*4.7. Cell Wall Swelling Measurements from Optical Density*

Cell wall fragments suspended in buffer were prepared from approximately 1 g of segments from the top 2 cm of sunflower hypocotyls or approximately 1 g of Arabidopsis seedling leaves and stems. The material was boiled for 90 s in distilled water to inactivate endogenous expansins in the plant tissues and then homogenized in 10 mL of MES buffer using a laboratory mixer/emulsifier at full speed for 5 min (Silverson Machines Ltd., Waterside, UK). The homogenized suspension was then centrifuged for 2.5 min at 650× *g* to remove large pieces of material and then centrifuged again for 10 min at 1450× *g* to pellet smaller cell wall fragments of cell wall. These were re-suspended in 10 mL MES buffer. Fragments from Arabidopsis seedlings were pelleted by centrifugation at 1450× *g* and resuspended in 10 mL MES buffer two further times to eliminate pigmentation in the suspensions (although there was negligible absorbance from them at a wavelength of 750 nm).

The optical density of the fragments suspended in buffer at a wavelength of 750 nm was determined using a spectrophotometer (Lambda 20, Perkin Elmer, Waltham, MA, USA). The initial optical density of 1.0 mL in a disposable plastic cuvette was recorded for 30 s, after which expansin (CsExp1) or snail powder extract was added to the suspension. The cuvette was then gently inverted to mix the solution and returned to the spectrophotometer, and the optical density was recorded for a further 120 s. The effects of horseradish peroxidase (Sigma-Aldrich, P6140) and H2O2 on the optical density of suspensions from sunflower hypocotyls were also tested.
