The isolation and characterization of cell wall proteins from Zea mays seedlings

The cell wall structure of plants is composed of a complex matrix of polysaccharides and protein. Controlled alteration of the cell wall matrix by enzymic hydrolysis allows cell enlargement in compliance to cell turgor. To investigate the metabolic events involved in cell elongation, proteins were extracted and characterized from Zea mays seedling cell walls. Wall proteins were extracted with lithium chloride and separated into several major fractions by using cation exchange chromatography. One fraction, which was not bound to the cation exchange column, elicited antibodies which inhibited auxin induced growth. Active subfractions were further resolved by using gel filtration, identified by bioassay, and characterized by immunoprecipitation. The active protein(s) were acidic in nature and had no hydrolytic activity against polysaccharides in isolated cell walls;A separate fraction investigated, bound tightly to the cation exchange column and contained endo-(beta)-D-glucanase activity. The endo-(beta)-D-glucanase was purified extensively using ion exchange and gel filtration chromatography. The enzyme is characterized by a molecular weight of 20-25 kD, an isoelectric point of greater than pH 9, and thermal stability to temperatures of 50 C. Hydrolytic activity against model substrates indicated a high specificity for (beta)-1,3;1,4-D-glucans. No hydrolysis of (beta)-1,3-D-glucans was observed. Hydrolytic activity against specific mixed-linked glucans appeared to be restricted to the hydrolysis of a (beta) 1--4 glucosyl linkage within regions enriched in (beta) 1--4 and (beta) 1--3 linkages. This restricted hydrolysis resulted in the release of uniform products containing 60 to 70 glucose residues. The endo-(beta)-D-glucanase has a putative role in the degradation of the mixed linked (beta)-D-glucan molecule during auxin induced growth

Biochemistry of Ensiling

The biochemistry of ensiling is essentially a simple process, which, however, can become complex when interactions among plant enzymes and the activities of numerous microbial species become involved. The desired effect is the conversion of simple plant sugars such as glucose and fructose to lactic acid by lactic acid bacteria (LAB) in an anaerobic fermentation. When sufficient lactic acid has been produced, all microbial activity is suppressed, primarily through the effect of undissociated lactic acid, and the silage can then be stored anaerobically until required for feeding. Complications arise because: 1. There are always aerobic periods at the start and end of the ensiling process. 2. Simple sugars are not the only substrates metabolized. 3. Plant enzymes and other microbial species apart from LAB compete for substrate. The complexity of ensilage is increased further when the difficulties of controlling large-scale processes like silage making on-farm are also considered

Cell wall composition throughout development for the model grass Brachypodium distanchyon

Temperate perennial grasses are important worldwide as a livestock nutritive energy source and a potential feedstock for lignocellulosic biofuel production. The annual temperate grass Brachypodium distanchyon has been championed as a useful model system to facilitate biological research in agriculturally important temperate forage grasses based on phylogenetic relationships. To physically corroborate genetic predictions, we determined the chemical composition profiles of organ-specific cell walls throughout the development of two common diploid accessions of Brachypodium distanchyon, Bd21-3 and Bd21. Chemical analysis was performed on cell walls isolated from distinct organs (i.e. leaves, sheaths, stems and roots) at three developmental stages of 1) 12-day seedling, 2) vegetative-to-reproductive transition, and 3) mature seed-fill. In addition, we have included cell wall analysis of embryonic callus used for genetic transformations. Composition of cell walls based on components lignin, hydroxycinnamates, uronosyls, neutral sugars, and protein suggests that Brachypodium distanchyon is similar chemically to agriculturally important forage grasses. There were modest compositional differences in hydroxycinnamate profiles between accessions Bd21-3 and Bd21. In addition, when compared to agronomical important C3 grasses, more mature Brachypodium stem cell walls have a relative increase in glucose of 48% and a decrease in lignin of 36%. Though differences exists between Brachypodium and agronomical important C3 grasses, Brachypodium distanchyon should be still a useful model system for genetic manipulation of cell wall composition to determine the impact upon functional characteristics such as rumen digestibility or energy conversion efficiency for bioenergy production