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Four growth-influencing compounds-hydroxyproline, 2,2'-dipyridyl, 2-chloroethylphosphonic acid, and indoleacetic acid-were used to examine the relationship between lignin formation and growth of wheat coleoptile sections. Hydroxyproline and 2-chloroethylphosphonic acid, at low concentrations, inhibited growth and increased lignin content. Dipyridyl, which promoted coleoptile elongation, decreased lignin content. Indoleacetic acid caused a 300% increase in growth at 0.1 mM but resulted in lignin content no different from controls with no auxin. Chemical and anatomical evidence is given which indicates that lignin is present in the epidermal cell walls of the wheat coleoptile. It is thus possible that bonding between lignin and hemicellulose may have some influence on coleoptile growth. Wardrop (20) has hypothesized that lignification may limit enlargement of plant cells by immobilizing the hemicellulose matrix, thereby preventing surface growth. His suggestion pertained to woody cells which undergo secondary wall thickening, and in which lignin forms a major component of the cell wall. Coleoptile cells, however, also have a secondary type of wall layer which is formed at the end of the extension phase (1). Experiments described in this report show that lignin may be synthesized in the epidermal cells of the wheat coleoptile. As a result of bonding between lignin and hemicellulose (5, 7), factors which control lignification may thereby influence the termination of elongation of coleoptile cells. The relationship between lignin biosynthesis and coleoptile elongation was investigated.MATERIALS AND METHODS Incubation and Nitrobenzene Oxidation. Wheat seeds (Triticunz vulgare cultivar Knox or Redcoat) were grown for 72 hr in the dark in vermiculite. Coleoptile sections 9 mm long were cut 3 mm from the tips, and the leaves were removed. The sections were floated in water for 1 to 2 hr before the treatment incubation. Twenty-five to 50 sections were incubated in 50-ml Erlenmeyer flasks in 2-to 4-ml solutions. The basic medium for incubation was 50 mm sucrose; 2.5 mm potassium maleate buffer, pH 4.8; the lignin precursor U-"C-Lphenylalanine, 15

The phenolic constituent of nonvascular cell walls of wheat (Triticum aestivum L.) coleoptiles, which yields vanillin upon nitrobenzene oxidation, is not lignin as I previously claimed. It seems to be mainly ferulic acid bonded to carbohydrate, probably by an ester linkage. The acid is associated with a fraction of the wall rich in arabinose and xylose, although it is not known whether it is esterified directly with these pentose residues. The phenolic-carbohydrate complex is released by cellulase, but not by pronase or a mixture of hemicellulases.In an earlier report (14) then extracted with ethyl ether. The ether phase was dried with anhydrous sodium sulfate, evaporated to dryness, and the phenolic acids dissolved in ethanol for TLC or in Tri-Sil BSA (Pierce) for gas-liquid chromatography.Enzyme Treatment. Samples of whole walls or hemicellulose were extracted with Pronase, Macerase, and Cellulysin, all from Calbiochem. Pronase was used at the concentration of 5 mg/ml, in pH 7.2 tris buffer, 0.05 M at 30 C. Macerase and Cellulysin were used in pH 5.4 acetate buffer, 0.05 M, 25 mg/ml, 18 hr at room temperature. After incubation with the enzymes, the mixtures were centrifuged and the supernatants were removed and heated for 5 min in a boiling water bath to precipitate the enzymes.Gel Filtration. Samples of cell wall constituents were separated on columns of Sephadex G-100 and LH-20 (Pharmacia). G-100 columns (2.5 X 20 cm) were eluted with 20 mM ammonium formate buffer, pH 7.0. Flow was controlled at 10 ml/hr with a peristaltic pump and 4-ml fractions were collected. Ethanol was the solvent used in the LH-20 column (1 x 20 cm), which was operated by gravity flow. Carbohydrate was determined in fractions by the phenol-sulfuric acid method (8), with glucose used for standards. Radioactivity was measured in fractions dried on planchets and counted with a gasflow detector.Paper and Thin Layer Chromatography. Identification of ferulic acid was done by TLC as described earlier (14). Ascending paper chromatography of the Cellulysin extract was done on Whatman No. 3MM strips in butanol-acetic acid-water (62:15:23, organic phase). RF zones of 0.1 were cut, counted in planchets on a gas-flow detector, then eluted with 50% ethanol. Sulfuric acid was added to the ethanol solutions to make a final concentration of 3 g acid/ 100 ml. The solutions were heated on a boiling water bath for 2 hr. with replacement of water to maintain the volume. The hydrolyzed solutions were neutralized with a saturated solution of barium hydroxide, centrifuged, and the supernatants were freeze-dried.