DEK1 regulates cell wall properties in Arabidopsis thaliana. Andrea M. Watt1 & 2, Roberta Galletti2, Kim Johnson1, Tony Bacic1 and Gwyneth Ingram2. 1ARC Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, Royal Parade, Parkville, Victoria 3010, Australia. 2Laboratoire de Reproduction et Développement des Plantes, Ecole Normale Supérieure de Lyon, CNRS UMR 5667, 46, allée d'Italie, 69364 LYON cedex 07, France. , Abstract The DEK1 protein is absolutely required for plant development. The study of mutant and transgenic lines with reduced DEK1 function has revealed roles in the control of epidermal cell-cell adhesion and in the promotion of the differentiated epidermal state (Galletti et al., 2015). Here we present compositional and structural studies in concert with comparative studies of the mechanical properties of epidermal cells between wild type plants and a partial loss of function mutant of AtDEK1 (dek1-4) (Roeder et al., 2012, Galletti et al., 2015). The epidermal pavement cells of dek1-4 mutants show less pronounced inter-digitation than in wild-type. Measurements of epidermal cell junction height showed that epidermal cell contact zones were less uniform in height in the mutant than in wild type seedlings. This variation may be explained by the later division of expanded cells which is consistent with a loss of the differentiated state. The application of mechanical forces to cotyledon surfaces by Atomic Force Microscopy revealed that the cell walls of mutant cotyledons are significantly less stiff than those of wild type plants, indicating a possible perturbation in cell wall structure or composition in these mutants. We observed that epidermal cell boundaries in the expanding cotyledons of dek 1-4 seedlings appeared to have an abnormal accumulation of callose which was not observed in the wild type seedlings (Galletti et al., , 2015). This led us to hypothesize that dek1-4 epidermal cells may tear apart during organ expansion. Immunolabelling studies, using a range of antibodies that recognize crystalline cellulose and the homogalacturonan domain of pectic polysaccharides indicate differences in the epidermal cell walls consistent with this hypothesis. For example, we observe less pectin and more cellulose in mutant epidermal cell-walls than in wild-type. Our results suggest that although DEK1 is expressed in most cell types, its function is particularly important in the epidermis. These results further support the hypothesis that AtDEK1 could play a key role in signaling necessary for the differentiation of the structural components of the epidermis and particularly for epidermal cell-cell adhesion. dek1-4 mutant epidermis shows protodermal characteristics Immunolabeling of pectin A B C A B C D * * * Figure 1. DEK1 promotes cell differentiation. (A-C) Confocal images of the adaxial side of 3-day-old Col-0 (A) and dek1-4 (B,C) Arabidopsis cotyledons stained with PI. Scale bars:50 μm. Asterisks show 4-way junctions in pavement cells. Asterisks show 4-way junctions in pavement cells. Figure 2. DEK1 promotes the production of pectin Confocal images of 4 day old cotyledons of Col-0 (A) and dek1-4 (B) and young flowers of Col-O (C) and dek1-4 (D) exposed to JIM5 which recognizes the homogalacturonan domain of pectic polysaccharides. An accumulation of callose was observed in mutant epidermal cell walls Epidermal cells of dek 1-4 mutants may have irregular cell-cell contacts A B Figure 3. continued (C and D) Adaxial epidermis of Col-0 and dek1-4 4 day old seedlings, respectively, stained with aniline blue and imaged either under either white light using DIC optics or using a DAPI filter. Callose is only present in epidermal cell junctions in dek1-4. Bar = 100 μm. Figure 3. Analysis of cell-cell junctions in the adaxial epidermis of wild-type and dek1-4 mutant plants. (A) Box plot chart of cell junction height showing median (central box) and interquartile range (box) n=16 for Col-O and n=18 for dek1-4. (B) Representative pictures of transverse sections of cotyledons from 10 day old Col-0 (left) and dek1-4 (right). Conclusions Epidermal cell walls of dek 1-4 mutants are less stiff A B C Although dek1-4 mutants have a continuous epidermis, the zone of cell-cell contact is perturbed and is less uniform in cell junction height compared to the wild type plants. This work has shown that fewer epitopes for pectin recognizing antibodies are produced in the epidermal cells of mutant cotyledons and flowers compared to wild type plants. In addition, the mutant has less stiff cell walls compared to the wild type plants. These results support the hypothesis that the epidermal cells of Arabidopsis thaliana are compromised in structure and mechanical strength when DEK1 function is reduced. References Galletti, R., Johnson, K. L., Scofield, S., San-Bento, R., Watt, A. M., Murray, J. A. H. and Ingram, G. C. (2015) DEFECTIVE KERNEL 1 promotes and maintains plant epidermal differentiation. Development 142, 1-6. Roeder, A. H., Cunha, A., Ohno, C. K. and Meyerowitz, E. M. (2012). Cell cycle regulates cell type in the Arabidopsis sepal. Development 139, 4416-27. Figure 4. Analysis of cell wall stiffness using Atomic Force Microscopy (AFM). Atomic Force Height sensor map of 4 day old cotyledons of (A) Col-O and (B) dek1-4 and (C) stiffness analysis (MPa) of 2 individual samples of Col-O and dek1-4. Red and white crosses indicate positions of stiffness measurements. A spherical 42 N/m cantilever tip with a 400 nm radius was used for the above measurements.