OsCSLD1, a cellulose synthase-like D1 gene, is required for root hair morphogenesis in rice

Abstract

Root hairs are long tubular outgrowths that form on the surface of specialized epidermal cells. They are required for nutrient and water uptake and interact with the soil microflora. Here we show that the Oryza sativa cellulose synthase-like D1 (OsCSLD1) gene is required for root hair development, as rice (Oryza sativa) mutants that lack OsCSLD1 function develop abnormal root hairs. In these mutants, while hair development is initiated normally, the hairs elongate less than the wild-type hairs and they have kinks and swellings along their length. Because the csld1 mutants develop the same density and number of root hairs along their seminal root as the wild-type plants, we propose that OsCSLD1 function is required for hair elongation but not initiation. Both gene trap expression pattern and in situ hybridization analyses indicate that OsCSLD1 is expressed in only root hair cells. Furthermore, OsCSLD1 is the only member of the four rice CSLD genes that shows root-specific expression. Given that the Arabidopsis (Arabidopsis thaliana) gene KOJAK/AtCSLD3 is required for root hair elongation and is expressed in the root hair, it appears that OsCSLD1 may be the functional ortholog of KOJAK/AtCSLD3 and that these two genes represent the root hair-specific members of this family of proteins. Thus, at least part of the mechanism of root hair morphogenesis in Arabidopsis is conserved in rice.

Figure 1.

Transcript analysis of OsCSLD1∷Ds. A, Genomic structure of OsCSLD1 showing the position of the trap Ds element. The open reading frame comprised two exons that were 2,676 and 708 bp in size and a short 87-bp intron. The black boxes indicate exons, while the eight small gray boxes indicate the sequences that encode membrane-spanning domains. The arrows correspond to three variable regions. B, Northern analysis of OsCSLD1 mRNA. Total RNA was extracted from the roots of wild-type and OsCSLD1∷Ds plants. The probe was from the 3′ UTR of the OsCSLD1 gene. C, Schematic depiction of the spliced junction of the OsCSLD1∷Ds fusion transcript. Shown are part of the first exon and the GUS of the trap Ds. int., A partial intron fused to the GUS coding region. The putative splicing donor (S.D.) and acceptor sites (S.A.) are indicated by the bent arrow. The primers used to clone the cDNA are indicated by the small arrows. D, The cDNA sequence of the spliced junction of the OsCSLD1∷Ds fusion transcript. The Ds sequence is underlined and S.J. indicates the splicing junction. The ATG codon in a box is the start codon of GUS.

Figure 2.

RT-PCR analysis of OsCSLD1 mRNA expression in seedling roots of wild type, mutant, and three revertant lines (Rev0, Rev6a, and Rev6b). Total RNA was extracted from roots of 15-d-old seedlings. RT-PCR (25 cycles) was conducted with oligo(dT) primed cDNAs using OsCSLD1 gene-specific primers. The probe was the 3′ terminal genomic DNA of the OsCSLD1 gene. Actin mRNA and genomic DNA (gDNA) were used as controls.

Figure 3.

Root phenotypes of wild type, OsCSLD1 mutants, and revertants. A, Gross root phenotype of soil-grown seedlings from wild-type, OsCSLD1∷Ds, and OsCSLD1-Rev0 plants. Germinated seeds were grown in soil for 10 d. B, Growth rates of seminal roots of MS-grown seedlings from wild type, the two mutants (OsCSLD1∷Ds and oscsld1-7a), and the two revertants (OsCSLD1-Rev0 and OsCSLD1-Rev6a). Sterilized seeds were germinated and grown in soil for 10 d. For measurements, germinating seeds that sprouted seminal roots to the same extent were used. oscsld1-7b and OsCSLD1-Rev6b which growth rates were similar to ones of OsCSLD1∷Ds and OsCSLD1-Rev0, respectively, were not shown. [See online article for color version of this figure.]

Figure 4.

SEM images of the seminal roots of wild-type, OsCSLD1∷Ds, Rev0, Rev6a, Rev6b, oscsld1-7a, and oscsld1-7 seedlings. Seedlings were grown vertically for 3 d on MS media. The seminal roots were fixed and observed by SEM. The photos show the root hair and elongation zones. Bar = 200 μm.

Figure 5.

Epidermal morphology of the seminal roots from wild-type, OsCSLD1∷Ds, and OsCSLD1-Rev0 seedlings in the hair and elongation zones. A to C, The hair zone in wild type, OsCSLD1∷Ds, and OsCSLD1-Rev0, respectively. D to F, Close ups of root hairs in the wild type (D) and OsCSLD1∷Ds (E and F). G and H, The elongation zone in wild type and OsCSLD1∷Ds. Bulges are where root hairs are emerging. Seedlings were grown vertically for 3 d on MS media and the epidermal cells were observed by cryo-SEM. Bars = 100 μm (A–C) and 50 μm (D–H).

Figure 6.

Expression patterns of the OsCSLD subfamily. Total RNA was isolated from the roots and leaves of 15-d-old plants and the flowering tissues of mature rice plants at two different stages (booting and heading). Total RNA (30 μg) was loaded in each lane. Equal loading was confirmed by ethidium bromide staining. Northern hybridizations were performed with gene-specific probes for OsCSLD1, OsCSLD2, OsCSLD3, and OsCSLD4, as described in “Materials and Methods.”

Figure 7.

GUS patterns in OsCSLD1∷Ds/+ roots. A, The root hair zone under the light microscope. B, A root hair cell under high magnification (400×). The GUS stain appeared to be contained in an internal compartment. C, The GUS staining of the whole seminal root. D, GUS staining between the elongation zone and the root cap was not detected. E, Below the root hair zone, GUS expression in the elongation zone and in the root cap. The roots of 5-d-old heterozygous seedlings were stained with 0.5% 5-bromo-4-chloro-3-indoyl glucuronide.

Figure 8.

In situ localization of OsCSLD1 mRNA and GUS staining of 5-d-old roots from wild-type and OsCSLD1∷Ds/+ seedlings, respectively. A, In situ hybridization with a medial longitudinal section. Sections of 5-d-old wild-type roots were probed with digoxigenin-labeled antisense RNA and viewed under a light microscope. The arrowhead indicates the signal in the epidermal cell layer of the medial longitudinal section. B, Higher magnification of this part of the epidermal layer. C, In situ hybridization with transverse sections. Signal-positive cells are marked with arrowheads. D, In situ hybridization with tangential sections. Sections through the epidermal cell layer were used for hybridization. E, GUS staining of a longitudinal section. GUS-stained roots of 5-d-old of OsCSLD1∷Ds/+ seedlings were embedded in paraffin and sections were viewed under a light microscope. The section shows GUS staining in root hair cells of the epidermis. F and G, GUS staining of transverse and tangential sections, respectively. GUS-positive cells were clearly distinguishable from GUS-negative cells along the epidermal cell layer. H and I, The GUS-positive epidermal cells in the root hair zone and the elongation zone, respectively. GUS-positive cells are indicated by blue shading. GUS-stained roots were fixed and examined by SEM.

Figure 9.

Seminal root morphology and expression of the OsCSLD1-overexpressing line grown in the presence or absence of NPA. A, The epidermal cells of the hair zone observed by cryo-SEM. An OsCSLD1-overexpressing line was grown vertically on MS media. B, Seminal roots grown without or with NPA. Germinated seeds were grown for 5 d in MS media without or with 10⁻⁵ m NPA. C, RT-PCR analysis of OsCSLD1 mRNA. The mRNAs of the roots shown in B were subjected to 25 cycles of RT-PCR using OSCSLD1 gene-specific primers.