REGENERATION OF CHLOROPLAST STRUCTURE IN TALBOTIA ELEGANS: A DESICCATION TOLERANT PLANT

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1 New Phytol. (1918)81, REGENERATION OF CHLOROPLAST STRUCTURE IN TALBOTIA ELEGANS: A DESICCATION TOLERANT PLANT By N. D. HALLAM and D. F. GAFF Botany Department, Monash University, Gay ton, Victoria 3168, Australia {Received 2 May 1978) SUMMARY Talbotia elegans (Velloziaceae) is a desiccation-tolerant plant whose dry leaves contain chlorophyll. The plastids of dry viable leaves retain some semblance of thylakoid structure and organization but are in a loose array. This contrasts with desiccation-tolerant plants that lose chlorophyll during dehydration and reduce the chloroplast to pro-plastid like bodies containing vesicles and numerous plastoglobuli. During rehydration, the existing thylakoid stacks are rehabilitated by the production of a 'chloroplast designate' volume surrounding the mass of thylakoids. This region is delimited by a double membrane and the thylakoids stack to form grana and intergranal lamellae by 24 h of hydration. Few organelles other than the chloroplast initials are easily recognizable in dry leaves but mitochondria, reduced to membrane-bound sacs in the dry state, reform cristae after full hydration has been achieved. INTRODUCTION Until recently there has been little interest in desiccation-tolerant angiosperms (as defined by Gaff and Churchill, 1976), although lichens and mosses that are able to tolerate extensive periods of drought are well known to most botanists. Some angiosperms that tolerate desiccation may lose their chlorophyll completely while other species retain amounts comparable to fully hydrated tissue. Data summarizing this information are included in Gaff and Hallam (1974) for two species, Xerophyta villosa, a chlorophyll loser, and Talbotia elegans, a species that retains its chlorophyll on drying. Xerophyta villosa, when dry, is pale yellow and, during rehydration, regreening occurs as chloroplasts are developed from organelles containing vesicles which expand to form the thylakoid stacks (Hallam and Gaff, 1978). Both X. villosa and Talbotia elegans are members of the Velloziaceae and this paper describes changes occurring in T. elegans during rehydration. The desiccation tolerance of these plants is extreme: specimens of both Talbotia ^nd Xerophyta have been held over concentrated sulphuric acid (0% RH) for as long as 6 months and on rehydration begin photosynthesizing within 48 h. MATERIALS AND METHODS Plants, grown in plastic pots under glasshouse conditions at Melbourne (approximately 25 C with natural light) from seed collected at Marieskop in South Africa, were watered with a complete nutrient solution each week. Well-hydrated plants were selected and the pots X/78/ $ Blackwell Scientific Publications 657

2 658 N. D. HALLAM and D. F. GAFF allowed to dry out in growth cabinets (28 C, 18-h photoperiod; 'GrowLux' fluorescent tubes; 120 jue m~^ s~'). Central pieces of mature leaf were selected for electron microscopy. The techniques of fixation of dry leaf tissue using dimethylsulphoxide as a solvent, rather than water for fixative solutions, have been discussed elsewhere (Hallam and Capicchiano, 1974; Hallam, 1976). In the fixation of dry tissue, the use of non-aqueous methods is necessary as physical hydration of leaf material occurs prior to fixation if conventional aqueous fixatives are employed for either light or electron microscopy. Dry plants of Talbotia elegans (relative water content = 3%) were rehydrated by total immersion and leaf samples taken at 1,2,4,8,24 and48 h of hydration. Pieces of comparable rehydrating leaves were sliced in 6% of glutaraldehyde in 0.1 M sodium cacodylate buffer at a ph of 6.8 and fixed for 2 h. The slices were washed for a further 4 h in buffer and then post-fixed in 2% osmium tetroxide in 0.1 M sodium cacodylate buffer for 2 h. The leaf slices were dehydrated in an ethanol series and transferred to acetone for 1 h, then for 24 h on a rotary mixer in a 1:1 acetone : Spurr's resin mixture. After final infiltration into Spurr's resin for 3 days, polymerization was carried out at 60 C for 16 h. Sections, cut either with a diamond or glass knife on a Reichert 0MU3 ultramicrotome, were stained in lead citrate for 5 min and then aqueous uranyl acetate for 15 min. They were examined with a Hitachi 1 IE electron microscope. Neutral Red and Evans Blue (see Gaff, 1971) were used to verify that the rehydrated tissue had survived. Relative water contents (RWC; Barrs, 1968) were estimated from the fresh weight of fully turgid leaves (at 24 h rehydration) and from dry weights (70 C oven). Insoluble protein was determined from the nitrogen content of the residue from repeated macerations of leaves in borate buffer, estimated by microkjeldahl digestion and Nesslerization (see Gaff, Zee and O'Brien, 1976). RESULTS Since Talbotia e/e^flws retains its chlorophyll during dehydration, leaves dried in the laboratory are usually a dull green, resembling a well-prepared herbarium specimen, but occasionally the green colour is masked by purple anthocyanins. During desiccation, there is a reduction in complexity of the fine structure of the cytoplasm so that only chloroplast membranes are readily recognizable. During rehydration this complexity is restored. The insoluble protein content of the tissue remains similar to that in fully hydrated tissue (Table 1). In Xerophyta Table 1. The changes in insoluble protein (TCA precipitable materials) of leaves of Talbotia elegans plants, indicated by the ratio of the final level to the initial level during the processes of; (a) dehydration and (b) rehydration while immersed in water (a) Protein content during dehydration (b) Protein content during rehydration Protein content of dry tissue Protein content of rehydrated tissue X 100 :.. Protein content of hydrated tissue Protein content of dry tissue X % 118% 102% 68% Average 109% 93%

