THE CHLOROPLASTS OF EQUISETUM TELMATEIA ERHR.: A POSSIBLE DEVELOPMENTAL SEQUENCE

Transcription

1 Keu- Phytol. (1971) 70, THE CHLOROPLASTS OF EQUISETUM TELMATEIA ERHR.: A POSSIBLE DEVELOPMENTAL SEQUENCE BY JEAN M. WHATLEY Botany Department, University of London, King's College, Strand, London, W.C.2 (Received 20 April 1971) SUMMARY Parenchyma cells in the leaf sheath of Equisetum telmateia form linearly arranged series of maturing and senescing cells. These series of cells contain chloroplasts showing a progressive sequence of developmental stages easily recognizable because of the spatial relationships involved. The cell sequence may be classified into four successive zones, characterized by the typical structure of the chloroplasts present in the cells of each zone. Zone I. Meristematic cells contain immature chloroplasts with little internal lamellar structure. Zone II. Adjacent to the above are ceils containing mature chloroplasts with a well-developed internal lamellar system. Zone III. Next in sequence are cells in which the chloropl^ts retain a mature internal lamellar system but are distinguished from the previous group by the presence of irregular osmiophilic deposits around the outer double membranes. ZoTie IV. Finally there occur cells with usually rounded chloroplasts which show the osmiophilic deposits described above, but in which the outer double membrane is often difficult to distinguish. Internally the lamellar system is highly sinuous, and contains many swollen locules. In spite of a general similarity in appearance between chloroplasts of Equisetum and those of angiosperms, Eguisetttm plastids are characterized by a distinctive feature more commonly associated with algae than with higher plants. Young chloroplasts are almost completely sur.- rounded by rough endoplasmic reticulum. This ver)' close relationship between reticulum and chloroplast is retained as the chloroplast matures, though the degree of envelopment becomes less with increasing age. INTRODUCTION There is little published information about the fine structure of chloroplasts of the genus Equisetum. Sun (1963) attempted a description of chloroplasts of 'E. hiemale'. Manton (1966) in her article on structural relations between chloroplasts and other cell components is concerned for the most part with algal material. Illustrations of chloroplasts from sporogenous tissue of Equisetum are included in the study as representatives of higher plants (in the widest sense) but some interesting aspects of the structural relationships shown in the micrographs are not discussed. Although chloroplasts of higher plants (in the wide sense used by Manton) do not show the variety of structure shown by the highly variable algae, nevertheless the chloroplasts of Equisetum show several noteworthy features which do not seem to be generally found at least among the angiosperms. Furthermore, the mode of development of the leaf sheath, which produces relatively well-defined rows of cells representing a time sequence, makes possible a comparison of cells representing developmental stages. 109s

