μm (D–I), and 0.1 μm (J–L).

Figure 4.

Microtubule and F-actin organization is disrupted in living csld2-1 root hairs. A to D, F-actin (A and B) and microtubules (C and D) in the base of wild-type Col-0 (A and C) and csld2-1 (B and D). E to J, F-actin organization in the tip regions of wild-type Col-0 (E) and csld2-1 (G and I), and microtubule organization in the tip regions of wild-type Col-0 (F) and csld2-1 (H and J). F-actin and microtubules were visualized by GFP-ABD2-GFP and GFP-MBD2 fusions, respectively. Bars = 20 μm.

Figure 5.

The altered root hair phenotypes in csld2-1 and csld3-2 are partially rescued by growth at reduced temperature. A to E, csld2-1 (A and B) and csld3-2 (C and D) were grown at 25°C (A and C) or 15°C (B and D). Growth at 15°C resulted in partial rescue of root hair phenotypes in both csld2-1 and csld3-2. However, the phenotype of a double mutant with lesions in CSLD2 and CSLD3 (csld2/3) was not rescued by growth at 15°C (E). The inset in E shows a high-magnification image of a ruptured root hair of the csld2/3 double mutant. F, Quantification of root hair length in csld2-1, csld3-2, and csld2/3. The graph shows average root hair length of 10 seedlings. WT, Wild type. Error bars indicate se. Bars in A to E = 100 μm.

Figure 6.

Effect of reduced temperature on CSLD2 and CSLD3 expression. A, RT-PCR analysis of CSLD2 and CSLD3 genes in the wild type (WT), csld2-1, csld3-2, and csld2/3 mutants. RNA was isolated from roots of 6-d-old seedlings grown at 25°C or 15°C. Arabidopsis translation initiation factor EIF4A2 was used as a constitutive expression control. B, Histochemical analysis of GUS expression in roots of 6-d-old wild-type seedlings carrying constructs of GFP∷GUS driven by the CSLD2 or CSLD3 promoter and grown at 25°C or 15°C.

Figure 7.

The altered root hair phenotype of csld2-1 was restored by transformation with wild-type CSLD2 driven by its own promoter. Images show the root hair morphologies in csld2-1 (A), csld2-1 transformed with the wild-type CSLD2 gene driven by its own promoter (csld2-1+WTD2; B), and wild-type Col-0 (C). All images are of roots from 10-d-old seedlings. Bars = 50 μm.

Figure 8.

csld4-1 pollen has a normal morphology but reduced pollen tube germination. A and C, Resin-embedded pollen from csld4-1 (A) and qrt (C) was sectioned and stained with toluidine blue. B and D, Images from in vitro germination assays showing quartets of pollen grains and pollen tubes from csld4-1 (B) and qrt (D). c, Cytoplasm; i, intine; pc, pollen coat. Bars in A and C = 1 μm.

Figure 9.

Quantification of pollen tube germination rates in CSLD mutants. A, Quantification of the in vitro germination rate of pollen tubes from csld1-1, csld2-1, and csld4-1 compared with wild-type (WT) Col-0 and qrt. Note that pollen tube germination was normal in csld2-1. B, Quantification of the number of pollen tubes germinating from quartets. In csld4-1, the maximum number of pollen tubes germinating from a single quartet was two, whereas up to four tubes germinated from the qrt quartet. Error bars indicate se among three independent experiments with 80 samples each.

Figure 10.

Subcellular localization of CSLD1 and CSLD4. Fluorescently tagged versions of CSLD2 (A), CSLD3 (C), and CSLD4 (E) in which YFP was fused to the N termini were expressed in Nicotiana benthamiana leaves and visualized using confocal laser-scanning microscopy. The Golgi apparatus marker STtmd-GFP (ST-GFP) was also expressed in N. benthamiana leaves (B, D, and F). Bars = 15 μm.