3 Chloroplast regeneration in Talbotia 100 r- 659 m o 50 I 0 10 Time (h) 20 Fig. 1. Time course of water intake by air-dry leaves of Talbotia elegans floated on water. RWC - Relative Water Content. villosa, however, there is a reduction of about 40% in the insoluble proteins (Hallam and Gaff, 1978). Rehydration takes 8 h (Fig. 1) during which the leaves expand and flatten. In the dry state, the cell walls are convoluted. The chloroplasts are seen as loose stacks of thylakoids that have separated from one another. This characteristic thylakoid disorganization is present in anhydrously fixed material (Hallam and Capicchiano, 1974) and is present immediately on wetting and after 1 h (Plate 1, No. 1). Some membrane-bound structures of mitochondrial dimensions are visible, these being distinct from numerous larger vacuolar vesicles and osmiophilic lipid bodies. In this earliest stage of hydration, there is little evidence of organized structure within the cytoplasm. The rehydration ofthe walls is rapid. Hand-cut sections of dry tissue, placed on a microscope slide and rehydrated by running water under the cover slip, expand within 1-2 s. By 2 h of wetting (Plate 1, No. 2), consolidation of cytoplasm has occurred, particularly that surrounding chloroplast lamellae, and the cytoplasm appears granular. The granules are of dbosomal dimensions except in areas around the thylakoid stacks. In these areas the granules are finer and less dense. After 4 h of hydration (Plate 2, No. 3), the changes evident at 2 h are further advanced with the chloroplasts becoming defined. Osmiophilic granules appear within the thylakoid stacks at this stage. By 8 h of wetting (Plate 2, No. 4), the cytoplasm appears to be more organized and the plasmalemma is now mostly applied along the cell wall. The obvious chloroplasts are delimited by a distinct double membrane. After 12 h of hydration (Plate 3, No. 5), membrane-bound organelles of mitochondrial dimensions are recognizable, but lack cristae. Extensive growth of the area occupied by the thylakoids takes place within the chloroplast and there is a better organization of thylakoids into grana and intergranal lamellae. The area of piastids not occupied by the internal membrane system now has the fme granulation typical of chloroplast stroma. By 24 h (Plate 3, No. 6), fully structured chloroplasts are developed. The grana are tightly compacted and the thylakoid system now fills much of the volume of the plastid. Starch

