Planta (1985)165:469~76
P l a n t a 9 Springer-Verlag 1985
Chloroplast development and the synthesis of chlorophyll and protoehlorophyllide in Zostera transferred to darkness H. Adamson, N. Packer and J. Gregory School of Biological Sciences, Macquarie University, North Ryde, N.S.W. 2113, Australia
Abstract. Intact plants and isolated leaves of Zostera capricornii Martens ex Aschers were transferred from daylight to darkness. Substantial amounts of chloropyll a and b continued to accumulate in immature and mature tissue in the same ratio as in the light and were incorporated into chlorophyll-protein complexes in the thylakoids. A small amount of protochlorophyllide also accumulated in immature tissue in the dark. Proplastids and immature chloroplasts continued to develop into mature chloroplasts in the dark in the normal manner but prolamellar bodies, which are a conspicuous feature of immature chloroplasts, took longer to disperse than in the light. Protochlorophyllide accumulation and prolamellar-body formation were not correlated. The results indicate that Zostera has a genetic capacity for dark chlorophyll synthesis which is expressed in immature and mature leaf tissue and enables this plant to continue synthesising chlorophyll and assembling chloroplasts at night. Key words: Chlorophyll synthesis - Chloroplast development - Light (chloroplast development without ) Zostera.
Introduction There have been several reports of chlorophyll (Chl) synthesis continuing when angiosperms are transferred from daylight to darkness. Godnev et al. (1959) extracted labelled chlorophyll from a number of plants which had been exposed to 14CO2 in darkness and interpreted this as evidence of light-independent chlorophyll synthesis. How-
ever, because they were unable to demonstrate a net accumulation of chlorophyll in the dark they were unable to eliminate the possibility of label being incorporated into existing chlorophyll molecules. Popov and Dilova (1969) and Adamson (1982a) observed that when etiolated barley seedlings which had been exposed to light for several hours were returned to darkness, their chlorophyll content continued to increase. Barley seedlings which had been germinated and grown under diurnal light-dark conditions also continued to accumulate chlorophyll when transferred from daylight to darkness (Adamson 1982a). The total amount of chlorophyll which accumulated in barley in the dark was small (5-7 gg Chl/leaf) compared to that reported for Tradescantia albiflora (up to 60 gg/leaf) (Adamson et al. 1980). Chlorophyll continued to accumulate in Tradescantia for days rather than hours as in barley and was less susceptible to destruction in prolonged darkness. Zostera is another monocotyledon with the capacity for chlorophyll synthesis in darkness 1(Adamson and Hiller 1981). Like Tradescantia it grows well at low light fluxes (Dennison and Alberte 1982) and, as we show in the present paper, is capable of withstanding prolonged darkness and accumulating large amounts of chlorophyll in the absence of light. Since, like barley, Zostera has elongated, strap-like leaves which grow as a result of the elongation of cells produced in an intercalary meristem at the leaf base, it is a very suitable plant in which to study the effect of tissue age on the capacity for dark chlorophyll synthesis. In this paper we describe the changes in levels of chlorophyll a and b and protochlorophyllide in tissue at specified distances from the base of isolated and intact Zostera leaves after prolonged
Abbreviations: Chl = chlorophyll; To = time of transfer to
darkness
I Henceforth, briefly "dark chlorophyll synthesis"
470 d a r k n e s s . C h a n g e s i n c h l o r o p l a s t size, e x t e n t o f thylakoid development and appearance of p r o l a m e l l a r b o d i e s are also d e s c r i b e d . A p r e l i m i n a r y a c c o u n t o f this w o r k w a s p r e s e n t e d b e f o r e ( P a c k e r et al. 1984).
Material and methods Plant material. Zostera capricornii Martens ex Aschers was collected from Cared Bay in Pittwater, north of Sydney, N.S.W., Australia. This site was chosen because it was not polluted. The ability of Zostera from polluted estuaries in industrial locations to withstand prolonged darkness is impaired. Plants which were still covered by water at low tide were collected and transferred within 2-3 h to tanks with aerated seawater in complete darkness at 25 ~ All manipulations of dark-treated plants were performed under a dim green safelight (Wratten filter No. 54; Kodak, Sydney, Australia; 40W incandescent green lamp, Philips, Sydney). The effectiveness of the safelight was evidenced by its failure to convert protochlorophyllide to chlorophyllide in etiolated seedlings used in other experiments. All analyses were carried out on the youngest leaf visible above the sheathing leaf bases of surrounding older leaves.
