THE CUP PLASTID OF NICOTIANA RUSTICA

WEIER, T. E., and C. R. STOCKING. (U. California, Davis.) The cup plastid of Nicotiana rustica. Amer. Jour. Bot. 49(1): 24-32. Illus. 1962.-In situ and isolated chloroplasts of Nicotiana rustica have been studied by light and electron microscopy. Under certain conditions, notably of low light intensity, the starch-free plastid forms a cup. In isolated plastids the form may be modified by the tonicity of the isolation medium. In situ, the cup always faces the cell wall. Electron micrographs show the cup to be formed of compartmented grana connected at irregular intervals by flexuous channels known as frets. The interior of the cup is filled with a finely granular stroma whiclh also forms the material surrounding the grana and the frets in the body of the cup. The grana radiate outward from the central stroma. They may be considered as cylinders. They are fairly rigid, as curvatures to form the cup-shape occur only in the interconnecting fretwork. The compartments may have a limited movement with reference to their axis. These evidences of movement of the part of the ultramicro plastid structures thought to contain chlorophyll suggest that the movement may be related to changes in light intensity or other factors influencing the rate of photosynthesis. CUP-SHAPED chloroplasts occur in homogenates of leaf mesophyll tissue (Weier, 1958). Mudrack (1956) has shown that in Viola tricolor the condition was due to the hypertonicity of the sugar solution in which they were suspended. This observation was extended by Kern (1959) who described the appearance of chloroplasts in hypotonic, isotonic, and hypertonic solutions. The side views of plastids under these 3 conditions were as follows: "hypotonic form," spindle-shaped plastid with an extended membrane on one side; "isotonic form," spindle-shaped plastid; "hypertonic form," cupshaped plastid with or without membrane destruction. Senn (1908) described cup plastids in living leaves of Funaria hygrom.etrica and one of Meyer's drawings indicates that he was aware of their occurrence in 1883. Weier (1958) observed cup-plastids in fixed and stained leaves of Nicotiana rustica. The chloroplasts in these cells were oriented so that the cup generally faced the cell wall. The interior of the cup stained with iron alum hematoxylin after fixation in potassium dichromate and formaldehyde, while only the margin of the cup stained if a fixing solution containing acetic acid was used. The cup plastids were always starch-free and occurred only in leaves taken from plants that had been in darkness 36 hr or longer. 1 Received for publication May 28, 1961. This project was supported in part by the National Science Foundation (Grant G-9885) and in part by the United States Public Health Service (Grant RH -5868). The implication of these observations, as well as of others, going back to Meyer in 1883, is that the grana complex may, under appropriate conditions, move as a unit within the stroma in response to changes in the metabolic state or environmental conditions. The observations of Mudrack and Kern indicate that the tonicity of the ambient medium influences the distribution of the grana within the plastid. Conversely, it seems possible that the pressence of a cup plastid in intact leaves may give some indication of the relationship between the tonicity of hypaloplasm and the plastids. This paper reports the distribution of chloroplasts in starch-free leaves of Nicotiana rustica subjected to 36 hr darkness and briefly compares this with their distribution in chloroplasts in which carbohydrate is being deposited. MATERIAL AND METHODS-The plants of Nicotiana rustica were greenhouse-grown and collections were made of fully expanded leaves from plants just ready to flower. Light intensity varied from about 8000 ft-c during the summer months to about 800 during cloudy days in winter. Previous to collection for cytological study, the plants were placed in a dark cabinet for 36 hr to allow for complete dissolution of starch. Plants were then returned to light and collections made after 1 and 2 hr in the light. For light microscopy, sections of leaves were killed in Regaud's solution (8 ml of 4%o K2Cr2O + 2 ml of formol) and in Allen's solution (75 ml satFig. 1-5.-Fig. 1. Chloroplast isolated from frozen dried leaves by the aqueous technique. Resuspended in water prior to photographing. X 1000.-Fig. 2. Chloroplasts fixed in Allen's solution, stained in hematoxylin. The margin of the cup is basophilic. X 1000.-Fig. 3. Chloroplasts fixed in Regaud's solution. The interior of the cup is basophilic. X 1000.Fig. 4. Chloroplasts in spongy parenchyma cells adhering to an epidermal peel. Fixed in 2.5% KMnO4 dehydrated and mounted in Euparal. The grana are the darkened spindle-shaped dots; they are aligned to form the cup proper. The interior of the cup is colorless and completely transparent. X 4000.-Fig. 5. Chloroplast fixed in Dalton's fluid, dehydrated and embedded in methacrylate. An electron micrograph. This is a section about 0.03A thick showing approximately a median section of cups. Note, as in Fig. 3, they face the cell wall, and as in Fig. 4, the grana form the cup proper. The interior of the cup is filled with a granular stroma. X 6000.