Elicitation of Lignin Biosynthesis and Isoperoxidase Activity by Pectic Fragments in Suspension Cultures of Castor Bean

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Abstract
Suspension cultures of castor bean (Ricinus communis L.) which have been treated with pectic fragment elicitor rapidly accumulate lignin as measured by derivatization with thioglycolic acid. The responsiveness of cultured cells to elicitor is dependent on the stage of culture growth. In 6-day (maximally responsive) cultures, increases in lignin are first evident 3 hours after addition of pectic fragment elicitor with maximal rates of lignin synthesis between 4 and 10 hours. The abundance of lignin in cultures after 12 hours of elicitor treatment is 10- to 20-fold higher than in untreated control cultures and can thereby account for as much as 2% of the dry cell weight. Only intermediate sizes of pectic oligomer are active as elicitors of lignin. Half-maximal accumulation of lignin occurs at 250 to 300 micrograms per milliliter of an optimal elicitor preparation with an average degree of polymerization of seven. We consider the synthesis of lignin in elicited cultures to be a mechanism of plant disease resistance which is induced by the elicitor. Plant peroxidases have been proposed to catalyze the last enzymatic steps in the biosynthesis of both lignin and hydrogen peroxide. Six extracellular isoenzymes of peroxidase (two anionic, designated A1 and A2, and four cationic, designated C2, C3, C4, and C7) are detectable in healthy suspension cultures of castor bean by native gel electrophoresis. Treatment of cultures with elicitor causes substantial changes in the activity of four of these species (A1, C2, C3, and C7). Elicitor treatment also results in the appearance of three new peroxidase isoenzymes that are not readily detectable in healthy cultures (C1, C5, and C6). Increases in the activities of these isoenzymes are concurrent with or slightly precede the accumulation of lignin in elicited 6-day cultures. By 12 hours after addition of elicitor, C1 becomes the most abundant extracellular isoperoxidase. The differential regulation of expression of peroxidase isoenzymes following elicitor treatment suggests that individual isoenzymes of peroxidase may have specific functional roles in the biosynthesis of disease-lignin.