Planta (Berl.) 126, i--10 (1975) 9 by Springer-Verlag 1975

Seasonal Changes in Structure and Function of Spruce Chloroplasts M. Senser, F. SchStz, and E. Beck Botanisches Institut der Universit/~t and Botanischer Garten, Menzinger Str. 67, D-8 Miinchen, Federal Republic of Germany Received March 21 ; accepted May 5, 1975

Summary. Seasonal changes of ultrastructure were studied by electron microscopy and by determining the chlorophyll and starch content of the plastids. Young plastids of spruce (Picea abies (L.) Karst.) first function as amyloplasts which store reserve material for the growth of the young needles. Then they develop a normal thylakoid system and produce assimilation starch during the day. In autumn, starch synthesis ceases and the plastids group together. In winter they swell and their membrane system becomes disorganized and reduced. With increasing temperatures in spring the chloroplasts recover, but then they accumulate large amounts of starch, which is not broken down during the night or even during a dark period of several days. As in the previous year they now function as amyloplasts providing reserve material for the new shoot. In summer these plastids are again converted into typical chloroplasts. The same seasonal changes of structure and function could be observed in chloroplasts from 2- or 3-year old needles. Thus these changes represent cyclic processes, which repeat each year. Features of slow aging are superimposed on to these cycles. Introduction Evergreen conifers are protected against frost damage during the winter period by hardening, a process which is induced by the decrease in temperature and length of day in autumn and is reversed in early spring. Many correlations exist between the seasonal cycle of hardiness and changes in metabolic activities or of the levels in chemical constituents, respectively (Larcher et al., 1973). However, there are only few and sometimes contradictory investigations dealing with correlations of the frost hardening cycle and seasonal changes in the fine structure of cell organelles. I n earlier studies Lewis and Turtle (1920), Zacharova (1929), and Schmidt (1936) described a partly reversible disintegration of conifer chloroplasts during the winter. The occurrence of free chlorophyll in the cytoplasm, especially in cells exposed to light, was also reported by Perry and Baldwin (1966). In contrast to these findings, Holzer (1958) could not discover a disintegration of the plastids although he found great changes in eytoplasmatic structures. Using electron microscopy Parker et al. (1961, 1963), also, did not observe significant differences between the ultrastructure of chloroplasts of P i n u s strobus (and Rhododendron catawbiense) in summer or in winter. However, a comparative investigation of frost-resistant and non-resistant grasses showed that the chloroplasts are the organelles which are most sensitive to freezing and that even in frost-resistant plants the fine structure of chloroplasts is altered by low temperature (Taylor et al., 1971; Kimball et al., 1973). The aim of the present work was to obtain exact information on the structural alterations of spruce chloroplasts during the whole year in order to differentiate between seasonal changes and normal aging 1 Planta(Berl.)

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processes. Th e i n v e s t i g a t i o n s were t h e r e f o r e started w i t h t h e buds an d e x t e n d e d to 3 needle generations. T h e electron microscopical o b s e r v a t i o n s were s u p p o r t e d b y t h e d e t e i m i n a t i o n of t h e c o n t e n t of chlorophyll a a n d b as well as of t h e a m o u n t of s t a r c h f o r m e d p e r plastid. Material and Methods Needles of Picea abies (L.) Karst. were harvested from a 50-year old tree growing in the Botanical Garden at Munich. Samples were taken from the buds and from the 1, 2 and 3 year old shoots at distinct intervals which were suggested by concurrent light microscopic observations. All collections were made in the morning between 10.00-10.30 a.m. Electron Microscopy. For electron microscopy the needles (the large ones halved lengthwise) were cut into 1-2 mm long slices. Fixation was carried out with 2% osOa (in 0.1 M SSrensen phosphate buffer pit 7.2) mixed 1:1 with 5% K~Cr~O~ (adjusted to pH 7.6 with 2.5 N KOH) for 3 h at 0 ~ C according to Diers et al. (1973). The fixed material was dehydrated with ethanol and propylene oxide and then embedded in the mixture described by Spurr (1969). Ultrathin sections were cut on an Ultrotome I I I (LKB) and viewed with a Zeiss EM GS. Fixation with OsO4 (2%) or glutaraldehyd (6%) and OsOt (2 % ) followed by staining with uranyl acetate (0.5 %) and, in a few cases, fixation with glutaraldehyd (6 % ) and poststaining with KMnO 4 (2.5 % ) gave no satisfactory results. Starch and Chlorophyll Estimation. For the analysis of starch and chlorophyll content per plastid, the needles (2-4 samples each of 5 g) were homogenized in 30 ml pyrophosphate medium pH 6.5 (Cockburn et al., 1968) containing 0.33 M sorbitol and 20% polyethylene glycol 6000. The slurry was poured through 6 layers of gauze, centrifuged at 100 g to remove rough materials and subsequently at 2400g. The pellet containing the chloroplasts was suspended in Hepes-buffer pH 7.6 (Jensen et al., 1966). Immediately after chloroplast isolation the chlorophyll content was measured according to Amen (1949) and the number of plastids in an aliquot volume was determined using a light microscope. For starch estimation another aliquot part of the chloroplast suspension was used. The polysaccharide was solubilized by boiling with 1.1% HCI for 15 min. After centrifugation enzymatic hydrolysis with amyloglucosidase (Boehringer Mannheim GmbI-I) was carried out at a pH of 4.6 and 30 ~ C for 12 h. Glucose was determined enzymatically using hexokinase plus glueose-6-P-dehydrogenase.

