ORGANIZATION AND BREAKDOWN OF THE PROTOPLAST DURING MATURATION OF PINE TRACHEIDS

The protoplast of maturing axial tracheids in the secondary xylem of shortleaf pine (Pinus echinata Mill.) was studied by transmission and scanning electron microscopy. The mature protoplast is differentiated into two interconnected components: (1) the commonly observed peripheral layer lining the secondary cell wall, and (2) an elaborate reticulum of cytoplasmic filaments and placoids within the central vacuole. The reticulum provides an extensive surface area of vacuolar membranes for rapid exchange of nutrients and metabolites with the vacuolar sap, which is envisaged to function as a vital medium during the period of secondary cell wall synthesis. The breakdown of the protoplast which terminates tracbeid maturation is associated with poorly defined alterations of the vacuolar membranes. This is indicated by increased formation of cytoplasmic spherules and membraneous vesicles which may be portions of separated vacuolar membrane during early stages of degradation. Autolysis is supposed to occur when the cytoplasm is exposed to the vacuolar sap after rupture and separation of the vacuolar membranes. The Gomori acid phosphatase technique as combined with electron microscopy produced no evidence of autolysosomal segresomes in strands of intravacuolar reticulum of the cytoplasm. DIFFERENTIATION OF cambial derivatives into axial tracheids involves several steps, including the maturation phase during which the secondary cell wall is deposited (Wilson, Wodzicki, and Zahner, 1966). This phase begins near the completion of radial enlargement of the cell and is terminated upon disintegration of the protoplast. Lysis of the protoplast in differentiating xylem elements is rapid (Cronshaw, 1965). Skene (1972) found the average time of protoplast lysis in stem tracheids of Tsuga canadensis to be 4 days. In the tracheids of Pinus sylvestris stem, autolysis of the protoplast occurs in 2-5 days, however the exact duration of the process is not known. Earlier or later termination of the maturation phase significantly affects the thickness of the tracheid secondary cell wall through its direct effect on the duration of cell wall deposition (Wodzicki, 1971). The present paper describes changes in the structure of the protoplast in differentiating tracheids beginning with the early phases of secondary cell wall deposition and end' Received for publication 21 July 1972. 2 Visiting research professor in tree physiology, School of Forest Resources, University of Georgia. Permanent address: Faculty of Forestry, Agricultural University of Warsaw, Poland. Supported in part by McIntire-Stennis GEO 0025-A funds and Georgia Forest Research Council, Macon, Georgia. We wish to acknowledge the helpful advice and suggestions of Dr. W. J. Humphreys, Director, Electron Microscopy Laboratory, University of Georgia, and the excellent technical assistance of Mrs. Janet Johnson. ing with the final breakdown and maturation of the functional tracheid as viewed with scanning and transmission electron microscopy. MATERIALS AND METHODS-Small blocks of tissue (1 mm3) comprising phloem, cambial zone, and xylem, were isolated several times during the season from three 8-year-old stems of Pinus echinata Mill. growing near Athens, Georgia. The tissue blocks were fixed either in 2 % glutaraldehyde buffered at pH 7.2 with 0.1 M sodium cacodylate alone, or postfixed with 2 % osmium tetroxide 0.1 M cacodylate solution for 2 hr each at 0-4 C. Some of the double-fixed tissue blocks were immediately embedded by the maraglas technique of Freeman and Spurlock (1962), and sectioned on a Sorvall MT-2 ultramicrotome with diamond knives. Sections were picked up on copper grids (300 mesh), stained with uranyl acetate and lead citrate, and viewed in a Philips 200 electron microscope. Other cubes of tissue fixed by both methods were stored in cold 0.1 M sodium cacodylate, pH 7.2 and 5 % sucrose until used for scanning electron miscroscopy or histochemical studies following the Gomori (1952) technique of acid phosphatase as combined with electron microscopy (Ericsson and Trump, 1965). The tissue fixed in glutaraldehyde only was incubated and afterwards postfixed with osmium tetroxide. The double-fixed tissue for scanning and electron microscopy was washed for 1 hr in several changes of glass-redistilled water and processed in the following ways: (1) cubes were positioned in an