Plant Science

Arabidopsis seed coat epidermal cells deposit a significant quantity of mucilage, composed of the cell wall components pectin, hemicellulose, and cellulose, into the apoplast during development. When mature seeds are hydrated, mucilage extrudes to form a gelatinous capsule around the seed. Determining the monosaccharide composition of both extruded mucilage and whole seeds is an essential technique for characterizing seed coat developmental processes and mutants with altered mucilage composition. This protocol covers growth of plants to produce seeds suitable for analysis, extraction of extruded mucilage using water and sodium carbonate (used for mutants with impaired mucilage release), and extraction of alcohol insoluble residue (AIR) from whole seeds. The prepared polysaccharides are then hydrolyzed using sulfuric acid, which hydrolyses all polysaccharides including cellulose. Sensitive and reproducible quantification of the resulting monosaccharides is achieved using high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

Lignocellulosic biomass is a versatile renewable resource for fuels, buildings, crafts, and biomaterials. Strategies of molecularly designing lignocellulose for industrial application has been developed by the discoveries of novel genes after the screenings of various mutants and transformed lines of Arabidopsis whose cell walls could be modified in the inflorescence stem, a model woody tissue. The mechanical properties are used as a quantitative index for the chemorehological behavior of the genetically modified cell wall in the tissue. This parameter can be measured with tensile or bending tests of tissue explants, the vibration analysis of tissue behavior or using atomic force microscopy to probe the tissue surface. Here, we describe in detail the procedure to determine the stiffness of methanol-fixed, rehydrated and pronase-treated inflorescence explants with a tensile testing machine based on classical methods for the determination of cell wall extensibility.

Estimation of stomatal aperture using low viscosity silicone-base impression material has the advantage of working with the whole leaf. The developmental stage and the environment strongly affect the stomatal aperture. Therefore, it is mandatory to have accurate estimations of the stomatal aperture of intact leaves under different situations. With this technique, it is possible to get the real picture at any moment. The outputs of the data include studies on cell area and morphology, epidermis cell and stomata lineages, among others. This protocol is useful for the accurate estimation of stomatal aperture in many samples of intact leaves in Arabidopsis thaliana.

Cellulose is synthesized by Cellulose Synthase A proteins at the plasma membrane using the substrate UDP glucose. Herein, we provide a detailed method for measuring the incorporation of radiolabeled glucose into the cellulose fraction of the cell wall. In this method Arabidopsis seedlings are treated for 2 h with a cellulose biosynthesis inhibitor in the presence of radiolabeled glucose, and are subsequently boiled in acetic-nitric acid to solubilize non-cellulosic material. The radiolabeled glucose detected in the insoluble fraction indicates the amount of cellulose synthesized during the experimental timeframe. The short-term nature of this method is a useful tool in determining if inhibition of cellulose biosynthesis is the herbicides primary mode of action.