UPTAKE BY AND FATE OF LYSOZYME IN ROOTS OF IASIONE MONTANA (CAMPANULACEAE)

A basic protein, lysozyme, labeled with fluorescein isothiocyanate, is readily taken up by roots of lasione montana. Most of the protein taken up is tightly bound to cell walls of the roots. Fluorescent protein is diluted in the growing region of a root as cells elongate and divide. Fluorescence remains in mature nongrowing regions and root cap cells for one to two weeks. Redistribution and translocation of the protein within the root is minimal or nil. A layer of chloroform-soluble material that prevents lysozyme from interacting with stem cell walls exposed to fluorescent lysozyme was found on stems of germinating lasione montana. SOME RHIZOSPHERE MICROORGANISMS secrete exoenzymes that can induce pathological conditions in plant roots (Jones, 1909; Ulrich and McLaren, 1971). Not all proteins taken up by roots are enzymes, and nonenzymatic proteins can influence root growth and cell function. Lysozyme (as a protein; there is no known substrate for this enzyme in roots) has been found to inhibit root growth and has minimal nutritional value as a nitrogen source (Ulrich, Luse, and McLaren, 1964). Uptake of basic proteins by plant roots has been studied with barley, onion, tomato, and vetch (Seear, Bradfute, and McLaren, 1968). Penetration is rapid and the amount of lysozyme taken up can exceed 1 mg per g of fresh barley roots. About 90 % of the protein is simply adsorbed by ion exchange in root cell walls (McLaren, Jensen, and Jacobson, 1960). Location of the remaining 10 % of the lysozyme taken up is less precisely known; by means of autoradiography, radioactive lysozyme has been found in cytoplasm and nuclei of cells several layers beneath the epidermis (Jensen and McLaren, 1960). Translocation of lysozyme to leaves has also been observed (Ulrich and McLaren, 1965). With fluorescence microscopy, fluorescent proteins have been found in cytoplasm of damaged cells (Drew, Seear, and McLaren, 1970). By working with a plant with thin roots (only 14 cells across in the zone of elongation), the pattern of lysozyme uptake in living roots can be examined under a microscope without resorting to fixation and sectioning. Thus the time sequence of events at any interval of an experiment can be followed. This study concerns the uptake of lysozyme, labeled with fluorescein isothiocyanate, by roots of lasione montana (Burstr6m and Gabrielsson, 1964) and what becomes of the Received for publication 21 May 1973. 2 Present address: Department of Biology, M. I. T., Cambridge, Mass. 02139. protein as roots continue to grow. Some observations on the lack of penetration of labeled lysozyme into shoots have been interpreted in terms of shoot anatomy. MATERIALS AND METHODS-Seeds of lasione montanta L., Campanulaceae, (a gift from Professor H. Burstr6m), were surface sterilized with 1 % sodium hypochlorite in soapy water for 30 min on a shaker. The sterile seeds were then washed with sterile distilled water 4-5 times before being placed on an agar in disposable box type petri dishes, 50 x 12 mm. Each dish was set on edge by taping against a vertical wall so that germinating radicals would grow downward along the agar surface rather than into the agar. The agar, 1 %, contained Ca(NO3)2 and KH2PO4, each at 1/5000 M. Seedlings were allowed to germinate for 50 hr in the dark at 78 F. All solutions and glassware were previously autoclaved to minimize contamination by microorganisms. Twelve seedlings with approximately equal lengths (about 1 mm) were picked from the germinating plate and lined in a row upon a new agar plate. The growth rate of each root was measured with the aid of a microscope. Plants with roots growing at the same rate were chosen for experiments and only normal plants were used. All treatments were performed with an individual seedling suspended in a drop of test solution on a slide. The slide was placed in a petri dish containing two layers of wet filter paper to keep the atmosphere moistened. After the treatment, seedlings were rinsed with control solution and returned to the standing petri dish. Root growth was followed during and after the treatment. The control solution consisted of Ca(NO3)2 and KH2PO4, both 1/5000 M, at pH 7.2. Test solutions contained in addition egg white lysozyme or a fluorescent lysozyme (Fl-lys).