2 1096 JEAN M. WHATLEY In their studies on the development of the vegetative shoot of E. arvense L. Golub and Wetmore (1948), describing development of the leaf sheath, state, 'In the lower portion of the free teeth and down the entire length of the sheath, in the same vertical line the parenchyma cells radially peripheral to the vascular strands tend to divide peril clinally. The resulting short, radial rows of cells become hypertrophied, tend to round off and separate from one another, forming a loose, densely chlorophyllous tissue'. The structural pattern descrihed appears similar to that observed in E. telmateia. MATERIALS AND METHODS Stems of Equisetum telmateia were collected in the field about mid-day. Both immature and mature plants were included in the collection. Material from mature stems and from leaf sheaths from different positions on both young and mature stems were prepared for examination under an A.E.I. E.M. 6B electron microscope. Segments were fixed in glutaraldehyde, stained with osmic acid, dehydrated in an alcohol series and embedded in araldite. Most preparations were subsequently post-stained with uranyl acetate and lead citrate. OBSERVATIONS Examination of the parenchyma cells showed their linear arrangement to be as illustrated in Fig. I and in Plate i, Nos. i, 2 and 3. For convenience these cells were considered to belong to four distinct zones representing a series of developmental stages from meristematic tissue in Zone I through to senescing cells in Zone IV. The linear arrangement of the cells (and hence their relative ages) is clear in the earlier developmental stages. Older cells tend to separate off from each other and, with the loss of linear alignment, age determination becomes more difficult. However, examination of older leaf sheaths confirmed that the cells classed as belonging to Zones III and IV were in fact older cells. The chloroplasts of meristematic cells showed little variation in structure and their general similarity suggested a considerable degree of developmental synchrony. However, as distance from meristematic tissue increased the range of variation in chloroplast structure became greater and, in Zones II and III in particular, different chloroplast types suggesting several probable stages in chloroplast development, could be found within an individual cell. This is consistent with a progressive decrease in synchrony of development. Zone I (Plate 2, Nos. i and 2) Meristematic cells contained chloroplasts which were small and rounded. Their internal lamellar system was sparse and granal stacking minimal. Even in these relatively immature chloroplasts small osmiophilic globules were present. A very close association existed between all these immature chloroplasts and rough endoplasmic reticuium. This was consistently noted and the closeness of the association suggested the presence of rough endoplasmic reticuium as an unbranched sheath surrounding each chloroplast. The distance between the chloroplast outer membrane and the reticuliun appeared to be constant, at about 25 run. Gibbs (1962) described smooth endoplasmic reticuium occurring in algae as an outer envelope around chloroplasts and distinct from the remaining endoplasmic reticuium of

3 Equisetum chloroplasts 1097 the cell. The chloroplast endoplasmic reticulum was observed in most algal groups but appeared to be absent from the Rhodophyta and Chlorophyta. The envelope was described as an outfolding of the outer nuclear membrane. Bouck (1965) confirmed its Fig. 1. Part of the parenchyma from the leaf sheath of Equisetum showing the linear arrangement of cells. Numbers I, II, III, and FV indicate zones of cells of increasing maturity'. occurrence in Phaeophyta and, in addition, described the occurrence of villi protruding from the chloroplast endoplasmic reticulum into the space adjacent to the chloroplast bounding membrane. Manton (1966) discussed the close relationship between endoplasmic reticulum and algal chloroplasts, but did not draw attention to such a relationship in Equisetum, although the illustrations in her paper do suggest that Equisetum chloroplasts may be bounded by endoplasmic reticulum. Similar relationships have also been noted by Kawamatu (1963) for green chloroplasts from the root hairs of Azolla imbricata and by Bong Yul Yoo (1970) for some plastids of dormant peas. In peas the chloroplast endoplasmic reticulum was branched and smooth. Wooding and Northcote (1965) observed endopiasmic reticulum occurring as a sheath around immature plastids of companion cells and developing sieve tubes of Acer phloem and of resin canal and leaf callus cells of Pinus. These sheaths, as in Equise-