4 660 N. D. HALLAM and D. F. GAFF grains are present within the chloroplast and fully structured mitochondria are present within the cytoplasm. DISCUSSION The retention of the bulk of the internal chloroplast membrane systems in dry Talbotia plants, together with the maintenance of chlorophyll content (G. McGregor, private communication), contrasts with the situation in Xerophyta villosa (Hallam and Gaff, see Introduction). Dry plants of X. villosa (Velloziaceae) and Borya nitida (Liliaceae) both lose the intemal membrane system of the chloroplast during desiccation. In the dry state, the plastids of these plants contain numerous plastoglobuli and small vesicles (Hallam and Gaff, 1978). By contrast, the chloroplasts of Talbotia elegans retain intemal membranes, have an ill-defined bounding membrane and lack plastoglobuli. The contrast in fine structure between chlorophyll-retaining species (e.g. T. elegans) and species that lose chlorophyll on drying (e.g. Xerophyta villosa and^orya nitida) is also indicated by the insoluble protein contents of the leaf tissue, which are maintained undiminished in Talbotia (Table 1) but fall by about 40% in Xerophyta villosa and Borya nitida (Gaff and Hallam, 1974; Hallam and Gaff, 1978; Gaff er al., 1976). The rehydration processes occur in four reasonably well-defined phases. The first is apparently a physical wetting of the wall tissue as soon as the plant is immersed or heavily hosed. This, at a cellular level, results in the walls rapidly returning to their original position. The cytoplasm frequently remains attached to one part of the wall, often, but not always, on a wall with pit fields and extensive plasmadesmata. These cell-to-cell connections are presumably restored in the later stages of hydration, i.e. after 8 h of wetting. Physical hydration is complete within seconds in hand-cut sections of dry tissue when observed hydrating on a slide with a light microscope. There then follows a longer period of cellular organization which occupies the next h when mitochondria, initially present in thin sections of dry tissue as ill-defined circlets of membrane, finally develop cristae as respiration starts. The chloroplasts commence as disorganized arrays of thylakoid stacks with an illdefined membrane around them. The chloroplasts develop as hydration proceeds to completion and, by 8 h, the cytoplasm is applied to the wall for most of the cell and similar to other fully turgid tissues. Ribosomes are easily seen by this stage of development and the thylakoid system is well defined. It appears that the delimiting membrane of the presumptive chloroplast then moves in, condensing the granular material contained within into the granular matrix ofthe stroma. This is in contrast to the development of chloroplasts from pro-plastids or etioplasts of other angiosperms and is also different to the changes that occur in Xerophyta villosa. Osmiophilic granules or plastoglobuli, initially missing in the first 2 h of hydration, appear within the thylakoid stacks by 4 h and are an obvious feature of the developing chloropiast. By 24 h a fully organized chloroplast is developed and starch is present. Attempts to follow Hill activity during rehydration of Talbotia were unsuccessful; no activity was obtained even in plastids from hydrated controls (G.R. McGregor, personal communication), possibly owing to contamination by polyphenols. From our investigations on T. elegans and preliminary examination of other chlorophyllretaining desiccation plants, it appears that this pattern of reorganization is common to all. The interesting paper of WeUburn and Wellburn (1976) on the desiccation-tolerant angiosperm, Myrothamnus flabelufolia (Myrothamnaceae), at 1 h of hydration, suggests that

