Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Summary

We demonstrate the use of a constant-force extensometer to measure long-term extension (creep) of plant cell wall specimens induced by acidic buffers and expansin protein.

Abstract

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (< 5) ¹. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism ^{2, 3}. Auxin activates a H⁺ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins ⁴, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This 'acid growth' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins ⁵. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.

To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 °C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.

Protocol

Part 1: Growing and storing suitable plant material

1. In our experience, young hypocotyls from etiolated cucumber seedlings serve as a convenient source of cell wall material for these experiments. Cucumber seeds are sown on wet paper in a light-proof box, which is keep in a darkened cabinet in a constant temperature room set at 26 °C. The exact temperature is not critical, as anything between 22° and 30 °C should be fine, but the temperature will determine how fast the seedlings reach an appropriate stage of development. The warmer the temperature, the faster the seedlings will develop. We typically use seedlings when they have grown to about 5 cm in lengt....

Discussion

For this demonstration we used cell walls from cucumber hypocotyls because they have proved to be a reliable source of wall samples that are easy to handle and that respond with good sensitivity. We have also had good success with walls from other seedlings as well as some materials from the supermarket, such as young spinach leaves and celery stalks. Basically, young, soft, rapidly growing plant tissues are likely to be easily measured with this technique, but tough, old, oxidized or nongrowing plant tissues are unlike.......