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Sugar and uronic acid residues were derived from wall polysaccharides of oat (Avena sativa, var. Victory) coleoptiles by means of 2 N trifluoroacetic acid, 72% sulfuric acid, or enzymic hydrolysis. The products of hydrolysis were reduced and acetylated to form alditol acetates which were analyzed using gas chromatography. Time-course studies of auxin-promoted changes in Wiegand and Schrank (31). Seeds were dehulled, placed in distilled water at room temperature for 4 hr, then spread on the surface of distilled water-saturated vermiculite in plastic vegetable crispers with lids. Crispers with seeds were placed in dim red light for 48 hr, then transferred to darkness for 24 hr. Temperature ranged from 22 to 24 C. After a total of 73 to 76 hr, 25-to 30-mm coleoptiles were selected, and the primary leaf was removed. Beginning 3 mm from the tip, a 10-mm section was cut using a double-bladed cutting device. All manipulations were performed in dim green light. After cutting, sections were floated on distilled water for 30 min prior to treatment.Treatment of Sections. Treatments involved incubating 10 to 30 coleoptile sections in the dark in 43-X 20-mm Stender dishes containing 5 or 10 ml of 2.5 mM potassium citrate, pH 5.4. In some experiments, the dishes also contained either 50 mM glucose or 12.5 /M IAA, or both IAA and glucose. The rims of the dishes were coated with petrolatum to prevent evaporation. The dishes were placed in a Dubnoff shaker at 26 C, 60 oscillations per min. After treatment, sections were measured to the nearest 0.1 mm using a 10 x dissecting microscope fitted with an ocular micrometer and stored at -24 C. For a single experiment coleoptile sections from two or three replicates run on different days were pooled for preparation of wall samples and analyses.Preparation of Wail Samples. Wall samples were prepared using a technique adapted from that of Ray (19). Frozen sections were placed between two 10-X 10-cm pieces of plate glass, thawed, then crushed by a force of approximately 0.5 kg per cm2 applied manually. The crushed material was washed into a fritted glass funnel (medium porosity) with 10 ml of distilled water at room temperature. The water was rapidly sucked off, and the water wash was repeated twice more. Five ml of acetone were added to the sample, allowed to stand with occasional swirling for 5 min, and then removed by suction. The acetone extraction was repeated twice more. The residue was then similarly extracted three times, 5 min each, with 5 ml 556 www.plantphysiol.org on May 12, 2018 -Published by Downloaded from

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The proteins dissociated from isolated Zea seedling cell wall using high-ionic-strength salt solutions have been found to include a number of enzymes which appear to participate in autolytic reactions of the cell wall. These enzymes caused extensive degradation of enzymatically inactive cell wall, liberating as much as 100 μg/mg dry weight over a 48-h period. Lithium chloride (3M) was shown to be most effective in yielding protein and wall-degrading activities.Molecular-sieve chromatography of the cell-wall protein resolved endo-β-D-glucanase and exo-β-1,3-glucanase (EC 3.2.1.58) activities when Avena glucan and laminarin, respectively, were employed as substrates. The exoenzyme (molecular weight around 60,000) was strongly inhibited by inorganic mercury at a concentration which suppressed the release of monosaccharide from autolytically active cell wall. The endo-β-D-glucanase (MW around 26,000), which showed a marked preference for substrates of mixed-linkage, exhibited features indicating that it initiates the autolytic solubilization of wall glucan.Cell-wall β-D-glucan, recovered as a component of an alkali-soluble cell-wall fraction, served as a substrate for the purified glucanases. Their hydrolysis pattern, assessed using gel exclusion chromatography and product analysis, confirmed that they hydrolyze β-D-glucan. The products generated by the endoglucanase were similar in molecular-size distribution to those liberated from autolytically active-wall. Exoglucanase activity was required for extensive hydrolysis of β-D-glucan in vitro.During coleoptile development the autolytic activity of the cell wall increased dramatically. This increased activity, however, did not parallel the growth potential of the tissue, but more closely reflected an increase in cell-wall β-D-glucan, the primary substrate for autolytic reactions.

Cell wall isolation procedures were evaluated to determine their effect on the total pectin content and the degree of methylesterification of tomato (Lycopersicon esculentum L.) fruit cell walls. Water homogenates liberate substantial amounts of buffer soluble uronic acid, 5.2 milligrams uronic acid/100 milligrams wall. Solubilization appears to be a consequence of autohydrolysis mediated by polygalacturonase 11, isoenzymes A and B, since the uronic acid release from the wall residue can be suppressed by homogenization in the presence of 50% ethanol followed by heating. The extent of methylesterification in heatinactivated cell walls, 94 mole %, was significantly greater than with water homogenates, 56 mole %. The results suggest that autohydrolysis, mediated by cell wall-associated enzymes, accounts for the solubilization of tomato fruit pectin in vitro. Endogenous enzymes also account for a decrease in the methylesterification during the cell wall preparation. The heat-inacffvated cell wall preparation was superior to the other methods studied since it reduces,-elimination during heating and inactivates constitutive enzymes that may modify pectin structure. This heat-inactivated cell wall preparation was used in subsequent enzymatic analysis of the pectin structure. Purified tomato fruit polygalacturonase and partially purified pectinmethylesterase were used to assess changes in constitutive substrates during tomato fruit ripening. Polygalacturonase treatment of heat-inactivated cell walls from mature green and breaker stages released 14% of the uronic acid. The extent of the release of polyuronides by polygalacturonase was fruit development stage dependent. At the tuming stage, 21% of the pectin fraction was released, a value which increased to a maximum of 28% of the uronides at the red ripe stage. Pretreatment of the walls with purified tomato pectinesterase rendered walls from all ripening stages equally susceptible to polygalacturonase. Quantitatively, the release of uronides by polygalacturonase from all pectinesterase treated cell walls was equivalent to polygalacturonase treatment of walls at the ripe stage. Uronide polymers released by polygalacturonase contain galacturonic acid, rhamnose, galactose, arabinose, xylose, and glucose. As a function of development, an increase in the release of galacturonic acid and rhamnose was observed (40 and 6% of these polymers at the mature green stage to 54 and 15% at the red ripe stage, respectively). The amount of galactose and arabinose released by exogenous polygalacturonase decreased during development (41 and 11% from walls of mature green fruit to 11 and 6% at the red ripe stage, respectively). Minor amounts of glucose and xylose released from the wall by exog- ' Supported, in part, by a gift from Chesebrough-Ponds Inc.