Experiments with isolated leaves. In these experiments the youngest emergent leaves of 90 plants were isolated and divided at random into three equal groups. The chlorophyll content of pooled successive 10-ram segments measured from the base was determined on one group at the time of transfer of the leaves to darkness (To), and on the other groups of leaves after 22 h and 47 h of darkness, respectively. Any leaf growth during this dark treatment was monitored by determining the mean leaf length of each group at To and at harvest.
Experiments with intact plants. The youngest emergent leaf on 60 intact plants was marked by a V-shaped cut close to the apex. The pigment content was analysed in successive 10-mm segments measured from the base of 30 marked leaves removed and pooled from the whole plants at To, and after 9 d of total darkness in aerated seawater at 25 ~C. Leaf growth was determined on a parallel sample of 120 plants. The youngest emergent leaf was cut at an angle of 45 ~ above the top of the leaf sheath. Sixty plants were chosen at random and the trimmed leaves carefully removed and measured (To). The remaining plants were transferred to darkness and measured after 9 d. The zone of cell division and cell elongation at the base of the youngest emergent leaf was located by carefully removing the leaf from the plant and observing it with a light microscope (450-fold magnification) equipped with a mechanical stage and vernier. Zostera leaves contain elongated air chambers subdivided by transverse septa. The distance between successive septa from the bottom to the top of a single chamber and the number of mesophyll cells in a single file from one septum to the next were determined and mean mesophyll cell length calculated.
Analysis ofporphyrins. Pigments were extracted quantitatively from frozen tissue samples by grinding with acid-washed sand in 85% acetone (10 ml) and approx. 10 mg of magnesium carbonate (Arnon 1949). The absorbance of the acetone extract was recorded at 626, 645 and 663 nm and the chlorophyll concentration calculated using the simultaneous equations of Brouers and Michel-Wolwertz (1983). There was no significant change in the porphyrin composition upon freezing, or after storage of frozen samples over three to four weeks.
H. Adamson et al.: Dark synthesis of chlorophyll in Zostera Protochlorophyllide was separated from chlorophyll a and b by a modification of the phase partition method of Treffry (1970). The acetone extract was adjusted to a volume of 10 ml. Ammonia (0.2 ml, 16 N) and petroleum ether (40-60 ~ b.p., 3.4 ml) were added and the mixture was shaken vigorously. The upper and lower phases were separated and the lower phase containing the unesterified pigments was re-extracted twice with 2 ml of petroleum ether. The combined upper phase, containing the esterified porphyrins was backwashed twice with 2 ml of ammoniacal acetone (0.02% ammonia in 85% acetone). The concentrations of chlorophyll a and b in the upper layer were calculated from their absorbance values using the equations of Anderson and Boardman (1964). An equal volume of saturated sodium chloride was added to the combined lower phase and the unesterified porphyrins were extracted three times into an equal volume of diethyl ether. The concentration of protochlorophyllide (excitation 436 nm, emission 626 nm) was estimated by low-temperature (77 K) fluorimetry using a fluorescence spectrophotometer (MPF-448; Perkin-Elmer, Norwalk, Conn., USA). Protochlorophyllide extracted from etiolated barley plants was used as reference standard.
Isolation of chloroplasts. Young emergent leaves were removed from intact plants, chilled in ice-cooled seawater, chopped into segments and blotted dry. Approximately 10 g of tissue was homogenised in 100ml of the ice-cold extraction medium (pH 7.6) of Grant and Wright (1980) using a Sorvall Omnimixer (Dupont Instruments, Newtown, Conn., USA) for 2 x 20 s at maximum speed. The ratio of tissue to extracting medium was 1: 10 (w/v). The extract was filtered through three layers of muslin and two layers of Miracloth (Calbiochem. San Diego, Cal., USA) and centrifuged at 2 500 g at 4 ~C for 10 rain. The supernatant was discarded and the pellet washed twice by resuspending it in 20 ml of buffer (pH 7.6) and recentrifuging. The pellet (chloroplast fraction) was finally resuspended in sufficient buffer to give a final volume of 5 ml.
