The Occurrence of Auxin-induced Pectin Methylation in Plant Tissues

Abstract

Treatment of coleoptile tissues with indoleacetic acid (IAA) lead to an enhanced incorporation of methyl groups into both the cold-water-soluble uronic acids (C-Pectin) and the hot-water-extractable fraction of the cell wall (HWV-Pectin). This promotion of pectin methylation is one of the most extensively studied of the biochemical effects of auxin (4, 5, 7, 8, 9). There are 2 reasons for this particular attention. First, it is one of the few biochemical effects which hlas been shown to occur in response to auxin rather than to cell elongation. This is indicated by the fact that IAA still enhances pectin methylation even wheni all cell elongation is osmotically inhibited (9). Secondly, it was thought that this reaction might be important in the loosening of the cell wall andl, thus, in auxin-induced cell elongation (8,9). This possibility has been eliminatedl by the finding that auxin-induced cell elongation can occur even when auxin-induced methylation is completely block-e(l by ethionine (5). Although this auxin-induced pectin methylation (loes not lead to cell elongation, it may still be im-portanit in the growth or development of cells. If this is so, one might expect to find this particular biochenmical effect in most primary plant tissues. At present, auxin-induced pectin methylation has only been found in one highly specialized organ, the cole-optile of Aveina and maize (4). The following investigation was carried out in order to (leternmine whether auxin-induced pectin metlhylation is of coIml-mon occurrence in plant tissues. Selected for studly were tissues in which elongation is promoted by IAA (Avena coleoptile, maize coleoptile and mesocotyl, Helianitlhus hypocotyl, andl pea epicotyl), insensitive to IAA (Avena leaf), or inhlibited by IAA (pea root). Materials and Methods Planit Mllaterial. Seedings of Avena sativa var. Victory were grown as described earlier (5). When the coleoptiles were 2.5 to 3.25 cm in length, the cole-optile and leaf were carefully separated. Seedlings of Zea(nzavs var. Asgrow were grown in a similar mannier. Coleoptiles 2 to 4 cm in length were selected and the leaf was removed. Mesocotyls were used wlhich were 2 to 4 cm in length. They were severed froml the coleoptile and leaf by an incision at the node. Seeds of Helianthus anntus var. AMammoth Russiain and Pisusn sativumst var. Alaska were soaked for 2 hours in distilled water, planted in moist ver-miculite, and allowed to germinate at 250 under a dinm red light. Helianthus hypocotyl sections were I Received May 6, 1963. taken from 5-day-old seedlings after the apex hadl been removed by an incision at the base of the crook. Seven-day-old pea seedlings were used in which the third internode was 2 to 4 cm. in length. The apex was removed in a similar manner. Pea roots were obtained as follows. Alaska pea seeds were surface sterilized with Chlorox, and after soaking for 2 hours in distilled water, were laid out on moist cheesecloth which was suspended on a screen over distilled water. After 80 hours in the dark, roots 2.0 to 3.5 cm in length were collected. The apical 2 mm of eaclh tissue were excised and the next 10 mm were then removed as one section. The sections were collected in lots of 50 (maize coleoptile and mesocotyl, Helianthus hypocotyl), 100 (pea epicotyl and root) or 150 (Avena coleoptile and(l leaf). This was sufficient plant material to give 25 to 50 mg of dry cell wall. Incubation. Each lot of sections was place(d in 10 ml of 0.0025 m K-Alaleate buffer (pH 4.8) whiclh contained, as the methyl donor, 1.0 stc of L-methion-ine-methyl Cl4 (2 mc/m3,i). The appropriate solutions also contained 28.5 /A, IAA (5 ppm). In the pea root experiments the concentration of IAA was j.7p,u-r. The sections were incubated for 3 or 4 hours under a diim red light. At the end of the incubation the length of the sections was measured. Each treatment was run in duplicate; each experiment was repeated at least three times. Preparation of the Pectini Fractions. The procedure for isolation of the pectin fractions has been described inl detail elsewhere (5). Briefly, the tissues were ground in ice-cold buffer and the cell contents were separated from the walls by filtration. MIicroscopic exanmination showed that less than 2 % of the cells remained intact after homogenization. Cold-water-soluble uronic acids (C-Pectin) were precipitated from the filtrate with cold 70 % ethanol. The washed precipitate was resuspended and the nmethyl ester and uronic acid content of this suspension was (letermined. Cell wall pectic substances were extracted in 2 steps. First, the walls were extracted twice for 30 minutes with 3 ml of boiling water. This extract contained the hot-water-soluble pectins. (1HW-Pec-tin). The residual pectin (R-Pectin) was removed by extracting the walls twice for 30 minutes with 3 ml of boiling 0.05 N HCl. The methyl ester and uronic acid content of each extract was determined. Methods of Analysis. The content of radioactive methyl groups was measured by determining the loss of radioactivity from each fraction upon saponifica-tion for 1 hour with 0.1 N NaOH. The freed metlh-anol was lost during the drying of the sample for counting. Radioactivity waS deteriiiined Nvith saii-738