Phenolic Acids in Wheat Coleoptile Cell Walls

Abstract

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.

The composition of the cutin of the caryopses and leaves ofTriticum aestivum L.

“…Ferulic acid is present in many cutins and suberins (Holloway 1981(Holloway , 1984 but it is not a characteristic constituent of plant cutins. Ferulic acid has been found in leaf (Hartley 1973;Hartley and Jones 1977), coleoptile (Whitmore 1974), and scutellure (Smart and O'Brian 1979) cell walls, and in lignified tissues (Schwarz et al 1989) of Gramineae. It is thus likely that substantial portions of the amounts of ferulic acids found in the depolymerisates did not originate from cutin or suberin but rather from cell walls and lignin.…”

Control of diferulate formation in dicotyledonous and gramineous cell-suspension cultures

“…Initially, it was thought that this ferulic acid was ester-bonded only to lignin (Higuchi et al 1967;Whitmore 1974). However, further studies showed that it is also ester-bonded to wall polysaccharides (Hartley 1973;Hartley and Jones 1976) and that such feruloylation occurs also in some dicots (Fry 1979;Hartley and Harris 1981) and non-Poaceae monocots (Harris and Hartley 1980).…”

Incorporation of Proline and Aromatic Amino Acids into Cell Walls of Maize Coleoptiles

“…Hydroxyproline-rich glycoproteins are abundant in walls of dicots (10,22), but are minor constituents of the walls of cereals and other grasses. On the other hand, phenolic substances have been observed by fluorescence microscopy throughout the primary walls of grass tissues (17,19); ferulic acid has been identified unequivocally as a major phenolic compound (12,13,19,21,27,29,31), and its esterification to hemicellulosic arabinoxylans has been documented (21,27). Moreover, Markwalder and Neukom (23) have also identified diferulic acid in wheat flour and implicated this ferulate biphenyl dimer in crosslinking the water-soluble pentosans into an insoluble matrix.…”

“…The proportion and composition of these fractions varied markedly during coleoptile development (7). In related studies, treatment of walls of maize coleoptiles with acidic sodium chlorite to oxidize phenolic substances rendered much ofthe hemicelluilosic material soluble in as little as 0.02 N KOH (6 (29). Much more concentrated alkali is required to solubilize most of the hemicellulosic polysaccharide before sodium chlorite treatment, so many of these polymers may be cross-linked by phenolic linkages other than esterified diferulic acid.…”