5 Oiloroplast regeneration in Talbotia 661 thylakoid stacks within grana are offset relative to adjacent thylakoids. This is not a feature of Talbotia. Considering the remarkably rapid structural and biochemical changes that take place during rehydration, it is worthwhile remembering that it is fully-differentiated vacuolate, mature tissue that undergoes these processes. Presumably, changes taking place at the shoot and root apex are similar to those occurring in the embryo of a seed during germination. ACKNOWLEDGMENTS The authors are indebted to the Australian Research Grants Commission (Grants D72/15043, D66/16137) for financial support. Collection of plant material was made possible by funds received from the Rural Credit Fund of the Reserve Bank of Australia and the Water Research Foundation of Australia. We thank Ms S. E. Luff and Ms R. C. Jackman for skilled technical help. REFERENCES BARRS, H. D. (1968). Determination of water deficits in plant tissues. In; Water Deficits and Plant Growth (Ed. by T.T. Kozlowski). Academic Press, New York. GAFF, D. F. (1971). The desiccation-tolerant higher plants of Southern Africa. Science, 174, GAFF, D. F. & CHURCHILL, D. M. (1976). Borya nitida Labill - an Australian species in the Liliaceae with desiccation-tolerant iqzves.aust. J. Bot, 24, 209. GAFF, D. F. & HALLAM, N. D. (1974). Resurrecting desiccated plants. R. Soc. N.Z. Bull., 12, 389. GAFF, D. F., ZEE, S.-Y. & O'BRIEN, T. P. (1976). The fine structure of dehydrated and reviving leaves oi Borya nitida Labill. - a desiccation-tolerant plant. Aust. J. Bot., 24, 225. HALLAM, N. D. (1976). Anhydrous fixation of dry plant tissue using non-aqueous fixatives./. Microsc, 106(3), 337. HALLAM, N. D. & CAPICCHIANO, P. (1974). Studies on desiccation tolerant plants using non-aqueous fixation methods. Proc. 8th Intemat. Congress of Electron Microsc. Canbena, 2, 612. HALLAM, N. D. & GAFF, D. F. (1978). Reorganization of fine structure during rehydration of desiccated leaves of Xerophyta villosa. New Phytol, 81, 349. WELLBURN, F. A. M. & WELLBURN, A. R. (1976). Novel chloroplasts and unusual cellular ultrastructure in the 'resurrection' plant Myrothamnus flabellifolia Welw. (Myrothamnaceae). Bot. J. Linn. Soc., 72, 51. EXPLANATION OF PLATES All plates - mesophyll leaf tissue of Talbotia elegans. Scale marker 1 Mm. PLATE 1 No. 1. Fixation at 1 h of hydration. The cell walls rapidly expand and the disorganized cytoplasm contains chloroplast initials (C) made up of disorganized thylakoid membranes which aie not well defined by an outer membrane. Much of the cytoplasm is made up of large vacuolar vesicles (V) and lipid bodies (L). Pit fields containing plasmadesmata (P) can also be seen, often with residual cytoplasmic connections from cell to cell. Organelles of mitochondrial dimensions (M) are also present. X No. 2. Fixation at 2 h of hydration. As early as 2 h, thylakoid stacks (C) have areas of lesser granularity around them and may be termed 'chloroplast designate' areas (Cd). Vacuoles (V) contain dense osmiophilic materials and the walls (W) are fully expanded. X 5908.

6 662 N. D. HALLAM and D. F. GAFF PLATE 2 No. 3. Fixation at 4 h of hydration. At this stage thylakoid stacking is well advanced in many chloroplasts (C). In addition the 'chloroplast designate' areas (Cd) are more granular. Vacuoles (V) and osmiophilic globules (O) within the chloroplast areas are well defined at this stage. X No. 4. Fixation at 8 h of hydration. By this stage of development the organization of the cytoplasm is similar to a normal cell with cytoplasm applied to the cell walls (W). Most chloroplasts (C) show granal stacking and are well defined by an outer membrane, this containing granular 'chloroplast designate' material (Cd). X PLATE 3 No. 5 Fixation 12 h after hydration. The cytoplasm shows greater compartmentalization at 12 h with easily recognizable mitochondria (M). Plasmadesmata (P) are connected from cell to cell and the 'chloroplast designate' (Cd) areas around the chloroplast internal membrane systems (C) show a fine granularity similar to that of the chloroplast stroma. X No. 6. Fixation 24 h after hydration. By this stage the outer chloroplast membrane has retracted around the grana and intergranal lamellae to form a normal chloroplast containing starch (S) and numerous plastoglobuli or osmiophilic granules (O). Mitochondria (M) contain cristae and the plasmalemma is generally well applied to the cell wall (W). X

7 Tl E NEW PHYTOLOGIST, 81, 3 E>, HALLAM and D. F. GAFF CHLOROPLAST REGENERATION IN TALBOTIA PLATE 1 (facing p. 662)

8 THE NEW PHYTOLOGIST, 81, 3 N. D. HALLAM and D. F. GAFF CHLOROPLAST REGENERATION IN TALBOTIA H 2

9 HE NEW PHYTOLOGIST, 81, 3». HALLAM and D. F. GAFF CHLOROPLAST REGENERATION IN TALBOTIA PLATE 3

THE BEHAVIOUR OF CHLOROPLASTS DURING CELL DIVISION OF ISOETES LACUSTRIS L.

New Phytol (1974) 73, 139-142. THE BEHAVIOUR OF CHLOROPLASTS DURING CELL DIVISION OF ISOETES LACUSTRIS L. BY JEAN M. WHATLEY Botany School, University of Oxford (Received 2 July 1973) SUMMARY Cells in