4 1098 JEAN M. WHATLEY tum, lay at a constant distance from the plastids of about 25 nm. However, the Acer and Pinus sheaths were associated with ribosomes only on the side of the reticulum awav from the plastids whereas in Equisetum, ribosomes were present on the external faces of both membranes. Wooding and Northcote suggested that a direct connection existed between the chloroplast endoplasmic reticulum and both the c)ftoplasmic endoplasmic reticulum and the nuclear envelope. In Equisetum no such connections were observed nor were villi (as described by Bouck, 1965) present. However invaginations of the inner chloroplast membranes were common (Plate 2, No. 3). Zone II As described above, the cells of Zone II contained chloroplasts showing a considerable range of variation in structure, which are thought to represent different development stages. However, there was no recognizable progression of chloroplast structure which could be correlated with distance from the meristematic tissue. Individual cells were likely to contain several variants within their chloroplast populations. Five chloroplast types are described below and the order of listing may broadly represent a developmental sequence. Type a (Plate 2, No. 4). Most common were mature rounded chloroplasts with a well-developed internal lamellar system. Granal stacking was more advanced than m chloroplasts in Zone I, though stacks seldom comprised more than ten compartments. The close association with endoplasmic reticulum was retained although the sheath-iiie appearance was generally less complete. The constant distance between reticulum and chloroplast was maintained. Type h (Plate 2, Nos. 5 and 6). Some mature chloroplasts contained configurations similar to the prolamellar bodies seen in etioplasts of angiosperms. Although after illumination prolamellar bodies of etioplasts disappear, they may be induced to reappear under conditions of low light intensity (Weier, Sjoland and Brown, 1970) or flashing red light (Bradbeer, personal communication). The occurrence of such structures at this developmental stage in Equisetum may perhaps represent an initial phase of chloroplast degeneration, possibly related to the change in the leaf sheath colour from green to black, as it becomes older. Thomson (1966) gives illustrations of figures similar in appearance to prolamellar bodies (which he calls 'membrane remnants') in chloroplasts associated with colour change in oranges. However, in this case large osmiophilic globules are also present, and the internal membrane structure shows signs of breakdown. Although no prolamellar bodies were seen in chloroplasts of Zone I, illustrations of these younger chloroplasts (Plate 2, Nos. r, 2 and 4) show that the internal lamellar system appears polarized, with the lamellae orientated in such a way as to suggest their initiation at centres of divergence, comparable at least in siting with prolamellar bodies. Type c (Plate 3, No. i). Elongated chloroplasts were also found within Zone II. These too had mature internal lamellar systems and sometimes prolamellar bodies. It is not possible to say whether the two chloroplast shapes (rounded and elongated) are distinct or result from the angle of sectioning. However, similar elongated chloroplasts are common in leaf cells of Phaseolus vulgaris at a developmental stage associated with chloroplast division. Type rf(plate 3, No. 2). Among both the rounded and the elongated chloroplasts individuals occurred within which the internal membrane system no longer maintained its overall parallel pattern but appeared to fold in on itself. The form is similar to that shown

5 Equisetum chloroplasts 1099 by Leech (1966) who suggests that this pattern may be produced as a result of prolonged dark treatment resulting in destarching, or the use of hypertonic media. Type e (Plate 3, Nos. 3, 5 and 7). Occasional elongated chloroplasts appeared pinched together in the middle, a configuration suggesting the possibility of chloroplast division. Such 'division' figures are not uncommon in higher plants, e.g. bean and spinach (Plate 3, Nos. 4 and 6). However, there appeared to be one major difference between the pattern observed in Equisetum and that in the angiosperms. As Plate 3, Nos. 4 and 6, shows, the internal lamellar system in these angiosperms appears to run continuously between the two chloroplast segments. However, in Equisetum the internal membrane system appeared to have separated into two distinct units, one on each side of the pinched area, and with no connecting strand running between the two segments (Plate 3, Nos. 5 and 7). Preliminary counts suggest that the cells of Zone III contain an average of nine chloroplasts per cell compared with six per cell for Zone II. Further counts are, however, required before a definitive statement can be made that chloroplast division occurs in Zone II. A possible example of a 'post-division' chloroplast in Zone III is shown in Plate 3, No. 8. The irregularity of the internal membrane system distinguishes these two small chloroplasts from those of similar size in Zone I. More suggestive, however, is the endoplasmic reticuium sheath which, in its upper portion, encircles both chloroplasts, a feature at variance with the one chloroplast-one sheath relationship previously noted. It is not, of course, possible to determine with certainty whether the picture represents two small chloroplasts or one long plastid folded back upon itself. All types of chloroplasts described for Zone II retained a close association with rough endoplasmic reticuium. Although the extent of envelopment of these chloroplasts by reticuium varied, a subjective impression was obtained of a progressive reduction in degree of envelopment from chloroplasts of Type a, through to Type d. The extent of envelopment became increasingly less for chloroplasts in Zones III and IV. Zone III (Plate 4, Nos. i and 2) There was no sharp distinction between Zones II and III. The distinguishing feature of the chloroplasts of cells from Zone III was the occurrence along the outer chloroplast membranes of irregularly spaced heavily staining deposits. It was confirmed that these deposits were osmiophilic, when post-staining by uranyl acetate and lead citrate was omitted and the deposits were found to be still present. As distance from meristematic tissue increased a gradual transition was observed from cells with chloroplasts lacking the osmiophilic deposit (i.e. those in Zone II) through those with chloroplasts showing occasional deposits until several cells away an area was reached in which staining was very heavy. Where staining was particularly intense, the outer chloroplast membranes often appeared to have broken down. The internal chloroplast membranes were not stained in this way. The bounding membranes of mitochondria also showed heavy staining, and the golgi bodies, the endoplasmic reticuium and the tonoplast membrane occasionally had similar deposits. During the course of a previous study (Whatley, 1971), similar deposits were observed in cells from young leaves of Phaseolus vulgaris suffering from extreme potassium deficiency. The pattern of the internal lamellar system of chloroplasts of Zone III was often similar to that observed in Zone 11, and prolamellar bodies also occurred (Plate 2, No. 6). However, in many cases the granal structure appeared diffuse. The lamellar system

