Soluble and Bound Apoplastic Activity for Peroxidase, β-d-Glucosidase, Malate Dehydrogenase, and Nonspecific Arylesterase, in Barley (Hordeum vulgare L.) and Oat (Avena sativa L.) Primary Leaves

Abstract

An intercellular washing solution containing about 1% of the soluble protein, 0.3% or less of the glucose-6-phosphate dehydrogenase activity, but up to 20% of the peroxidase and 6-Dglucosidase activity of barley (Hordeum vulgare L.) or oat (Avena sativa L.) primary leaves was obtained by vacuum infiltrating peeled leaves with pH 6.9 buffered 200 millimolar NaCI. After this wash, segments were homogenized in buffer, centrifuged, and the supernatant was assayed for soluble cytoplasmic enzymes. The pellet was washed and resuspended in 1 molar NaCI to solubilize enzymes strongly ionically bound to the cell wall. The final pellet was assayed for enzyme activity covalently bound in the cell wall. Apoplastic (intercellular washing solution, ionically bound, and covalently bound) fractions contained up to 76% of the ,6-D-glucosidase activity, 36% of the peroxidase activity, 11% of the nonspecific arylesterase activity, 4% of the malate dehydrogenase activity, but less than 2% of the glucose-6-phosphate dehydrogenase activity of peeled leaf segments. The partitioning and salt-solubility of the enzymes between the apoplast and symplast differed considerably between these two species. Intercellular washing fluid prepared by centrifuging unpeeled leaves had higher activity for glucose-6-phosphate dehydrogenase, less soluble protein, and less peroxidase activity per leaf than intercellular washing solution obtained by our peeling-infiltration-washing technique. The results are discussed in relation to the roles of these enzymes in phenolic metabolism in the cell wall.

Cell Wall Proteome in the Maize Primary Root Elongation Zone. II. Region-Specific Changes in Water Soluble and Lightly Ionically Bound Proteins under Water Deficit

“…In addition, increasing evidence suggests that proteins can be secreted into cell walls without having a signal peptide (Voigt and Frank, 2003;Slabas et al, 2004;Juarez-Diaz et al, 2006). Consistent with this model, 17 of the proteins were predicted to be so-called nonclassical secretory proteins (Bendtsen et al, 2004;Zhu et al, 2006), and among the 30 remaining proteins, 19 were previously identified in cell walls including two malate dehydrogenases, two glyoxalases I, three UDPGlc pyrophosphorylases, and 12 b-D-glucosidases (Gross, 1977;Fry, 1988;Li et al, 1989;Chivasa et al, 2002;Pitarch et al, 2002;Kleczkowski et al, 2004;Watson et al, 2004).…”

Immunolocalization of Antifreeze Proteins in Winter Rye Leaves, Crowns, and Roots by Tissue Printing

“…The apoplast comprises xylem tracheids and vessels, cell walls, and intercellular spaces. Proteins extracted from the apoplast are soluble and/or loosely bound to cell walls or the outer surface of the plasma membrane (Li et al, 1989;Brune et al, 1994 Extraction of apoplastic proteins from CA leaves decreased immunologically detectable AFPs in total protein samples of the remaining leaf material (Fig. 3, D, F, and H), which indicates that most AFPs in winter rye are secreted proteins.…”

Effects of the Air Pollutant SO2 on Leaves

“…The apoplastic peroxidases are involved in the formation of cross-linkings within cell wall constituents. Some of them are bound to the cell wall, whereas others are soluble (7). These enzymes may also scavenge hydrogen peroxide in a way similar to that employed by vacuolar peroxidases, which first oxidize phenolics (14).…”