The Receptor-Like Kinase AtVRLK1 Regulates Secondary Cell Wall Thickening

Abstract

During the growth and development of land plants, some specialized cells, such as tracheary elements, undergo secondary cell wall thickening. Secondary cell walls contain additional lignin, compared with primary cell walls, thus providing mechanical strength and potentially improving defenses against pathogens. However, the molecular mechanisms that initiate wall thickening are unknown. In this study, we identified an Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like kinase, encoded by AtVRLK1 (Vascular-Related Receptor-Like Kinase1), that is expressed specifically in cells undergoing secondary cell wall thickening. Suppression of AtVRLK1 expression resulted in a range of phenotypes that included retarded early elongation of the inflorescence stem, shorter fibers, slower root growth, and shorter flower filaments. In contrast, up-regulation of AtVRLK1 led to longer fiber cells, reduced secondary cell wall thickening in fiber and vessel cells, and defects in anther dehiscence. Molecular and cellular analyses showed that down-regulation of AtVRLK1 promoted secondary cell wall thickening and up-regulation of AtVRLK1 enhanced cell elongation and inhibited secondary cell wall thickening. We propose that AtVRLK1 functions as a signaling component in coordinating cell elongation and cell wall thickening during growth and development.

Figure 1.

Phylogenetic and expression analyses of AtVRLK1. A, Phylogenetic analysis of AtVRLK1 homologs from three representative species, poplar, Arabidopsis, and rice. CLV1 from a different subfamily of LRR-RLK genes was used as an outgroup reference. B, Relative expression levels of AtVRLK1 in various organs of 4-week-old Arabidopsis plants. The expression level in each sample was normalized using Arabidopsis ACTIN2 (At3g18780) as an internal control. The values are means ± se; n = 3. Mr, Mature root; Is, inflorescence stem; Le, leaf; Fl, flower; Si, silique. C, Fluorescence in an AtVRLK1pro:GFP transgenic Arabidopsis root. D to I, GUS staining in AtVRLK1pro:GUS transgenic Arabidopsis root (D), seedling (E), rosette leaf (F), flower (G), and vascular bundles of the inflorescence stem (H and I). Bars = 50 µm (C and D), 2 mm (E–G), 100 µm (H), and 20 µm (I).

Figure 2.

Analysis of AtVRLK1 in vitro kinase activity. A, VRLK1K-His recombinant protein produced in Escherichia coli was detected using anti-His antibodies. B, Coomassie Brilliant Blue (CBB)-stained proteins by SDS-PAGE and autoradiography (Autorad) of the kinase reaction products. MBP, Myelin basic protein. Different amounts of MBP are indicated as + and ++.

Figure 3.

AtVRLK1 expression regulation affected Arabidopsis phenotypes at different developmental stages. A, Western-blot analysis of AtVRLK1 in inflorescence stems of wild-type (WT), AtVRLK1 OE, and DN Arabidopsis plants. In DN plants, a full size (top band) and a truncated size (bottom band) of AtVRLK1 are detected by anti-AtVRLK1 antibodies. An anti-ACTIN antibody was used as a loading control. B to D, Phenotypes at the age of 2 weeks (B) and 5 weeks (C) and siliques (D). E, Leaf length and width of the plants in B. F, Fertility rates of the wild-type, OE, and DN plants. The values are means ± se; n = 20 in E and 30 in F. Significance was determined by Student’s t test: **, P < 0.01. Bars = 2 cm (B and D) and 5 cm in (C).

Figure 4.

Effects of AtVRLK1 overexpression and dominant-negative suppression on vascular cell elongation in the inflorescence stem. A, Schematic representation of the measurement scheme to assess inflorescence stem elongation. B, Basal inflorescence stem elongation of wild-type (WT), OE, and DN plants from day 1 to day 6. C, Length distribution of fibers from basal 1-cm inflorescence stems after elongation had stopped. The values are means ± se; n = 5 in B and 800 in C.

Figure 5.

