504 Cytologia7

The Structure of the Chloroplast of Pellionia pulchra1) By Elliot Weier Collegeof Agriculture, University of California ReceivedFehruavy 4, 1936

That the chloroplast has presented one of the most difficult cytolo gical structures in the cell to interpret is shown by the points of view of various workers. Without going into any great detail concerning the literature of chloroplast structure the following few papers may be of interest. In 1837 Meyen described the chloroplast as a body which showed dark spots on a light background. Meyer (1883) nearly 50 years later proposed his well known grana theory. According to this the dark spots of Meyen are in the nature of granules in which the green pigment is supposed to be dissolved. These grana are imbedded in a material which Meyer suggests is colorless. Schimper (1885) corroborated Meyer's idea of plastid structure. Priestly and Irving (1907) report that the chlorophyll is to be found only in the peripheral layer of the chloro plast. Lloyd (1924) working with fluorescent light concluded that the chlorophyll is in the back ground substance of the chloroplast rather than in the granules. Zirkle (1926) is of the opinion that the chlorophyll is rather evenly distributed throughout the plastid, that the grana are canals connecting the exterior of the plastid to a large interior vacuole. Starch always appears in this central vocuole. This recalls the old utricle concept of Nageli (1846) and others. Zirkle's description of the chloro plast has found considerable acceptance in recent text books (Miller, 1931; Barton-Wright, 1933; Smith, 1935). Guilliermond's work (1933) with dark field illumination suggests that the chloroplast is a homo geneous structureless body. The writer's (Weier, 1932) observations in connection with inves tigations on the Bryophyte chloroplast have not contributed to the support of the recent reports of either Zirkle or Guilliermond. In the first place structure of some sort is easily visible in the great majority of plastids that have been examined, thus making it impossible to accept Guilliermond's conclusions. Secondly this structure definitely does not conform to Zirkle's description of the plastid in that non-starch containing plastids when fixed and stained do not possess a large central vacuole. When starch does appear, at least in those chloroplasts which definitely

1) Paper from the Department of Botany, University of MichiganNo. 530. 1936 The structure of the chloroplast of Pellionia pulchra 505 elaborate starch, it is not limited to grains located in a single central vacuole but is divided into numerous grains scattered throughout the plastid. Fixed and stained plastids do not show the slightest evidence for the presence of canals though frequently they do contain numerous granules. It should be recalled that Zirkle's evidence for the canalicular structure is negative. Unable to obtain positive tests for fat, protein or starch in the granules he observed in the chloroplast he concluded that these granules were minute pores or canals. Since it has been possible to stain granules within the plastid with both haematoxylin and osmic acid it is scarcely possible to accept Zirkle's conclusions in regard to the nature of the pores or grana as applying to many Bryophyte chloroplasts. It seems that the plastid is a structure of sufficient importance to merit detailed cytological investigation. The writer undertook to initiate a series of studies on the chloroplast in the hope of arriving at some definite knowledge of its structure, chemistry and function. Pellionia pulchra, a trailing greenhouse plant belonging to the Urti eaceae family was chosen as likely experimental material for the initial observations. These first studies were largely concerned with chloroplasts containing no starch. They were observed either in the living condition, fixed in various fluids and stained in Heidenhain's haematoxylin or treated with osmium according to the technique of Kolatchev (Bowen, 1929).

Observations The Pellionia chloroplast is usually described in text books as con taining one or two large starch grains showing the typical concentric structure of storage starch grains. A small amount of plastid cytoplasm is usually condensed at one end of the grain and it is supposed that a thin sheath of cytoplasm surrounds it (Meyer, 1885; SchUrhoff, 1924, Smith, 1935). This is true, according to the writer's observations, only of those plastids which store starch. This storage type of chloroplast is located in the chlorenchyma cells of the stem and in the spongy paren chyma of the Pellionia leaf. The palisade cells contain three or four chloroplasts with a strikingly different structure. This difference may be observed in living preparations but is more easily seen in material which has been prepared by one of the standard mitochondrial techniques and which has been well stained with Heidenhain's haematoxylin. The chloroplasts in the palisade cells are large and composed mostly of cytoplasm in which approximately a dozen small temporary starch grains may be imbedded (Fig. 2). These plastids are many times richer in cytoplasm than those present in the nearby spongy parenchyma cells. Instead of one or two large starch grains showing a concentric structure the palisade chloroplasts contain starch grains showing no structure. The starch in the chloroplasts of the spongy parenchyma cells is anisotropic 506 E. WEIER Cytologia 7

