A continuous fluorometric assay for pectin methylesterase.

A continuous, fluorometric assay for pectin methylesterase (PME) activity is described. In this assay, methanol produced by PME hydrolysis of pectin methyl esters is oxidized to formaldehyde by alcohol oxidase, and the formaldehyde is continuously reacted with 4-amino-3-penten-2-one to create a stable, fluorescent product. The increase in fluorescence intensity is linearly proportional to PME activity. The assay can be used in crude plant or fungal extracts with relatively little interference with chemicals or buffers commonly used in PME purification. The fluorescence assay has a useful pH range, from pH 5.0 to 6.5, which overlaps pH optima for many bacterial and fungal PMEs, but which limits its usefulness in assaying plant PMEs with alkaline pH optima. Nevertheless, the method is valuable for rapid assay of plant PMEs during their purification or for comparison of plant tissue PME activities.

Methanol Emission from Leaves (Enzymatic Detection of Gas-Phase Methanol and Relation of Methanol Fluxes to Stomatal Conductance and Leaf Development).

We recently reported the detection of methanol emissions from leaves (R. MacDonald, R. Fall [1993] Atmos Environ 27A: 1709-1713). This could represent a substantial flux of methanol to the atmosphere. Leaf methanol production and emission have not been investigated in detail, in part because of difficulties in sampling and analyzing methanol. In this study we used an enzymatic method to convert methanol to a fluorescent product and verified that leaves from several species emit methanol. Methanol was emitted almost exclusively from the abaxial surfaces of hypostomatous leaves but from both surfaces of amphistomatous leaves, suggesting that methanol exits leaves via stomates. The role of stomatal conductance was verified in experiments in which stomates were induced to close, resulting in reduced methanol. Free methanol was detected in bean leaf extracts, ranging from 26.8 [mu]g g-1 fresh weight in young leaves to 10.0 [mu]g g-1 fresh weight in older leaves. Methanol emission was related to leaf development, generally declining with increasing leaf age after leaf expansion; this is consistent with volatilization from a cellular pool that declines in older leaves. It is possible that leaf emission could be a major source of methanol found in the atmosphere of forests.