Polyacrylamide gel electrophoresis. Chloroplasts (1 ml) were lysed in 30ml of 0.1M 2-amino-2-(hydroxymethyl)-l,3propanediol(Tris)-acetate buffer (pH 9.2). After centrifuging at 1000 g at 4 ~ for 10 rain to remove starch, the supernatant was centrifuged at 5 000 g for 15 min. The pellet was washed once in the same Tris-acetate buffer and finally resuspended to give a final concentration of 250 ~tg Chl m1-1. The membranes were solubilised by adding 1% sodium dodecyl sulphate (SDS) in 0.1 M Tris-acetate (pH 9.2) to give an SDS:Chl (w/w) ratio of 15. The chlorophyll-protein complexes were separated at 4 ~ on 8% polyacrylamide gels containing 0.15% SDS, as described by Hiller et al. (1977), except that the running buffer was 0.I M Tris-acetate (pH 9.2). The proportions of chlorophyll in each chlorophyll-protein complex were estimated by scanning at 670 and 650 nm (Genge et al. 1974). The complexes were named according to Anderson et al. (1978).
Electron microscopy. Transverse sections of tissue (approx. 0.75 ram) were removed at specified distances from the base of at least six newly-emergent Zostera leaves at To and after 9 d darkness. Tissue was fixed for electron microscopy in a mixture of 5% glutaraldehyde, 1% dimethylsulphoxide, 2% acrolein and 0.4 M Na-eacodylate buffer, pH 7.0, under vacuum for 2 h in darkness. The samples were washed in 0.4 M cacodylate buffer and post-fixed in 2% osmium tetroxide in darkness for 2 h at room temperature. The specimens were dehydrated in acetone and embedded in Spurr's (1969) epoxy resin. The sections were stained sequentially with potassium permanganate, uranyl acetate and lead citrate (Soloff 1973). Chloroplast dimensions were obtained from approx. 20 chloroplasts chosen at
H. Adamson et al.: Dark synthesis of chlorophyll in Zostera
471
random from each leaf position. Thylakoid length/chloroplast section was obtained by measuring between five and ten representative chloroplasts.
160(~
..E
Results
The experiments described in this paper have been repeated three to eight times and although the absolute values of the various parameters measured varied between experiments, the same pattern of response was always observed. Results from typical experiments are described.
Chlorophyll synthesis in the dark in isolated leaves. When entire young leaves were carefully removed from Zostera plants and floated on seawater in darkness they stopped growing and became distinctly greener. The results of an experiment comparing the distribution of chlorophyll from the base to the apex of Zostera leaves at the time of transfer of the leaves to darkness (To) and after 22 h darkness are shown in Fig. 1. There was a substantial increase in chlorophyll in mature tissue near the apex (approx. 500 ~tg Chl g-1 fresh weight) but little change near the base. Table 1 shows the effect of extending the dark period from 22 to 47 h. Tissue 40-50 mm from the base continued to accumulate chlorophyll; there was no further increase in the chlorophyll content of older tissue near the tip and a net chlorophyll breakdown occurred in immature tissue 10-30 mm from the base. Despite the substantial increase in the chlorophyll content of the 40-50 mm segment over 47 h there was very little change in the chlorophyll a/b ratio. This indicates that chlorophyll a and b were synthesised in approximately the same proportions in light and darkness. These results demonstrate that light is not essential for chlorophyll synthesis in mature isolated leaf tissue of Zostera and that the amount of chlorophyll formed via light-independent routes is substantial. Although immature tissue in isolated leaves failed to accumulate chlorophyll in the dark, we had noticed repeatedly that leaves of intact plants which were growing in the dark did not become noticeably paler at their base. This qualitative observation implied that chlorophyll was accumulating in the newly formed cells at the base of the leaves without exposure to light. Experiments were carried out to determine quantitatively the extent of this greening of intact plants in the dark. Chlorophyll synthesis in the dark in intact plants. Detecting changes in the chlorophyll content of different parts of Zostera leaves when intact plants
|
120{]
800
400
I
i
i
[
I
10
20
30
40
50
Distance
from
Tip
base (mml
Fig. 1. Chlorophyll content (lag g-1FW) at specified distances from the base of isolated Zostera leaves at time of transfer to darkness ( 9 and after 22 h ( 0 )
Table 1. Change in chlorophyll content (Agg g I FW) at specified distances from the base of isolated Zostera leaves after 22 and 47 h continuous darkness, and Chl a/b ratio in 40-50 mm segment at specified times Distance from base (mm)
Ag Chl g-1 FW
Chl a/b
22 h
47 h
To
22 h
47 h
10-20 20-30 30-40 40-50 Tip
-- 60 + 30 +310 + 450 + 530
- 150 -200 + 50 + 800 + 52O
2.47
2.13
2.18
are transferred to darkness is complicated by the continued growth of the leaves from an intercalary meristem at their base. However, the maximum number of mesophyll cells in single file between the transverse septa subdividing the elongated air chambers, and the maximum distance between these septa both occurred about 6 mm, and never more than 10 mm, from the leaf base and remained constant beyond this point. We therefore conclude that cell division and cell elongation are confined to the basal 10 mm of the leaf. This fact enables us to locate tissue further up the leaf and at a specified distance from the base at To, thereafter with great precision, provided the amount of growth which has occurred in the intervening period is known.