while that in the palisade cells is isotropic. This uniform difference between the chloroplast of cells located so close together is quite striking and is well illustrated by Figs. 1 and 2. In Pellionia plants which have been kept in the dark the starch disappears from the palisade chloroplasts first. In plants which have been exposed to continuous light, for 48 hours the starch grains in the chloroplasts of the spongy parenchyma and of the stem increase to an enormous size. No apparent increase in the size of the starch grains in the palisade plastids is visible. However, the num ber of grains increases and the cytoplasm of the plastid becomes stretched into thin layers separating the numerous grains (Fig. 10). Zirkle has recently suggested that certain chloroplasts may be con cerned mainly with a type of starch storage while photosynthesis is more active in others. The observations presented here would seem to support this contention. While both plastids are green, apparently containing chlorophyll, one plastid is much poorer in cytoplasm and possesses one or two starch grains whose structure is typical of that of normal permanent storage starch grains. The other plastid is rich in cytoplasm and possesses numerous starch grains of a temporary isotropic nature. Furthermore the storage starch grains do not disappear as quickly in darkness as do the temporary grains. These factors strongly support and extend Zirkle's contention that some chloroplasts may be mainly concerned with carbohydrate elaboration while others may store starch. The living starchless chloroplast in the palisade cells when viewed either in transmitted or oblique white light shows very definite globules or grana within the chloroplast (Fig. 4). They have a very real appear ance. They are round and rather highly refractive, passing in Pellionia from brilliantly green grana to dark spots with a change of focus. It is not possible to follow them as threads deep into the plastid as it should be were they canals. In other forms more recently observed, light of varying wave lengths and of better quality than was available for the Pellionia studies brings the grana into great prominence. Under certain conditions, possibly of a pathological nature, they show, in a few forms at least, a delicate Brownian movement. In white light both the grana and background substance of the chloroplast appear to be green. If the grana are canals it seems that they should disappear when the general structure of the plastid was destroyed. Upon application of sufficient pressure to the cover slip the chloroplast may be completely disorganised. It loses its shape, bursts and flows out of the cell as a bit of fluid protoplasm into the surrounding media. Even under these conditions the grana are very much in evidence (Fig. 5). In fixed and stained chloroplasts there is no evidence for either a central cavity or canals (Figs. 3, 6, 7, 8, 9, 10). In some plastids 1936 The structure of the chloroplast of Pellionia pulchra 507

Figs. 1-10. 1 . Typical starch storing chloroplast of Pellionia. Stippled region, cytoplasm; white region , starch grain. This grain is anisotropic. 2. Typical starch elaborating chloroplast of Pellionia . The clear cigar shaped regions are starch grains. The densely stippled regions are the grana. These starch grains are iso tropic. 3. Non-starch containing chloroplast of Pellionia fixed in Helly's fluid and stained in haematoxylin . Chloroplast is perfectly homogeneous and in this instance t ook very little stain . 4. Typical living non-starch containing chloroplast. The grana are apparent . 5. A portion of a ruptured chloroplast. The grana may still be observed 508 E. WEIER Cytologia7

