Untersuchungen zur Lokalisation von Glycerophosphatase- und Nucleosidtriphosphatase-Aktivität in Siebzellen von Larix

Abstract

The presence of comparatively high concentrations of ATP in sieve elements and the known energy-dependency of the transport of assimilates force to search for possible sites of energy dissipation within sieve cells and sieve tubes. One experimental approach is to look for the sites of enzymatic splitting of nucleoside triphosphates within these cells themselves. While P-protein associated ATP'ase has been found by some authors in sieve tubes of angiosperms almost no information is available for the sieve cells of gymnosperms which seem to lack typical P-protein. In the present investigation, therefore, the enzymatic splitting of glycerophosphate (GP), adenosine triphosphate (ATP), and uridine triphosphate (UTP) in the sieve cells of the secondary phloem of branches from adult trees of Larix leptolepis ( Sieb. et Zucc. ) Gord. has been studied cytochemically throughout the vegetation period. Cytochemical tests were performed with the Gomori lead salt method at pH 5.0 and with a modified Wachstein-Meisel method at pH 7.2. The effect of the non-enzymatic hydrolysis of the substrates, of unspecific lead or leadphosphate adsorption, and of fixatives on the cytochemical enzyme localization has been paid special attention. At acid pH, fully differentiated sieve cells show remarkably increased rate of splitting of both, GP and the nucleoside triphosphates. Addition of NaF (10 mM) to the substrate containing media inhibits almost completly positive enzyme localization. Only with ATP as substrate, NaF inhibition is incomplete. Addition of ouabain (1 mM) showed no inhibition. Fixation of sections in formalin or glutaraldehyde containing solutions caused considerable loss of staining, particularly in case of ATP and UTP as substrates, although the localization remained the same. Enzymatic activity was clearly localized at such structures of the sieve cell protoplast which are enriched at the ends of sieve cells and at sieve areas. At neutral pH, no such enzyme localization is obtained with GP, nor with UTP or ATP. ATP, however, now causes strong staining at the plasmalemma site. Because increased splitting of GP, ATP and UTP is only observed in fully differentiated sieve cells it is considered to be of functional significance.