H. Adamson et al.: Dark synthesis of chlorophyll in Zostera
472 Table 2. Chlorophyll content at specified distances from the base of Zostera leaves from intact plants at To and after 9 d darkness (T9d). The increase in Chl content is calculated taking into account the 10 mrn growth which occurred at the base of the leaves. The suffixes a, a'; b,b' etc. identify the same tissue at T O and T9d Distance from base (ram)
0-10 10-20 20-30 30-40 40-50 50-60 60-70 a) a'--a, etc.
Chlorophyll content (lag g - l FW)
To
%d
25 a 34 b 238 c 646 a 1 276 e 1974 f 2146g
10 58 a' 346 b' 521 c' 1059 d' 1668 e' 2270 f
Increase in Chl (lagg-I F W ) a
Change in Chl (%)b
33 312 283 413 392 296
132 917 119 64 31 15
b) a'-a • 100, etc. a
At To, the mean length of trimmed, newly emergent leaves was 35.7 :t: 1.2 mm (n = 60). After 9 d dark, the mean length was 45.2:t: 1.3 mm (n = 60). Therefore, approx. 10 mm growth had occurred in leaves of intact plants which had been in the dark for 9 d. The successive leaf segments at To and after 9 d shows very little difference in the distribution of chlorophyll from the base to the apex (Table 2). Chlorophyll synthesis occurred in the dark throughout the leaf. This is obvious when allowance is made for the 10mm growth which occurred. The appropriate comparisons are between tissue at specified distances from the base at To and the same distances + 10 mm after 9 d dark. Chlorophyll accumulation was most noticeable in the region which was 10-20 mm from the base at To. Although tissue in this region was initially very pale green (34 lag Chl g-1 fresh weight) after 9 d darkness it had accumulated almost as much additional chlorophyll (312 gg Chl g-1 fresh weight) as that accumulated by mature tissue. When differences in the extent of photosynthetic membrane development in different parts of the leaf were taken into account by expressing the increase in chlorophyll in darkness as a precentage of chlorophyll at To (Table 2), this tissue appeared to have the greatest capacity for light-independent chlorophyll biosynthesis.
PIastid development. We have two lines of evidence that the chlorophyll which is formed in the dark in intact Zostera plants is incorporated into thylakoids in the normal manner. (1) There was no significant difference in the proportions of the main chlorophyll-protein com-
Table 3. Percentage of total Chl in various chlorophyll-protein complexes in newly ermergent leaves of Zostera at time of transfer to darkness (To) and after 4 d darkness (T4d) Complex
% of total chlorophyll To
T4~
CPla CP1 CPa
24.5• 5.0 a 7.84-2.5 18.4 • 2.3
17.9• 14.7• 22.7 4- 3.4
Total CP1
50.6 ~ 4.8
56.3 + 4.8
LHCP 1 LHCP 3
38.8 -4-2.2 5.8 • 2.2
31.6 • 3.4 8.7 + 0.05
Total LHCP
44.5 • 4.4
40.2 9 2.9
Free pigment
4.6 • 0.3
6.0 -t=0.05
a Mean values -4- SE ( n = 3, where n is a separate experiment)
plexes separated by sodium dodecyl sulphatepolyacrylamide gel electrophoresis from chloroplasts isolated from normal plants and from plants which had been kept in darkness for 4 d (Table 3). Since the percentage of free chlorophyll associated with the chloroplasts in dark-treated plants was the same as that observed under normal growth conditions it would appear that chlorophyll which accumulates in the dark is complexed with chloroplast proteins in the usual way. (2) Chloroplasts continued to develop in the absence of light. Different stages in the normal development of chloroplasts are illustrated on the left-hand side of Fig. 2; immature plastids, at the base of the leaf, are at the bottom of the figure and mature plastids towards the apex are at the top. Proplastids visible in the region 0-10 mm from the base develop into the etioplasts with single lamellae and prolamellar bodies found 10-20 mm from the base. Expansion of the etioplast is accompanied by synthesis and stacking of thylakoids (20-30 mm). Finally, the prolamellar bodies disappear and a mature chloroplast is formed (30 mm from leaf base). Each micrograph on the right-hand side of the figure illustrates the changes which occurred in these plastids after 9 d without light. Proplastids at the base of the leaf developed into etioplasts. Etioplasts increased enormously in size and lamellar content. Mature chloroplasts remained virtually unchanged. Prolonged darkness did not induce the formation of prolamellar bodies in mature chloroplasts; however it did enhance their development in etioplasts. The changes in plastid dimensions and thylakoid development illustrated in Fig. 2 are quantified in Figs. 3 and 4.