Results T wo changes in s t r u c t u r e a n d f u n c t i o n of t h e spruce chloroplasts could be o b s e r v e d d u r i n g t h e course of one year. T h e characteristic u l t r a s t r u c t u r a l stages are s h o wn in t h e Figs. 1-8, a n d t h e chlorophyll an d starch c o n t e n t are demons t r a t e d b y Figs. 9 a n d 10.

Fig. 1. Young plastid (April) from a needle still totally enveloped by dark bud scales. The plastids contain one prolamellar body, few thylakoids and a starch grain (St) Fig. 2. Plastid from a 3-5 mm long needle (May). In spite of exposure to light the plastids do not contain a thylakoid system. The presence of large starch grains (St) which are not removed upon darkening suggests that the plastids function as amyloplasts Fig. 3. Plastid from a 10-15 mm long needle (May) showing a well developed thylakoid system. Frequently membranes are separated from the envelope into the stroma and many osmiophilic granules are connected to the thylakoids Fig. 4. Plastid from a fully developed 30 mm long neeele (May). The chloroplasts produce considerable amounts of assimilation starch (St) during the day, which results in a strong deformation of their thylakoid system Fig. 5. Plastid as in Fig. 4, after darkening of the needles during the night. The degradation of the assimilation starch allows a redistribution of the thylakoid system. Typical for this stage are thylakoid free stroma spaces which situate adjacent to the cell wall

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Fig. 1-5. Plastids from mesophyll cells o~ spruce needles at subsequen~ developmental stages. • 14000

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Fig. 6--8. Plas$ids from mesophyll cells of spruce needles a t different seasonal stages

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In April the young needles are still totally surrounded b y m a n y dark eolonred bud scales. Their mesophyll cells contain plastids in which only a few parallel rows of membranes are present originating from a small prolamellar body (Fig. 1). Connections between the inner plastid membrane and the thylakoid system cannot be observed at this stage. Frequently starch grains are to be found in the stroma between the membranes. The plastids containing starch are present throughout the whole mesophyll and also in the bundle sheath. I n the latter, however, they do not contain a prolamellar body, and their very few membranes are not clearly oriented. The mesophyll cells are also well characterized by an electron dense layer of presumably tanniniferous material around the vacuole (of. Harris, 1971). Following shoot elongation the needles break through the buds in May. They are then 3-5 m m in length, contain pla_stids which are about twice the diameter that they were in April (i.e. 5 ~m) and have a slightly higher chlorophyll content (Fig. 9). Although they are now exposed to light they do not develop t h e typical chloroplast membrane system but contain only large starch grains (Fig. 2 and Fig. 9). The starch does not disappear upon darkening of the buds; the plas~ids therefore function more as amyloplasts than as chloroplasts. When the needles are 10-15 m m long (at this time they are arranged very closely on the short axis of the young shoot) the plastids reach a new stage in development. They are completely free of starch and show the typical lamellar system with grana and stroma thylakoids (Fig. 3). Frequent connections between the thylakoids and the inner part of the chloroplast envelope demonstrate clearly the participation of the latter in thylakoid development. Numerous osmiophilie granules, smaller than the plastoglobuli, are closely associated with the thylakoids, containing probable stock material for membrane development (of. Kutzelnigg et al., in press). When the needles have reached their final length of 3 cm, chloroplasts again accumulate starch during the light period, but in contrast to the situation in May, this starch disappears during the night (Fig. 4 and Fig. 5). The starch content in the mesophyll decreases from the cells situated immediately beyond the epidermis to those near the vascular bundle, according to the gradient in light intensity. I n starch-free plastids, particularly after darkening of the needles, two different areas can be distinguished: one which contains thylakoids and another without any membranous structures. The membrane-free spaces are always found at t h a t part of the chloroplast which is adjacent to the cell wall (Fig. 5). The lack

