Biochemistry and cytochemical localization of acid phosphatase in the phloem of Nicotiana tabacum

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Abstract
A biochemical and cytochemical study has been made of the distribution of β-glycerophosphatase (EC 3.1.3.2) activity in mature and differentiating phloem cells of Nicotiana tabacum L. and the pH dependence and kinetics of β-glycerophosphate hydrolysis of homogenates of fresh leaf midveins and midveins fixed in formaldehyde-gluteraldehyde. β-glycerophosphatase showed two peaks of activity at pH 5.5 and 6.2. Enzyme saturation kinetics were exhibited by both fresh and fixed tissue homogenates. At a substrate concentration of 2 mM, 65% of the enzyme activity survived fixation. Specimens for cytochemical localization were incubated with 2 mM β-glycerophosphate at pH 5.5 and 6.2. Specimens showed consistent patterns of reaction product deposition. Little or no reaction product was deposited in controls incubated without substrate or with substrate plus 0.01 M fluoride. β-glycerophosphatase activity in the phloem and xylem is considerably higher than in surrounding tissue. Dense localization of reaction product was demonstrated on the vacuolar membranes, the inner membranes of mitochondria, and the dictysomes of phloem parenchyma and companion cells. The plasma membrane and endoplasmic reticulum cisternae of these cells were usually free of reaction products. Enzyme activity in mature sieve elements was associated with the parietal and stacked systems of endoplasmic reticulum and with the P-protein. There was inconsistency of staining of P-protein in mature sieve elements although the association of reaction products with the P-protein appeared to show a correlation with maturity and dispersal. The P-protein bodies of differentiating sieve elements showed no reaction product deposition. The distribution of β-glycerophosphatase activity has been compared with that previously recorded for ATPase activity in the phloem of Nicotiana tabacum.