Effects of AtVRLK1 overexpression and dominant-negative suppression on inflorescence stem vascular cell wall thickening in Arabidopsis. A to C, Cross sections of wild-type (WT), OE, and DN inflorescence stems stained with phloroglucinol-HCl. D to F, Transmission electron micrographs of interfascicular fibers in wild-type, OE, and DN inflorescence stems. Inf, Interfascicular fiber. G to I, Transmission electron micrographs of xylem vessels and fibers in wild-type, OE, and DN inflorescence stems. Ve, Vessel; Xf, xylem fiber. J, Quantification of secondary cell wall (SCW) thickness of walls seen in D to I. The values are means ± se of 50 independent cells. K and L, Lignin and crystalline cellulose contents of inflorescence stems from wild-type, OE, and DN plants. M, Expression of secondary cell wall thickening-related genes in inflorescence stems of wild-type, OE, and DN plants. Gene expression was measured in the basal region of the inflorescence stem by RT-qPCR. The expression level in each sample was normalized using Arabidopsis ACTIN2 (At3g18780) as an internal control. The values are means ± se; n = 3. Significance was determined by Student’s t test: *, P < 0.05 and **, P < 0.01. Bars = 50 μm (A–C), 1 μm (D–F), and 2 μm in (G–I).

Figure 6.

Effects of AtVRLK1 dominant-negative suppression on filament elongation. A, Inflorescences of wild-type and DN plants. Red arrows indicate the flowers used for observation. B, Flowers at stage 13 (S13), stage 14 (S14), and stage 15 (S15) from wild-type and DN plants. C to E, Anatomy and micrographs of flowers from S13 to S15 from an inflorescence of a wild-type plant. F to H, Anatomy and micrographs of flowers from S13 to S15 from an inflorescence of a DN plant. I, Ratios of stamen length (the average length of four high stamens in a flower) to pistil length of wild-type (WT) and DN plants. The values are means ± se of 10 independent flowers. J and K, Micrographs of filament epidermal cells of wild-type and DN plants. L, Quantification of the lengths of filament epidermal cells in J and K. The values are means ± se of 30 cells from three independent fully elongated filaments. Significance was determined by Student’s t test: **, P < 0.01. Bars = 2 mm (A and B), 500 µm (C–H), and 20 µm (J and K).

Figure 7.

Effects of AtVRLK1 overexpression on anther dehiscence in Arabidopsis. A, Wild-type anthers dehisced, and pollen grains were released to the stigma. B, OE line anthers failed to dehisce, and no pollen grains were released. The insets in the top right corner show magnified images of the boxed anthers in A and B. C and E, Transverse sections of wild-type anthers at stages 11 and 13, respectively. D and F, Transverse sections of OE anthers at stages 11 and 13, respectively. C, Connective; En, endothecium; Fb, fibrous bands; PG, pollen grains; St, stomium; T, tapetum; V, vascular tissue. G and H, Phloroglucinol-HCl staining of wild-type and OE inflorescences. I, Expression of secondary wall thickening-related genes in flowers of wild-type (WT) and OE plants. Gene expression was measured in the flowers at anther stages 11 to 13 by RT-qPCR. The expression level in each sample was normalized using Arabidopsis ACTIN2 (At3g18780) as an internal control. The values are means ± se; n = 3. Significance was determined by Student’s t test: **, P < 0.01. Bars = 0.2 mm (A and B), 100 μm (C–F), and 2 mm (G and H).

Figure 8.

Proposed model for the role of AtVRLK1 in the coordination of cell elongation and secondary cell wall formation. During growth and development, following expansion to a certain size, specialized cells, such as fibers and vessels, activate the program of secondary cell wall (brown) deposition inside the primary cell wall (pink). The plasma membrane-localized RLK, AtVRLK1, recognizes ligands (blue dots), which may be generated during cell expansion and inhibit secondary cell wall component deposition in both vascular cells and anther endothecial cells through a transcriptional regulatory network.