SUBMICROSCOPIC STRUCTURE AND DEVELOPMENT OF THE CHLOROPLASTS OF PSILOTUM TRIQUETRUM

SUN, C. N. (Washington U., St. Louis, Mo.) Submicroscopic structure and development of the chloroplasts of Psilotum triquetrum. Amer. Jour. Bot. 48(4): 311-315. Illus. 1961.-Aerial stems and stem tips of Psilotum triquetrum were used for the study of the fine structure and development of chloroplasts. The chloroplasts of Psilotum are ellipsoidal, with a principal axis of approximately 13 ,u and a short axis of approximately 3.6 . They are bounded by a well-defined outer membrane which consists of 2 layers. Within the laminar system of the stroma, the lamellated grana appear as sharply defined regions. The grana are about 1-1.6 . in diameter. They are distributed more or less uniformly throughout the entire chloroplast with the exception of a very narrow peripheral zone. Relatively large, osmiophilic globules occur in groups in the stroma. The development of the Psilotum chloroplast may be summarized as follows: (1) in the undifferentiated proplastid, vesicles occur; (2) lamellated layers are formed by the fusion of vesicles; (3) the lamellae multiply by a process of thickening and splitting; (4) the grana are differentiated within a certain area by heterogeneous deposition of material and by further cleavage of the lamellae. Osmiophilic globules are present throughout the developmental stages, and increase in number and size with increase in age of the chloroplast. FROM the morphological standpoint, there seem to be at least 2 types of chloroplasts: "granular," which appear to be typical of seed plants, and "lamellated," which are often found in algae. The "granular" chloroplasts are so called because they consist of laminated green granules, termed grana, embedded in an amorphous basis substance, the stroma. In the "lamellated" type, on the other hand, the lamina are not, as a rule, aggregated into grana, but are more or less uniformly distributed throughout the chloroplast. Among the more recent studies of the fine structure of pteridophyte chloroplasts are those of Miihlethaler (1955) on Selaginella watsonii and those of Kaja (1958) on Selaginella martensii. These investigators found that Selaginella chloroplasts have the same type of laminated structure as is found in algae. Since the structure of the photosynthetic apparatus may vary from species to species, it seemed desirable to make a detailed study of the chloroplasts of other pteridophytes. Another problem is that of the origin of chloroplasts. This has attracted the attention of plant scientists since the time of Meyer (1883) and Schimper (1885), who established the theory of the continuity of plastids. Since the application of electron microscopy to the problem, various theories and interpretations as to the origin of the chloroplast lamellae have been advanced, such as those of Heitz (1954), Leyon (1954), and Hodge and co-workers (1955, 1956). However, there is as yet no conclusive evidence as to the mechanism by which chloroplast lamellae arise. The present study, therefore, will consider not only the fine structure of Psilotum chloroplasts, but also their development and differentiation. MATERIALS AND METHODS.-Aerial stems and 1 Received for publication September 9, 1960. stem tips of Psilotum triquetrum were used for this study. The freshly collected material was cut into pieces 1 mm. thick, which were then fixed for 4 hr. at 50C. in Dalton's osmium tetroxide-bichromate mixture at pH 7.6. After dehydration they were embedded in a 6:1 n-butyl and methyl methacrylate mixture, and were finally sectioned according to the method described in an earlier paper (Sun, 1960). The sections were studied, and electron micrographs made with an RCA-EMU-2 electron microscope. STRUCTURE OF THE CHLOROPLAST.-Fully developed chloroplasts of Psilotum are ellipsoidal, with a principal axis of about 13 /4 and a short axis about 3.6,u. They are bounded by a well-defined outer membrane which consists of 2 layers (fig. 1). The very regular lamellated structure of the chloroplast is easily recognized in ultra-thin sections. Within the stroma the grana appear as sharply defined structures, some 1 to 1.6 I in diameter, with their broad faces parallel to the broad faces of the chloro. plast. The grana are distributed more or less uniformly throughout the chloroplast, except for a very narrow peripheral zone. In a section of a full. grown chloroplast, the grana appear to consist of some 10-20 lamellae, each about 80 A thick, and separated by interlamellar spaces about 160 A wide. Stroma lamellae are rather infrequent. Relatively large osmiophilic globules, with a diameter of 0.070.3 ,x, occur in groups in the stroma. DEVELOPMENT OF THE CHLOROPLAST. In the api. cal meristem cells of Psilotum one finds mostly undifferentiated proplastids, whereas mature chloroplasts predominate in the parenchyma cells of the stem, about 10 mm. below the apical meristem. The undifferentiated proplastids are small, round or oval bodies, 0.8-1.1 / or less in diameter, surrounded by an outer membrane, and either containing no discernible structure or filled with small vesicles or