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Cereal Grass Pulvini: Oat Shoot Pulvini Hey you doofus!

The pulvinus is the major organ involved in the graviresponse of Oat Shoots, Avena sativa. Enzyme and cell wall changes within the pulvinus occur during gravistimulation, as demonstrated in the experiments of Wu et al. (1992), and Gibeaut et al. (1990).

Wu et al. (1992) studied the location of invertase mRNA following gravistimulation of oat shoots, using partial cDNA cloning and the polymerase chain reaction. Their results indicate there is greater invertase expression in the lower half of the oat shoot pulvinus than in the upper half. Invertase catalyzes the hydrolysis of sucrose into fructose and glucose, increasing the solute concentrations of cells located in the lower half of the pulvinus. This increase in solute concentration in the lower pulvinus halves promotes the influx of water which, in turn, allows for asymmetric cell expansion. Because the cells located in the lower half of the pulvinus undergo cell expansion, while those on the upper half of the pulvinus do not, an upward bend results and the oat shoot continues vertical growth. The results of Wu et al. (1992) suggest gravistimulation regulates oat shoot invertase gene expression at transcriptional or post-transcriptional levels.

In correspondence with the results of Wu et al. (1992), Gibeaut et al. (1990) demonstrate there is strong asymmetry in invertase activity, starch degradation, and components of cell wall synthesis. Oat shoot pulvini were first gravistimulated, which consisted of cutting oat stem segments above and below the pulvini, then placing the pulvini horizontally between paper towels and glass plates, and allowing the pulvini to respond for an incubation period of 50 hours. Then, pulvini were cut into upper and lower halves, "upper" corresponding to the upper half during gravistimulation, "lower" following the same standard. Following gravistimulation and division of pulvini into upper and lower halves, cell wall analyses and invertase assays were run, exploring invertase activity, starch content, glucan synthase activity, and cell wall extensibility.

Invertase Assay: Pulvinus halves were homogenized in sodium phosphate buffer containing DTT, EDTA, PMSF, and benzamide. Sucrose hydrolysis was terminated using Somogyi reagent, and resulting sugars detected by Nelson reagent at an absorbance of 550 nm. The results demonstrate that invertase is more active in the lower half of the pulvinus than in the upper half, as more sucrose hydrolysis occurs in the lower region. The results further indicate this difference in invertase activity is detectable just 3 hours after gravistimulation, and the difference continues to increase with time. For example, 24 hours after gravistimulation, lower half invertase activity had increased 28-fold, while upper half invertase activity had only increased 7-fold, demonstrating asymmetric invertase activity. As invertase catalyzes sucrose hydrolysis, Gibeaut et al. (1990) hypothesized that the products of sucrose hydrolysis (fructose and glucose) would be used in the synthesis of new cell wall material during the graviresponse, necessitating cell wall analysis assays.

Cell Wall Analysis: A starch extraction assay was performed using DMSO, allowing researchers to quantify starch present. Starch grains are used by the oat shoot to determine its orientation in a graviresponse, but are then degraded during the growth that follows gravistimulation. Digestion of amylopectin allowed for the determination of Starch hydrolysis efficiency, while glucose analysis results were compared to D-glucose standards. The results of the starch extraction assay indicate that starch degradation occurs asymmetrically, with more starch degraded in the lower half of the pulvinus than in the upper.

Using various techniques developed by other biologists, Gibeaut et al. (1990) found that major cell wall polysaccharides increase proportionately in upper and lower halves of the pulvinus following gravistimulation, with the exception of β-D-Glucan. Subsequently, the researchers measured cell wall β-D-Glucan content over an incubatory gravistimulation period of 50 hours. Both upper and lower pulvinar halves were evaluated and the lower half of the pulvinus was found to contain increasing amounts of β-D-Glucan, while the upper half of the pulvinus demonstrated no clear change in β-D-Glucan content over the 50 hour period. Interestingly, the asymmetry of β-D-Glucan content was only detectable 6 hours after gravistimulation (likely corresponding to the need for invertase activity to occur prior to glucan synthesis). Glucan synthase activity was measured through the incorporation of a UDP-[14C] glucose label, and activity of glucan synthase was shown to increase significantly in lower pulvinus halves of gravistimulated oat shoots. Finally, use of the Instron technique demonstrated a clear asymmetric change in cell wall extensibility, showing greater cell wall extensibility in lower pulvinus halves than in upper halves.