numerous blue staining granules are observed but variations in this structure are common. Some chloroplasts do not stain at all. In others the staining of the granules varies from a mere suggestion to an intense blue color (Figs. 6 and 7). Irregular deeply staining regions may be observed in some plastids (Fig. 8). This type of coloration grades into an even blue or blue-black throughout the whole chloroplast (Fig. 9). A similar variation may be observed in material fixed and stained by the osmium method of Kolatchev. The inference is permissible that different physiological stages obtained in different chloroplasts at the time of fixaiton. Blackening with osmium is due to a chemical reduction. Chromati city of certain regions within the cell after bichromate fixation and followed by a haematoxylin stain may also depend upon the presence of an unsaturated valence (Lee, 1924). It is not illogical to connect this variation in staining with the photosynthetic reaction which is in itself a reduction process. If this assumption be allowed the photosynthetic reaction, like many other vital reactions must be of a rythmic nature. Since the variation in staining covers the whole plastid region, it must be that each step takes place simultaneously throughout the chloroplast. At one stage carbonic acid or a carbonate would diffuse rapidly through every part of the aqueous phase of the plastid. Conditions would then change and it would combine with its acceptor (Spoehr, 1926). Energy relations now entering in would build up the carbon dioxide and water into a carbohydrate throughout the chloroplast, which would now as a unit return to a state which would allow for the inward diffusion of carbon dioxide in some form. Recent evidence in forms other than Pellionia suggests very strongly that starch is first formed everywhere within the plastid and that it later condenses to form the typical grain. This concept of the location of the complete photosynthetic reaction is in direct contrast to theories calling for the definite localization of its various steps in different parts of the cell or chloroplast (Spoehr 1926). It seems possible that an accurate knowledge of plastid structure should materially aid in the formulation of chemical theories. Obviously these theories must comply with the physical conditions under which the reaction takes place. The relationship of the grana to the starch grains is not at present understood. The grana are visible but not so clearly in palisade chloro

6. Chloroplast in Helly's fluid, stained in haematoxylin. Some grana are intensely colored, others moderately so. 7. Chloroplast in samelsection as No. 6. Most of the grana are intensely stained. 8. Chloroplasts from the same section as Nos. 6 and 7. The staining material scattered irregularly throughout the chloroplast. 9. In same cell as No. 8. Chloroplast stains heavily and evenly. 10. Chloroplasts from leaves exposed to light for 48 hours. Note the large number of small starch grains and the honey comb state of the background substance. No grana are visible. 1936 The structure of the chloroplast of Pellionia pulchra 509 plasts containing starch. They may be observed in fixed and stained plastids supplied with starch (Fig. 2). In the process of starch formation the mass of chloroplast ground substance comes to form a delicate honey comb structure. As stored starch increases, the amount of material separating the grains becomes very much reduced, eventually consisting of a delicate film (Fig. 10). Grana are not apparent here.

Conclusions 1. There are at least two kinds of structurally different plastids present in the leaf of Pellionia. This is in support of an observation made by Zirkle on other forms. 2. One structural plastid type is located in the palisade tissue. It is concerned mainly with the elaboration of starch. The starch grains formed within it are isotropic. 3. The other structural type is located in the cells of the spongy parenchyma and stem. It contains a single large starch grain indicating that it serves as a storage organ. This starch grain is anistropic. 4. Structurally the elaborating chloroplast of Pellionia is composed of numerous grana dispersed in some continuous background substance. 5. The structure of the chloroplast of Pellionia definitely supports Meyer's concept of grana as opposed to Zirkle's canalicular-vacuolar theory and Guilliermond's report that the plastid is a homogeneous body. 6. Plastids vary in their ability to reduce osmium tetraoxide and to stain with Heidenhain's haematoxylin. 7. Certain assumptions based on number six suggest that the photo synthetic reaction is of a rhythmic nature similar to other vital reactions.

Literature Cited 1933. Barton-Wright, E. C. Recent Advances in Physiology p. 173-174. 1929. Bowen, R. H. Bull. Torrey Bot. Club 56: 33-52. 1933. Guilliermond, A., G. Mangenot et L. Plantefol. Traite de cytologie vege tale. Paris. p. 420. 1924. Lee, Arthur Bolles. Microtomist's Vade Mecumedited by J. B. Gatenby. p. 360. 1923. Lloyd, F. E. Science 58: 229-230. 1924. - Science 59: 241-248. 1837. Meyen,F. J. F. Neues System der Pflanzenphysiologie. Band 2 p. 219. 1883. Meyer, A. Das Chlorophyllkorn. 1846. Nageli, Carl von. Zeits. f. wiss. Bot. 3 and 4: 94-128. 1931. Miller, E. C. Plant Physiology. p. 21. 1907. Priestly,J . H. and A. A. Irving. Ann. Bot. 21: 407-414. 1885. Schimper, A. W. F. Jahrb. f. wiss. Bot. 16: 1-247. 1935. Smith, Gilbert M. A Text-book of General Botany. p. 137. 1926. Spoehr, H. Photosynthesis. 1932. Weier, T. E. Am. Journ. Bot. 19: 659-672.1926 . Zirkle, C. Am. Journ. Bot. 14: 301-341.