Fig. 6. Winter stage; the plastids are concentrated at one side of the cell. Mainly due to a swelling of the stroma areas the plastids are enlarged and pressed tightly together. At high resolution they sometimes even appear to be fused. Many plastoglobuli are found between the membranes. • 8500 Fig. 7. Plastid in the early spring. The thylakoid system is regenerated and the number of the plastoglobuli between the thylakoids is reduced. Buds are formed from the envelope, which is closely surrounded by the cisternae of the endoplasmic reticulum (ER). • 14000 Fig. 8. Plastid of a one year old needle during starch accumulation in May. The thylakoid system is strongly reduced and the whole plastid is occupied by one large grain of reserve starch (St). • 14000

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of membranes in large parts of the plastids could be due to the destruction of chloroplast membranes b y the photosynthetic starch accumulation as Barrels (1971) showed in Peperomia. The restoration of the membrane system starts in the dark with the degradation of starch and is completed during the first hours of the light period. When the temperature decreases in a u t u m n the synthesis of assimilation starch ceases and the membrane system of the chloroplasts becomes very compact. Then the chloroplasts, which are randomly distributed in the cytoplasm since the period of ceil elongation, begin to concentrate at one side of the cell. The other cell walls are then covered only by a very thin layer of cytoplasm, but plasmolysis was never observed. I n winter conspicuous changes of chloroplast structure take place: the membrane-free areas are strongly enlarged due to considerable swelling of the plastids. The latter are deformed and pressed together so tightly t h a t even a fusion could be suggested (Fig. 6). A disorganization of the thylakoid system is also connected with the proceeding of swelling and deformation. The membranes of the thylakoids separate, and bulges of low electron density appear along the stroma thylakoids. Then the number of stroma thylakoids as well as the size and number of the grana are reduced. Sometimes only single membrane fragments are to be found within the chloroplast. This disintegration of the membranes is accompanied by a decrease in the chlorophyll content (Fig. 10) and by the occurrence of plastoglobuli. Concurrently numerous similar but larger osmiophilie globules, which might represent reserve material, are found in the cytoplasm. I n early spring, with rising temperatures the chloroplasts separate

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from each other by shrinkage and are again distributed randomly at all cell walls. The membrane-free areas within the plastids are restricted to several peripheral buds and the membrane system is reconstituted (Fig. 7). During this phase the plastids are closely surrounded by some cisternae of the endoplasmic reticulum (Fig. 7) as also observed from the plastids of the resin canal cells of Pinus pinta (Wooding, 1965). This association of the endoplasmic reticulum with the plastids suggests an intensive protein synthesis. The restoration of the thylakoid system is correlated with an increase of the chIorophyll content. As in the proceeding year the chloroplasts during spring again produce starch which does not disappear during the night or artificial darkening. This starch accumulation is obviously accompanied by a new reduction of the thylakoid system, coincident with a decrease in the chlorophyll content and an increase in the number of plastoglobuli. The maximum starch accumulation is reached in May, when the new buds are sprouting. The plastids now reach their maximum size and each of them is occupied by an enormous starch grain (Fig. 8). Obviously the plastids function as amylop]asts for the second time and accumulate material for the next young generation of needles. In July, when the young shoots have totally developed, the one-year old plastids again become normal photosynthetic organelles which produce assimilation starch during the light phase and which are destarched during the night. The same seasonal changes in structure and function of the spruce chloroplasts could be observed when the age of the needles was 2 or 3 years. Thus these changes represent annual cyclic processes which are governed by climatic factors. Features of slow aging, however, seem to be superimposed on to this annual rhythm. This is suggested by a heavier disintegration of the thylakoids and by an increased production of plastoglobuli observed in chloroplasts of 2 or 3-year old needles during the winter or the starch accumulation phase in spring. Discussion Electron microscopic studies of 3 generations of spruce needles during a period of 2 years revealed a uniform picture showing an annual cycle of seasonal changes in chloroplast structure and function. The first alteration of the mature chloroplasts occurs in late autumn, when the synthesis of assimilation starch ceases in favour of the formation of the oligosaccharides raffinose and stachyose (Sensor et al., 1971). During winter the chloroplasts group closely together, a phenomenon also observed by Parker etal. (1961, 1963) in mesophyll cells of white pine. The reasons for this chloroplast movement are unknown. In contrast to Parker, who found no structural differences between summer and winter plastids in white pine, the present studies reveal marked changes in this respect during the cold season. Swelling of the plastids, membrane reduction and accumulation of plastoglobuli are phenomena which were also observed in chloroplasts of other plants upon onset of experimental stress (Taylor etal., 1971 ; Kimball etal., 1973 ; Fischer etal., 1973). While prolonged experimental stress, however, in the mentioned investigations caused disruption of the plastids, the chloroplasts of the hardened spruce needles are normally able to recover with increasing temperatures in spring. After this restoration phase they change to amyloplasts accumu-

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