Biochemical and molecular features of cell wall dynamics related to cell growth in grasses

Jong-Bum Kim, Purdue University

Abstract

Cell walls of grasses have two major polysaccharides that contain uronic acids, the hemicellulosic glucuronoarabinoxylans and the galactosyluronic acid-rich pectins. A technique whereby esterified uronic acid carboxyl groups are reduced selectively to yield their respective 6,6-dideuterio neutral sugars was used to determine the extent of esterification and changes in esterification of these two uronic acids during elongation of maize coleoptiles. The glucosyluronic acids of glucuronoarabinoxylans were not esterified at any time during coleoptile elongation. The galactosyluronic acids of embryonal coleoptiles were about 65% esterified, but this proportion rose to nearly 80% during the rapid elongation phase before returning to about 60% at the end of elongation. Methyl esters accounted for about two-thirds of the total esterified galacturonic acid in cell walls of unexpanded coleoptiles. The proportion of methyl esters fell throughout elongation and did not account for the increase in the proportion of esterified galactosyluronic acid units during growth. The results indicate that the galactosyluronic acid units of grass pectic polysaccharides may be converted to other kinds of esters or form ester-like chemical interactions during expansion of the cell wall. Hydrolysis of (1 $\to$ 3),(1 $\to$ 4)-$\beta$-D-glucan in the cell walls of growing maize seedlings is catalyzed by endo- and exo-$\beta$-D-glucanases. An exo-$\beta$-D-glucanase was purified from the cell walls of developing maize seedlings by cation-exchange, Concanavalin-A affinity, and hydroxylapatite chromatography. The enzyme was purified nearly 40-fold from a mixture of cell wall proteins and gave a single band 72 kDa in SDS-PAGE. The enzyme was specific for $\beta$-D-glucopyranoside, with only trace activity toward $\alpha$-D-glucopyranoside, and no activity against several other $\beta$-D- or $\alpha$-D-pyranosides or furanosides. Monospecific antisera were used in tissue prints to show that the enzyme is abundant in the growing portions of the mesocotyl and coleoptile. To obtain information about the structure of the gene encoding exo-$\beta$-D-glucanase, an expression library was constructed from mRNA of maize seedling and screened with antisera against exo-$\beta$-D-glucanase. Tryptic peptides were separated by reverse-phase HPLC and sequenced. Oligonucleotides were designed based on low degeneracy amino acid sequence of one of four peptides, and used as probes in Southern blot analysis of genes screened from the expression library. The two clones were identified as the same gene, and longer clone was completely sequenced. Translation of this partial clone contained 2 of the 4 peptides that were sequenced, confirming that the clone exactly encodes a portion of the exo-$\beta$-D-glucanase.

Degree

Ph.D.

Advisors

Carpita, Purdue University.

Subject Area

Botany|Biochemistry|Cellular biology

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