6 iioo JEAN M. WHATLEY was more commonly folded back on itself than in Zone II. Occasional elongated 'dividing plastids' with pinched in central portions were observed (Plate 3, Nos. 5 and~l More frequently the plastids were rounded in appearance with folded lamellae apparently separated into two portions by a central segment of incomplete double membrane stained with the osmiophilic deposit described above (Plate 4, Nos. 3 and 4). In most cases there was no sign of the 'pinching off' noted in chloroplasts of Zone II, but there did appear to be segregation of the internal lamellar system into two units. This configuration might perhaps represent a stage of incomplete division characteristic of degenerating chloroplasts. Several chloroplasts contained discrete circles or ovals ('stromal inclusions') (Plate A No. i) of this heavily stained double membrane apparently separating one part of the stroma from the remainder. These 'stromal inclusions' are not constituents of the internal lamellar system of the chloroplasts, because the internal lamellae do not become stained in this way. Further, the inclusions are not 'fingers' of cell cytoplasm penetrating into the chloroplast because the staining and dimensions of the granular contents of the inclusions are similar to those of the stroma and not to those of the cytoplasm (Plate 4, Nos. I and 5). Chloroplasts of Zone III retained their close association with rough endoplasmic reticulum but the encirclement of the chloroplasts usually appeared less complete than in previous zones. Zone IV (Plate 4, No. 5) In leaf sheaths from young stems of Equisetum, this zone was not always represented. However, in the blacker leaf sheaths from mature stems many cells of this type were found adjacent to those of Zone III type. Chloroplasts of Zone IV were usually rounded. The osmiophilic deposit described above was present, but the bounding membranes frequently appeared disrupted. Although stained, membrane-bounded 'stromal inclusions' were often observed, no bisecting membranes were seen, Occasional possible prolamellar bodies were present but always appeared very diffuse (Plate 4, No. 6). The stroma appeared granular and the internal lamellar system was sinuous, with diffuse grana, and many swollen locules. It is suggested that these are senescing chloroplasts associated with the blackening of the leaf sheaths. However, it should be noted that these chloroplasts, like those in all the zones previously described, contained only a few small osmiophilic globules. The very large globules commonly observed in senescing chloroplasts of angiosperms were completely absent. The appearance of osmiophilic globules in chloroplasts of senescing higher plants is frequently associated with disruption and diminution of the internal lamellar system. In the case of senescing Equisetum chloroplasts where osmiophilic globules are few and small, the internal lamellar system appears, if anything, to be more extensively developed than at earlier stages. Illustrations of Equisetum cells by Manton (1966) show starch grains present in plastids of sporogenous tissue. The paper does not mention whether starch also occurs elsewhere. Examination of material from both stems and leaf sheaths revealed starch grains in plastids from only three types of cells; (i) guard cells; (2} some cells of the vascular system; (3) in two pairs of cells in which nuclear division had been completed but in which median wall formation was still in progress. The general absence of starch provides a point of contrast between chloroplasts of

