Lectins in Castor Bean Seedlings

Abstract

The amounts of the two lectins (ricin and Ricinus communis agglutinin) in tissues of castor bean seedlings were followed during germination and early growth. For measurement, lectins in extracts were separately eluted from Sepharose columns; an antibody to the agglutinin was also used to detect the lectins by immunodiffusion. The endosperm of the dry seed contains 3.5 mg total lectin (5.6% of the total seed protein), which declines by 50% by day 4 and more rapidly thereafter as the tissue is completely consumed. The cotyledons of the dry seed also contain lectins but the amounts are less than 1% of those in the endosperm, and, as in the endosperm, they are constituents of the albumin fraction of the isolated protein bodies. No lectins were detected in the green cotyledons of 10-day seedlings that had been exposed to light from day 5. The embryonic axes of 2-day seedlings contained very small amounts of lectins but they were not detectable in the aerial parts of seedlings grown for 3 weeks or in cells from endosperm grown in tissue culture. matrix and the globulin proteins of the crystalloid. The lectins are components of the matrix proteins (44,46) and are the only glycoproteins detectable in the protein bodies (8,44,46 Endosperm tissue was extracted with 1 N NaOH, using 2 ml per endosperm, by grinding in a mortar and pestle with sand. The homogenate was centrifuged at 20,000g for 15 min. The supernatant was decanted through two layers of Miracloth and the pellet re-extracted with 1 N NaOH. One ml of extract was mixed with 1 ml of 1 N HCI and 1 ml of 20% (w/v) TCA and held on ice for 5 to 10 min. The precipitated protein was pelleted and the pellet dissolved in 0.5 N NaOH. An appropriate dilution was assayed for protein (18), using BSA as the standard. The protein content of other extracts was assayed in a similar manner.Quantification of the Lectin Levels. All operations were conducted at 4°C. Twenty seedlings of known age were harvested and their endosperms extracted with 25 to 40 ml 0.1 M Na phosphate (pH 7.2), 0.15 M NaCl, 3 mM NaN3 by grinding in a mortar and pestle with sand. The extract was centrifuged at 20,000g for 15 min, the supernatant decanted through two layers of Miracloth, and the pellet re-extracted. The combined supernatant was brought to 65% saturation with (NH4)2SO4. A crude lectin fraction precipitated and was collected by centrifuging at 20,000g for 15 min. The (NH4)2SO4 pellet was suspended in PBS; 10 mm Na phosphate (pH 7.2), 150 mM NaCl, 3 mM NaN3, dialyzed against PBS, and loaded onto a Sepharose 4B-200 (Sigma) column equilibrated with PBS. The column was washed with PBS to remove contaminating proteins. Ricin was eluted www.plantphysiol.org on April 4, 2019 -Published by Downloaded from

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