H. Adamson et al.: Dark synthesis of chlorophyll in Zostera
473
Fig. 2. Stages in the normal development of Zostera plastids and changes occuring in darkness. Plastids at specified distances up from the base of Zostera leaves from intact plants at To (left-hand side of the figure) are compared with plastids from the same distance + 10 m m after 9 d growth in the dark (right-hand side). 1 = 0-10 m i n x 10.000; 2 = 10-20 m m x 10.000; 3 = 20-30 m m • 10.000; 4 = > 3 0 m i n x 10.000
474
H. Adamson et al. : Dark synthesis of chlorophyll in Zostera
5
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160
r 0
140
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I
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120
2 la m e~ O h. O
e-
1
0
I
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10
I 20
I 30
mm
I 40
I 50
I 6O
f r o m base
e~
100
80
e"
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>. e-
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20
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20 mm
I 30 from
I 40
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0 ~
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10
20
30
40
50
60
m m from base
base
Fig. 3. Mean length and breadth of chloroplast sections obtained at specified distances from the base of Zostera leaves at time of transfer to darkness ( 9 and after 9 d ( e ) . Error bars = • S E ( n = 20)
Fig. 4. Mean thylakoid length per chloroplast section for chloroplasts at specified distances from the base of Zostera leaves at the time of transfer to darkness ( 9 and after 9 d darkness ( e ) . Error bars = ~: SE (n = 5-10)
Immature chloroplasts in the first 30 mm of the leaf increased significantly in length in the dark. The breadth of the chloroplast sections also increased but overall the effect was not significant (Fig. 3). Chloroplast expansion in the dark was accompanied by a substantial increase in mean thylakoid length per chloroplast section (Fig. 4). Thylakoids formed in the dark became appressed in the usual manner. There was no difference in the percentage of thylakoid per chloroplast section in grana in chloroplasts in the regioin 20-30 mm from the base at To (60 + 4) and in the same chloroplasts after 9 d darkness (70 • 6) even though total thylakoid length per chloroplast section had almost doubled. Prolamellar-body formation did not depend on the accumulation of either protochlorophyllide or protochlorophyll. Protochlorophyllide was present in trace amounts 5-10 mm from the base of Zostera leaves under normal diurnal conditions but prolamellar bodies were observed 20 mm from the base in the absence of any detectable protochlorophyllide (Table 4). Chlorophyll synthesis in the dark was accompanied by the accumulation of very small amounts ofprotochlorophyllide in tissue
Table
4. Protochlorophyllide (Pchlide) content and presence or absence ( + or - ) of prolamellar bodies (PLB) in plastids at specified distances from the base of Zostera at times of transfer to darkness (To) and after 9 d darkness (T9d) Distance from base (mm)
0-10 10-20
20-30 3040
To
~9d
Pchlide PLB (ggg ]FW)
Pchlide PLB (~tgg-l FW)
9" 10 .3 nd a nd nd
-}-
1.6
+
+
0.4
+
-
nd nd
+
a Not detectable by fluorescence spectroscopy
up to 20 mm from the base but again prolamellar bodies were observed in plastids further up the leaf. Using low-temperature fluorimetry we could not detect any protochlorophyll among the esterified pigments extracted from either normal or darktreated plants. Discussion
Protochlorophyllide reduction to chlorophyllide is an essential step in chlorophyll formation. Light is
H. Adamson et al.: Dark synthesis of chlorophyll in Zostera
required for this reduction in dark-grown angiosperm seedlings but not in some gymnosperm seedlings (see review by Castelfranco and Beale 1983). This difference has led to widespread acceptance of the view that light is essential for chlorophyll synthesis in angiosperms (Kirk and Tilney-Bassett 1978, pp. 627, 722). Our results indicate that Zostera capricornii is an exception to this general rule. When intact Zostera plants were transferred from daylight to darkness, substantial amounts of chlorophyll accumulated in both immature and mature tissue (up to 400 gg Chl g-~ fresh weight). Chlorophyll also accumulated in isolated leaves deprived of light, but only in mature tissue. The reason for this difference is not clear. Possibly chlorophyll synthesis in immature isolated leaves was inhibited by a shortage of nutrients and-or plant growth-regulating substances normally supplied by the rhizome and older leaves. This could also account for the failure of the isolated leaves to grow, although the possibility that the basal meristern was damaged when the leaves were excised cannot be excluded. In growing leaves the capacity of immature chloroplasts in young tissue for dark chlorophyll