CELLULOSE SYNTHASE9 serves a nonredundant role in secondary cell wall synthesis in Arabidopsis epidermal testa cells

Abstract

Herein, we sought to explore the contribution of cellulose biosynthesis to the shape and morphogenesis of hexagonal seed coat cells in Arabidopsis (Arabidopsis thaliana). Consistent with seed preferential expression of CELLULOSE SYNTHASE9 (CESA9), null mutations in CESA9 caused no change in cellulose content in leaves or stems, but caused a 25% reduction in seeds. Compositional studies of cesa9 seeds uncovered substantial proportional increases in cell wall neutral sugars and in several monomers of cell wall-associated polyesters. Despite these metabolic compensations, cesa9 seeds were permeable to tetrazolium salt, implying that cellulose biosynthesis, via CESA9, is required for correct barrier function of the seed coat. A syndrome of depleted radial wall, altered seed coat cell size, shape, and internal angle uniformity was quantified using scanning electron micrographs in cesa9 epidermal cells. By contrast, morphological defects were absent in cesa9 embryos, visually inspected from torpedo to bent cotyledon, consistent with no reduction in postgermination radical or hypocotyl elongation. These data implied that CESA9 was seed coat specific or functionally redundant in other tissues. Assessment of sections from glutaraldehyde fixed wild-type and cesa9 mature seeds supported results of scanning electron micrographs and quantitatively showed depletion of secondary cell wall synthesis in the radial cell wall. Herein, we show a nonredundant role for CESA9 in secondary cell wall biosynthesis in radial cell walls of epidermal seed coats and document its importance for cell morphogenesis and barrier function of the seed coat.

Figure 1.

Examination of cesa9-1 and cesa9-2 plants imply that phenotypes are restricted to the seed. A, Analysis of seed weight was performed on 100 individual seed. Error bars are se of three replicates for two alleles (P > 0.01 ANOVA). B, Light microscopy was used to image wild-type (WT), cesa9-1, and cesa9-2 seed (scale bar = 100 μm). C, Phenotypic examination of rosette leaves (scale bar = 1 cm), dark-, and light-grown seedlings (scale bar = 7 mm) comparing cesa9-1 and cesa9-2 with wild type. Col-0, Columbia-0. [See online article for color version of this figure.]

Figure 2.

Mutations in CESA9 cause distorted cell shape and morphogenesis in seed coat epidermal cells. A, Nomarski optics were used to visualize the epidermal cells of seed excised from siliques at the 14th silique posterior to gynoecium protrusion (scale bar = 50 μm). B, SEMs of seed coat epidermal cells in mature wild-type, cesa9-1, and cesa9-2 seed (scale bar = 50 μm for high magnification [left sections] and 250 μm for image of entire seed [right sections]). C, SEM of wild-type epidermal seed coat (scale bar = 50 μm). Adjacent section is a schematic illustration of the cell boundary (purple), trough (blue), and columella (green; scale bar = 50 μm). D, SEM of cesa9 epidermal seed coat (scale bar = 50 μm). Adjacent section is a schematic illustration of the cell boundary (purple), trough (blue), and columella (green; scale bar = 50 μm). Arrowheads in C and D indicate radial cell wall. [See online article for color version of this figure.]

Figure 3.

Quantitative analysis of cell morphogenesis using SEM. A, Epidermal cell area (μm²) in seeds from cesa9-1 and wild type shows a greater variation in cell area in cesa9 relative to wild-type seed. Area measurements on 300 cells in 10 different seeds displaying significantly smaller cell size. B, Columella area measurements of the cesa9 mutant columella relative to wild type (n = 300). C, Analysis of columella area relative to the cell size. Columella area was approximately 14% of the wild-type cell area, and 20% of the cesa9 mutant. D, Analysis of the internal angles between the cell sides (n = 1,080). Statistical significance of distribution shifts was calculated by using the Wilcoxon rank sum test for cesa9 relative to wild type (P < 0.01). Wild-type and cesa9 cell area (P > 0.001), wild-type and cesa9 columella area (P > 0.008), wild-type and cesa9 cell area/columella area (P > 0.001), and wild-type and cesa9 internal angle (P > 0.5). E and F, Provides an example of morphological variation between wild-type and cesa9 epidermal testa cells (scale bars = 30 μm). Std Er, Standard error. [See online article for color version of this figure.]

Figure 4.

Analysis of the structure and development of wild-type and cesa9 seed coats. A, Epidermal cell morphology of wild-type and cesa9 toluidine blue-stained sections of cryrofixed 4 DPA, 7 DPA, and 10 DPA seeds. Arrows on 10 DPA images indicate the location of the radial wall where secondary cell wall synthesis is occurring (scale bar = 10 mm, all images same magnification). B, Epidermal cell morphology of wild-type and cesa9 toluidine blue-stained sections of aqueous (3% v/v) glutaraldehyde fixed mature seeds (scale bar = 10 mm). C, Average height of the radial wall (RW) of wild-type and cesa9 seed coat cells (mm). D, Average width of the radial wall of wild-type and cesa9 seed coat cells (mm): error bars are se from the mean. The asterisk (*) in C indicated significant difference from wild type based on one-way ANOVA at P > 0.05 (Student's t test). Col-0, Columbia-0. [See online article for color version of this figure.]

Figure 5.

Composition of wild-type (WT) and cesa9 seed cell walls. A, Acid-insoluble crystalline cellulose content of various tissues was determined colorometrically for mature seed, leaves, and stems. Error bars are ses of three technical replicates from three independent batches of seed as biological replicates. CW, Cell wall. B, Calcofluor staining and subsequent illumination with UV light (scale bar = 250 μm). C, GC analysis of cell wall neutral sugars. Error bars are se of three replicates. D, Mucilage weights determined for wild-type and cesa9 seed, error bars are se of three replicates. E, Ruthenium red-stained seed visualized by light microscopy (scale bar = 200 μm). Col-0, Columbia-0. [See online article for color version of this figure.]

Figure 6.

Polyester and aliphatic monomer composition for wild-type (WT) and cesa9 seed. GC and GC-MS analysis of mature cesa9 seed assessed lipid polyester monomers from seeds of wild-type and cesa9 plants. The insoluble dry residue obtained after grinding and delipidation of tissues with organic solvents was depolymerized by acid-catalyzed methanolysis and aliphatic and aromatic monomers released were analyzed by GC-MS. Error bars are the se of four replicates. Black bar is wild type and red bar is cesa9. PAs, Primary alcohols; br., branched. Inset documents tetrazolium salt uptake into the cesa9 seed relative to wild type (scale bar = 250 μm). [See online article for color version of this figure.]