The cell wall hydroxyproline-rich glycoprotein RSH is essential for normal embryo development in Arabidopsis

Abstract

Although a large number of embryo mutants have been studied, mostly at the morphological level, the critical molecular and cellular events responsible for embryogenesis are unknown. Here, we report that using an enhancer-trap embryo mutant of Arabidopsis, we identified a gene, ROOT-SHOOT-HYPOCOTYL-DEFECTIVE (RSH), that is essential for the correct positioning of the cell plate during cytokinesis in cells of the developing embryo. We traced the earliest point of influence of RSH to the first asymmetrical division of the zygote. Homozygous rsh embryos were defective morphologically, had irregular cell shape and size, and germinated to form agravitropic-defective seedlings incapable of further development. The RSH gene encodes a Hyp-rich glycoprotein-type cell wall protein. RSH localized to the cell wall throughout the embryo and to a few well-defined postembryonic sites. Although several lines of evidence from previous work suggest that the cell wall is involved in development, the protein(s) involved remained elusive.

Figure 1.

Phenotypes of rsh/rsh Mutant Seedlings and Embryos Compared with the Wild Type. (A) to (C) Phenotypes of wild-type seedlings. (D) to (F) Phenotypes of rsh/rsh mutant seedlings. (A) and (D) show 5-day-old seedlings; (B) and (E) show 13-day-old seedlings; and (C) and (F) show 21-day-old seedlings. All of the seedlings except the one shown in (C) were grown on sterile media; the seedling shown in (C) was transferred to soil. Arrows indicate root tissue. Bars in (A), (B), and (D) to (F) = 1 mm; bar in (C) = 1 cm. (G) Immature silique of a self-fertilized RSH/rsh plant. Arrows indicate seed with rsh/rsh embryos.

Figure 2.

Morphology of rsh/rsh Mutant Embryos Compared with Wild-Type Embryos during Development. Stained sections (rows 1 and 2) and Nomarski images (rows 3 and 4) of wild-type (rows 1 and 3) and rsh mutant (rows 2 and 4) embryos at comparable stages of development (columns). Column 1-C, one-cell stage; column 2-C, two-cell stage; column 4-C, four-cell stage; column 8-C, eight-cell stage; column G, globule stage; column E-H, early-heart stage; column B-C, bent-cotyledon stage; column M-C, mature-cotyledon stage. Note the large apical cell in 1-C rsh, the abnormal cell shapes in 2-C and 4-C rsh (arrowheads show the dividing line in rsh), the absence of normal protoderm (p), O-ring (o), and hypophysis (h) in G rsh, and the small abnormal embryo within the normal seed coat in M-C rsh. Bar in 1-C = 20 μm for 1-C to E-H; bar in B-C = 80 μm for B-C and M-C. WT, wild type.

Figure 3.

Comparison of Cell Shapes in Root Sections of Young Seedlings. (A) and (C) Wild type transverse (A) and longitudinal (C) sections. (B) and (D) rsh homozygous mutant transverse (B) and longitudinal (C) sections.

Figure 4.

Genetic Map of the Mutated Region in the rsh Mutant. (A) Cloned 6483-bp EcoRI DNA fragment. The predicted amino acid coding sequence of RSH is from 2940 to 4220 bp. The transcription start nucleotide is marked TS (+1). The translation start nucleotide (A of ATG) is at +119. The transposon insert (Tn) mapped at −109 bp from the transcription start nucleotide. Arrows represent transcripts of RSH (1.5 kb) and uidA, which encodes the GUS reporter. CTGA are the deleted nucleotides in the rsh mutant. The scheme is not drawn to scale. (B) Predicted bZIP response element at −149 to −196 nucleotides from the transcription start nucleotide. (C) Amino acid sequence of RSH. The asterisk indicates the most likely cleavage site of the signal peptide (Nielsen et al., 1997). Note the 13 nearly identical repeats of 28 amino acids (nonidentical amino acids are underlined). (D) RNA gel blot of RSH mRNA in total RNA from wild-type roots (right lane) and RNA molecular mass markers (kb) (left lane).

Figure 5.

Alignment of RSH and EXT1. Pairwise alignment of the deduced amino acid sequence of EXT1 with that of RSH using the ClustalV (Higgins and Sharp, 1989) method in Megalign (DNAStar, Madison, WI) with PAM250 residue weight tables. Identical amino acids are shaded black.

Figure 6.

GUS Reporter Gene Expression Patterns during Development Viewed by Light Microscopy, and Tissue Distribution of RSH mRNA. (A) to (K) Embryos dissected from seed coats at different stages of development. (A) to (F) Heterozygous rsh at progressive stages of development: globular (A); heart (B); early torpedo (C); late torpedo (D); early bent cotyledon (E); and late bent cotyledon (F). (G) to (I) Homozygous rsh at progressive stages of development: heart (G); torpedo (H); and nearly mature cotyledon (I). Compare with rsh heterozygous equivalents (B), (D), and (F), respectively. (J) and (K) Wild-type globular and bent-cotyledons stages, respectively (negative controls). Note that blue color did not develop after the GUS assay. (L) Homozygous rsh seedling 2 weeks after germination. Note that the root of the mutant is GUS positive. (M) to (T) Postembryonic heterozygous rsh: seedling 2 weeks after germination (M); roots at bolting (N); roots at harvest (O); stipules (P); nodes (Q); prepollination stylar transmitting tissue (R); postpollination stigma (S); and wounded leaf edge (T). Blue color indicates GUS positive. (U) Tissue distribution of RSH mRNA and 18S rRNA in wild-type plant tissue as indicated and in 12-day-old rsh homozygous mutant seedlings (total tissue). Note that the detected RSH mRNA coincided with GUS reporter gene expression and that the rsh homozygote had no detectable RSH mRNA. The images shown in (A) to (T) are not to scale; sizes can be estimated from Figures 1 and 2.

Figure 7.

Electron Micrographs of RSH-GFP Localization. (A) Localization of RSH-GFP to the cell wall of a 3-day-old root section. Note the cell wall (arrow) between two cells with a concentration of immunogold particles (sharp black spots). Arrowheads denote the plasma membrane. (B) Golgi and trans-Golgi network in a 3-day-old root section. Note the concentration of immunogold particles. (C) and (D) Localization of RSH-GFP to the cell walls of heart-stage embryo cells. Note the immunogold particles in the thin walls (arrows) of young, rapidly dividing cells. (E) Fusion of the cell plate to the cell wall in a dividing heart-stage embryo cell. Two new cells (c1 and c2) with a cell plate (pp) between them. c3 indicates an adjacent cell. Note the cell wall connections between the edge of the cell plate and the swollen regions of the mother cell wall. (F) Localization control. This embryo section was prepared like the other sections except that preimmune serum was substituted for the first antibody (anti–RSH-GFP antibody). Note the absence of immunogold particles in the three cells (c1, c2, and c3), the cell wall (arrow), and the middle lamella (ml). (G) Enlargement of (E). Localization of RSH-GFP as seen by the concentration of immunogold particles in swollen regions (sw) of the mother wall and new connecting walls (arrows). Bar in (A) = 100 μm for (A) to (D), (F), and (G). Bar in (E) = 25 μm.