synthesis exceeded that of mature chloroplasts in older tissue. Young tissue also accumulated small amounts of protochlorophyllide in the dark. Protochlorophyllide accumulation and prolamellar-body formation, however, were not always associated. Michel-Wolwertz and Bronchart (1974) also noted that there was no correlation between the formation of prolamellar bodies in gymnosperms greening in the dark and the accumulation of protochlorophyllide. In general, the pattern of chlorophyll synthesis and chloroplast development established in Zostera under normal growth conditions was maintained in darkness. Chlorophyll a and b were synthesised in the dark and distributed among the various chlorophyll-protein complexes in essentially the same proportions as in the light. This indicates, and our electron micrographs confirm, that chlorophyll synthesised in the dark is incorporated into thylakoids in the usual manner. These findings with Zostera are remarkably similar to our earlier findings with Tradescantia (Adamson etal. 1980). In both species, dark chlorophyll synthesis is a major process which enables normal chloroplast development to continue in the absence of light. Although small prolamellar bodies sometimes form when darkness is prolonged, in each case this is only occasionally accompanied by the accumulation of protochlorophyllide. Unlike many gymnosperms,both Zostera and
475
Tradescantia require prior exposure to light before their dark chlorophyll biosynthetic pathways become operational. In both species, as in all angiosperms, leaves which have been initiated in the dark remain devoid of chlorophyll until they are exposed to light. We do not know how light induces the capacity for dark chlorophyll synthesis and have not investigated which wavelengths are effective. Phytochrome may be involved but since short periods of exposure to daylight (minutes to a few hours) are not sufficient to develop the capacity for light-independent chlorophyll synthesis in etiotated seedlings (Adamson 1982a), other factors must also be operating. Light-independent chlorophyll synthesis has also been reported in other monocotyledons. When Maitra and Mukherji (1983) exposed etiolated rice seedlings to light for different lengths of time and then returned them to darkness they observed an increase of 30 gg Chl g-1 fresh weight during a 24h dark period. Their findings are reminiscent of those of Popov and Dilova (1969) who observed increases of 50 gg Chl g-~ fresh weight following the return of partially greened, etiolated barley seedlings to darkness. Adamson (1982a,b) confirmed the findings of Popov and Dilova (1969) and extended them to barley seedlings grown under glasshouse conditions. Apel et al. (1984), however, were not able to confirm these findings. When they exposed etiolated barley seedlings to light and then returned isolated leaf pieces to darkness in the presence of radioactive amino levulinic acid (ALA) they observed that only protochlorophyllide became highly labelled. Since they did not present data on the chlorophyll content of their tissue at the beginning and end of the dark period it is not clear whether their failure to incorporate labelled ALA into chlorophyll was because chlorophyll was not being synthesised in the leaf pieces they used or because exogenous ALA was not readily incorporated into the light-independent pathway. In addition to the monocotyledonous plants already discussed, there is some evidence for lightindependent pathways of chlorophyll biosynthesis in dicotyledons. We have observed an increase in the chlorophyll content of young expanding pea leaves when glasshouse-grown seedlings are transferred to darkness (Adamson and Packer 1984). The amount of chlorophyll which accumulates in the dark varies with leaf age and rate of growth but is similar to that formed in barley and rice. Ikegami etal. (1984) have also reported very small (0.0037gg g-1 fresh weight) increases in the chlorophyll content of tobacco tissue cultured on agar in the dark and interpreted as these evidence
476
that tobacco is genetically capable of reducing protochlorophyllide in darkness. The published evidence for dark chlorophyll synthesis in other light-grown angiosperms is sparse and requires further confirmation, and the contribution of the dark pathway, except in Zostera and Tradescantia is small. However, these observations when taken together, imply that lightindependent pathway(s) of chlorophyll biosynthesis have not been entirely lost in the course of evolution of the angiosperms. This is an important idea because it raises the possibility that the failure of some plants or tissues to form chlorophyll in the dark might be the consequence of repression rather than absence of the necessary genetic information. This project was funded by the Australian Research Grants Scheme and Macquarie University.