7 Equisetum cuoroplasts IIOI Equisetum and those of most dicotyledons though most monocotyledons do not accumulate starch. CONCLUSIONS The basic developmental sequence of chloroplast structure in Equisetum seems similar to that in angiosperms. Small chloroplasts with immature lamellar structure are succeeded by larger chloroplasts with more complex internal membrane systems. Division of chloroplasts in Equisetum appears to result after elongation and 'pinching off', as is the case among angiosperms. However, in Equisetum the internal lamellar system apparently segregates into two discrete units prior to division which is unlike the pattern in angiosperms. The succeeding degenerative stages also appear to be quite distinctive. The osmiophilic deposits are highly unusual, as are the 'stromal inclusions' and bisecting membranes. The nature of the deposit is not known but may perhaps be associated with the breakdown of the external membrane system which was observed to occur at the same developmental stage. Although the deposits observed in Equisetum and in potassium-deficient Phaseolus are similar only in superficial appearance, senescence of Equisetum chloroplasts might well be associated with a deficiency of potassium. The absence of large osmiophilic globules, the more extensive lamellar development, and the pattern of locular swelling distinguish the senescing chloroplasts of Equisetum from those of most angiosperms. The most distinctive feature of Equisetum chloroplasts is their close relationship with enveloping sheaths of rough endoplasmic reticulum. Some examples of chloroplast endoplasmic reticulum are known among higher plants but in the case of dormant pea plastids the enveloping reticulum is smooth and branched, and in Acer and Pinus ribosomes are present on only one face, whereas in Equisetum both faces possess ribosomes. Although a similar relationship is found in several algal groups, the reticulum in the case of algae is also smooth, and, as in Acer and Pinus, but unlike Equisetum, is connected directly with the outer nuclear membrane. The chloroplast endoplasmic reticulum is apparently absent from the Chlorophyta, the algal group with presumably the closest affinities to Equisetum. ACKNOWLEDGMENTS This investigation was supported by a grant from the Science Research Council. Mrs R. P. Shellis, Mr H. Edge and Mr K. Maybury have provided excellent technical assistance. It is a pleasure to acknowledge valuable discussions with Professor C. R. Stocking and Professor F. R. Whatley. REFERENCES BONG YUL YOO (1970). Ultrastructural changes in cells of pea embryo radicles during germination. J. CeHBiol.,4S, 158. BoucK, G. B. (1965). Fine structure and organelle associations in brown algae. J^. CellBiol., 26,523. GiBBS, S. P. (1962). Nuclear envelope-ehloroplast relationships in algae, j'. CellBiol., 14, 433. GoLUB, S. J. & WETMORE, R. H. (1948). Studies of development in the vegetative shoot of Equisetum arvense L. Am. J. Bot., 35, 755. KAWAMATU, S. (1963). Electron microscope observations on the root hair cell of Azolla imbricata Nakai. Cytologia, a8, 12. LEECH, R. M. (1966). Comparative biochemistry and comparative morphology of chloroplasts isolated by different methods. In: Biochemistry of Chloroplasts, I (Ed. by T. W. Goodwin), p. 65. Academic Press, London and Nevf York.

