An examination of the peroxidases from Lupinus albus L. hypocotyls

Abstract

Abstract. Peroxidases (EC 1.11.1.7) from hypocotyls of Lupinus albus L. cv. Rio Maior have been characterised using one-and two-dimensional, native electrophoretic techniques. Data are presented showing the complexity in charge and molecular size or shape of these peroxidases. We report the finding of a new acidic peroxidase and several new basic peroxidases in these hypocotyls, and of their stability to treatments considered to break ligandinduced variants and conformational variants derived from differences in polypeptide folding. Densitometric data demonstrate that these new peroxidases contribute up to 60% of the total peroxidase activity in hypocotyls. Studies of intercellular fluid, cell-wall and soluble fractions, with assays of purity were conducted in an attempt to define the subcellular locations of these additional peroxidases. The acidic form (pI 4.1) is greatly enriched in soluble fractions, three of the basic peroxidases (pls 9.5, 9.7 and > 9.7) are strongly associated to the cell wall, ad a minor, basic component (pI 9.7) is enriched in the intercellular fluid. Individual peroxidase activities with the substrates coniferyl alcohol, ferulic acid or indole acetic acid were compared by densitometric analysis of zymograms with those for guaiacol, and notable differences between these peroxidases in their capacity to oxidise indole acetic acid in vitro were identified. The possible functions of these peroxidases in vivo and their implications to current understanding of peroxidases in L. albus are discussed.Key words: Cell wall -Conformer (peroxidase) -Indole-3-acetic acid (oxidases) -Leguminosae -Lupinus (hypocotyl) -Peroxidase (acidic, basic) Abbreviations: APAGE = anionic polyacrylamide gel electrophoresis; CA = coniferyl alcohol; CPAGE = cationic polyacrylamide gel electrophoresis; IEF = isoelectric focusin; NEIEF = non-equilibrated isoelectric focusing; 2D=two dimensional; pI=isoelectric point; RCPAGE=reversed current polyacrylamide gel electrophoresis

Study of the Intercellular Fluid of Healthy Lupinus albus Organs (Presence of a Chitinase and a Thaumatin-Like Protein)

“…It was surprising that some of the prominent IF polypeptides had identity to PR proteins, which have been reported in healthy plant tissues, but only at specific physiological stages such as flowering and senescence (Legrand et al, 1987). These observations lead us to conclude that several proteins are constitutively present in the IF of healthy lupin tissues (leaves, stems, and roots) including PR-like proteins (at least a chitinase and a thaumatin-like protein) in addition to the expected enzymes such as the peroxidases already reported for this plant (Jackson and Ricardo, 1994).…”

Rapid Deposition of Extensin during the Elicitation of Grapevine Callus Cultures Is Specifically Catalyzed by a 40-Kilodalton Peroxidase

“…Gels were run at 4°C with the constant power of 0.15 W cm Ϫ1 width of gel and with a limiting voltage at 420 V for 1 h. All zymograms were developed using guaiacol as substrate as before (Jackson and Ricardo, 1994). SDS-PAGE was performed as described by Laemmli (1970), using 12% T and 3% C 0.75-mm polyacrylamide gels run in the Mini-Protean II electrophoresis apparatus (Bio-Rad, Hercules, CA) at 50 mA per gel and were stained with Coomassie blue R-250.…”

Lupinus albus L. Pathogenesis-Related Proteins That Show Similarity to PR-10 Proteins

“…Proteins from the intercellular fluid were obtained according to Parent and Asselin (1984), and proteins from the cell walls were extracted as described by Jackson and Ricardo (1994).…”