References Adamson, H. (1982a) Evidence for a light-independent protochlorophyllide reductase in green barley leaves. In: Progress in clinical and biological research, vol. 102B: Cell function and differentiation, pp. 33-41, Akoyunoglou, G., Evangelopoulos, A.E. Georgatsos, J., Palaiologos, G., Trakatellis, A., Tsiganos, C.P., eds. Alan R. Liss, New York Adamson, H. (1982b) Chloroplast development in green barley leaves transferred to darkness. In: Progress in clinical and biological research, vol. 102B: Cell function and differentiation, pp. 189-199, Akoyunoglou, G., Evangelopoulos, A.E., Georgatsos, J., Palaiologos, G., Trakatellis, A., Tsiganos, C.P., eds. Alan R. Liss, New York Adamson, H., Hiller, R.G. (1981) Chlorophyll synthesis in the dark in angiosperms. Proc. Vth Int. Congr. on Photosynthesis, vol. 5: Chloroplast development, pp. 213-221, Akoyunoglou, G., ed. Balaban Services, Philadelphia, Pa., USA Adamson, H., Hiller, R.G., Vesk, M. (1980) Chloroplast development and the synthesis of chlorophyll a and b and chlorophyll-protein complexes I and II in the dark in Tradescantia albiflora (Kunth). Planta 150, 269-274 Adamson, H., Packer, N. (1984) Dark synthesis of chlorophyll in vivo and dark reduction of protochlorophyllide in vitro by pea chloroplasts. In: Protochlorophyllide reduction and greening, pp. 353-363, Sironval, C., Brouers,M., eds. Martinus Nijhoff/Dr. W. Junk, The Hague Anderson, J.M., Boardman, N,K. (1964) Studies on the greening of dark-grown bean plants. II Development of photochemical activity, Aust. J. Biol. Sci. 17, 93-101 Anderson, J.M., Waldron, J.C., Thorne, S.W. (1978) Chlorophyll-protein complexes of spinach and barley thylakoids. FEBS Lett. 92, 222233 Apel, K., Motzkus, M., Dehesh, K. (1984) The biosynthesis of chlorophyll in greening barley (Hordeum vulgare). Is there a light-independent protochlorophyllide reductase? Planta 161, 550-554
H. Adamson et al.: Dark synthesis of chlorophyll in Zostera Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1-15 Brouers, M., Michel-Wolwertz, M. (1983) Estimation of protochlorophyll(ide) contents in plant extracts; reevaluation of the molar absortption coefficient of protochlorophyll(ide). Photosynth. Res. 4, 265 270 Castelfranco, P.A., Beale, S.I. (1983) Chlorophyll biosynthesis: recent advances and areas of current interest. Annu. Rev. Plant Physiol. 34, 241 278 Dennison, W.C., Alberte, R.S. (1982) Photosynthetic responses of Zostera marina L. (eelgrass) to in situ manipulations of light intensity. Oecologia 55, 137-144 Genge, S., Pilger, D., Hiller, R.G. (1974) The relationship between chlorophyll b and pigment-protein complex II. Biochim. Biophys. Acta 347, 22-30 Godnev, T.N., Shlyk, A.A., Rotfarb, R.M. (1959) The synthesis of chlorophyll in the dark in angiosperms. Faziol. Rast. 6, 33-37 Grant, B.R., Wright, S.W. (1980) Purity of chloroplasts prepared from the siphonous green alga, Caulerpa simpliuscula, as determined by their ultrastructure and enzymic content. Plant Physiol. 66, 130-138 Hiller, R.G., Pilger, T.B.G., Genge, S. (1977) Effect of lincomycin on the chlorophyll-protein complex I content and photosystem I activity of greening leaves. Biochim. Biophys. Acta 460, 431M44 Ikegami, I., Kamiya, A., Hase, E. (1984) Dark formation of chlorophyll in cultured tobacco cells. Plant Cell Physiol. 