CINEMATIC OBSERVATIONS ON THE GROWTH AND DIVISION OF CHLOROPLASTS IN NITELLA

As it elongates from about 0.2 to 80 mm, the Nitella internodal cell shows an increase in plastid number from a few thousand to about 4 million. The increase takes place by plastid division. A continuous motion picture record followed a population of 8 plastids in an elongating cell until their progeny numbered 18, a span longer than 1 fission cycle for some of the plastids. One complete fission-fission cycle was about 22 hr. The highly directed nature of chloroplast expansion (elongation) is lost when cell wall strain (expansion) is mechanically inhibited by pressing the cell between glass plates. The plastids then expand about equally in all directions in the plane of the cell surface. When a new direction of maximum strain is introduced by the mechanical induction of a lateral in the cell, the plastids elongate in this new direction. The direction of the protoplasmic stream does not show this striking response to strain but tends to follow the lines of the chloroplast chains, not the long axis of individual plastids. THE UNDERSTANDING of the growth of cell organelles in terms of ultrastructure is facilitated by certain information difficult to obtain except from long-term observations on living material. The site of expansion, nature of the effective growth axis, the drift of proportions with time, the generation time, etc. are broad questions of organelle development which can, in favorable material, be unambiguously answered through time-lapse motion pictures. This article presents data of the above sort for the chloroplasts of growing Nitella cells. The chloroplasts of Nitella internodal cells lend themselves well to direct observation because they have strong natural pigmentation, are confined to a monolayer at the extreme periphery of a cell of large radius, and do not circulate in the protoplasmic stream. These plastids elongate and divide as the cell-and the cortical gel to which they are attached-increases its surface area. I Received for publication August 18, 1963. Supported by NSF Grant G-12919. The technical contributions of Mrs. Mary Eager are gratefully acknowledged. This activity produces long helical files of chloroplasts which lie along the lines of maximum surface expansion of the cell. These lines are offaxial because the cell twists as it elongates (Green, 1954). Growth in cell surface area takes place evenly over the cell surface (no localized growth zone). Because of the great length of the mature cells (20-60 mm), it is possible to follow the chloroplast activities in an already sizable cell (2 mm long) throughout a many-fold increase in cell surface area and a many-fold increase in chloroplast number. MATERIALS AND METHODs-A clone of N. axillaris Braun grown in autoclaved soil extract was employed throughout. Motion pictures were taken through a 16-mm Bolex camera provided with a motor and timing box designed by Dr. Ralph 0. Erickson of the University of Pennsylvania. Timing was for 1 exposure every 2 min. The camera was mounted horizontally to photograph through a Spencer microscope fitted with a 23 X oil immersion lens (Leitz) and a Wild viewing attachment. The first major technical difficulty was that of holding the specimen firmly while the This content downloaded from 207.46.13.52 on Fri, 21 Oct 2016 04:28:10 UTC All use subject to http://about.jstor.org/terms March, 1964] GREEN-CHLOROPLASTS OF NITELLA 335 objective viewed it through a glass window thin enough to permit good resolution of the plastids. This was solved by drilling a small tapered hole in a sturdy glass vessel (Bausch and Lomb) and then covering the hole with a # 1 coverslip glued to the cell with glassblower's pitch. The smaller the hole, the less was the bowing of the coverslip in response to the increase in the Nitella cell diameter. The oil immersion objective had a convenient taper, so the hole could be reduced to about 9 mm diameter (Fig. 1). A second difficulty concerened holding the Nitella cell. If surface expansion is to be observed, the area of interest must slide across the field of view. This tends to confound efforts to keep the chloroplasts in focus for many hours at a time. The most suitable arrangement was to clamp an area of the Nitella cell between 2 glass surfaces. One surface was the above-mentioned coverslip, the other a thick glass clamp ultimately held to the sturdy glass vessel by clothespins. Glass shims prevented excessive compression of the internode cell. To observe chloroplast divisions, the immediately adjacent area was photographed. In cases where cell expansion was to be mechanically inhibited, a flat glass bar lightly pressed the cell against the coverslip and the clamped area was observed directly. Friction between the cell wall and glass was great and the observed cell surface of the clamped area did not expand or expanded extremely slowly. A microscope condenser focussed continuous light from a Unitron K6hler-illuminator onto the cell.