8 1102 JEAN M. WHATLEY MANTON, 1. (1966). Some possibly significant structural relations between chloroplasts and other cell components. In: Biochemistry of Chloroplasts, I. (Ed. by T. W. Goodwin), p. 23. Academic Press, London and New York. SUN, C. N. (1963) Submicroscopic structure and development of chloroplasts in Equisetum hietnak. Protoplasma, 56, 346, THOMSON, W. W. (1966). Ultrastructural development of Chromoplasts in Valencia oranges. Bot. Gaz., WEIER, T. E., SJOLAND, R. D. & BROWN, D. L. (1970). Changes induced by low light intensities on the prolamellar body of 8-day, dark grown seedlings. Am. J. Bot., 57, 276. WHATLEY, J. M. (1971). Chloroplasts in nutrient deficient leaves. New Phytol., 70, 725. WooDiNO, F. B. P. & NoRTHCOTE, D. H. (1965). Association of the endoplasmic reticuium and the plastids in Acer and Pinus. Am. J. Bot., 52, 526. EXPLANATION OF PLATES Key to lettering: BM: bisecting membrane; C: centre of lamellar alignment; CER: chioroplast endopvasmic reticulmm; ER: endoplasmic reticuium; I: invagination,m.mitochondrion; O: osmiophilic globules; OM: osmiophilic deposits on membranes; P: 'prolamellar body'; R: ribosomes; SI: 'stromal inclusions'. TTie numbers I, II, III and IV represent zones of cells of increasing age. I. Composite pictures showing the zonal arrangement of cells. No. I. Zones I and 11. x No. 2. Zones I, II and III. x No. 3. Zones I, 11 and III. x PLATE 2. No. I. Immature plastid with limited lamellar system, showing chloroplast endoplasmic reticuium distinct from cytoplasmic endoplasmic reticuium. x 24,000. No. 2. Immature plastid with limited lamellar system which shows polarity of alignment. X 48,000. No. 3. The chloroplast endoplasmic reticuium has ribosomes on both membranes. The inner chloroplast membrane shows invaginations. x 90,000. No. 4. Mature chloroplast showing granal development, and polarity of lamellar alignment. X 24,000. No. 5. Mature chloroplast with a possible 'prolamellar body', x 24,000. No. 5. Mature chloroplast with osmiophilic deposits on the bounding membranes, and a possible 'prolamellar body^ x 120,000. PLATE 3. No, I. Elongated chloroplast. x 15,000. No. 2. Elongated chloroplast with partial central folding of the internal latnellar systenri. X 15,000. No. 3. Possible dividing chloroplast in Equisetum. x 17,500. No. 4. Possible dividing chloroplast in Phaseolus. x 10,000. No. 5. Possible dividing chloroplast in Equisetum showing segregation of internal lamellae into two discrete units, x 22,500. No. 6. Possible dividing chloroplast in Spinacia. x 12,500. No. 7. Possible dividing chloroplast in Equisetum showing two discrete lamellar units. Note the osmiphilic deposits associated with the membranes, x 22,500. No. 8. Possible post-division chloroplasts showing the chloroplast endoplasmic reticuium partiallj' encircling both plastids. X 15,000. PLATE 4 No. I. Mature chloroplast showing osmiophilic deposits on bounding membranes and 'stromal inclusions', x 27,000. No. 2. Part of a chloroplast showing the osmiophilic deposit associated with the bounding membranes, x 54,000. No. 3. Mature chloroplast showing osmiophihc deposit associated with bounding membranes and a bisecting membrane. The internal lamellar system forms two units, x 18,000. No. 4. Detail of the osmium-stained bisecting membrane, x 54,000. No. 5, Senescing chloroplast showing its sinuous, swollen lamellar system, granular cytoplasm and 'stromal inclusions'. Osmiophilic deposits occur on both chloroplast and mitochondrial bounding membranes, x 21,000. No. 6. A similar senescent chloroplast which contains a possible 'prolamellar body', x 72,000.

9 THE NEW PHYTOLOGIST, 70, 6 PLATE 1 ^^m H :^'^ JEAN M. WHATLEY EQUISETl'M 2 Li CHLOROPLASTS {facing page 1102)

10 THE NEW PHYTOLOGIST, 70, 6 PLATE 2 ^. : M '. ^ f. r;' *-. cr.r 1 : jf, _,. CER JEAN M. WHATLEY EQUISETUM CHLOROPLASTS

11 HE NEW PHYTOLOGiST, 70, 6 PLATE 3 ^ ^ ^ ^ ^ CER JEAN M, WHATl^EY EQUISEITM CHLOROPLASTS

12 THE NEW PHYTOLOGIST, 70, 6 PLATE OM I \ l r. ;i OM -t*--, i GB J.» ^^ JEAN M. WHATLEY EQCISETUM CHLOROPLASTS

13