25, 343-348 Kirk, J.T.O., Tilney-Bassett, R.A.E. (1978) The plastids. Elsevier, North Holland Biomedical Press, Amsterdam New York Oxford Maitra, P., Mukherji, S. (1983) Dark conversion of protochlorophyllide to chlorophyllide following brief illumination and temperature dependence of photo-induced carotenoid synthesis in rice (Oryza sativa L.) seedlings. Biochem. Physiol. Pflanzen 178, 207 211 Michel-Wolwertz, M.R., Bronchart, R. (1974) Formation of prolamellar bodies without correlative accumulation of protochlorophyllide in pine cotyledons. Plant Sci. Lett. 2, 45-54 Packer, N., Adamson, H., Gregory, J. (1984) Chloroplast development and associated changes in protochlorophyllide and chlorophyll levels in seagrasses transferred to darkness. In: Advances in photosynthesis research, vol. 4: pp. 745-748, Sybesma, C., ed. Martinus Nijhoff/Dr. W. Junk, The Hague Boston Lancaster Popov, K., Dilova, S. (1969) On the dark synthesis and stabilisation of chlorophyll. In: Progress in photosynthesis research, vol. 2, pp. 606-609, Metzner, H., ed. Int. Union Biol. Sci., Tfibingen, FRG Soloff, B.L. (1973) Buffered potassium permanganate-uranyl acetate-lead citrate staining sequence for ultrathin sections. Stain Technol. 48, 159-165 Spurr, A.R. (1969) A low viscosity Epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43 Treffry, T. (1970) Phytylation of chlorophyllide and prolamellar body transformation in etiolated peas. Planta 91, 279-284 Received 27 February 1984; accepted 24 February 1985
P l a n t a 9 Springer-Verlag 1985
Chloroplast development and the synthesis of chlorophyll and protoehlorophyllide in Zostera transferred to darkness H. Adamson, N. Packer and J. Gregory School of Biological Sciences, Macquarie University, North Ryde, N.S.W. 2113, Australia
Abstract. Intact plants and isolated leaves of Zostera capricornii Martens ex Aschers were transferred from daylight to darkness. Substantial amounts of chloropyll a and b continued to accumulate in immature and mature tissue in the same ratio as in the light and were incorporated into chlorophyll-protein complexes in the thylakoids. A small amount of protochlorophyllide also accumulated in immature tissue in the dark. Proplastids and immature chloroplasts continued to develop into mature chloroplasts in the dark in the normal manner but prolamellar bodies, which are a conspicuous feature of immature chloroplasts, took longer to disperse than in the light. Protochlorophyllide accumulation and prolamellar-body formation were not correlated. The results indicate that Zostera has a genetic capacity for dark chlorophyll synthesis which is expressed in immature and mature leaf tissue and enables this plant to continue synthesising chlorophyll and assembling chloroplasts at night. Key words: Chlorophyll synthesis - Chloroplast development - Light (chloroplast development without ) Zostera.
Introduction There have been several reports of chlorophyll (Chl) synthesis continuing when angiosperms are transferred from daylight to darkness. Godnev et al. (1959) extracted labelled chlorophyll from a number of plants which had been exposed to 14CO2 in darkness and interpreted this as evidence of light-independent chlorophyll synthesis. How-
ever, because they were unable to demonstrate a net accumulation of chlorophyll in the dark they were unable to eliminate the possibility of label being incorporated into existing chlorophyll molecules. Popov and Dilova (1969) and Adamson (1982a) observed that when etiolated barley seedlings which had been exposed to light for several hours were returned to darkness, their chlorophyll content continued to increase. Barley seedlings which had been germinated and grown under diurnal light-dark conditions also continued to accumulate chlorophyll when transferred from daylight to darkness (Adamson 1982a). The total amount of chlorophyll which accumulated in barley in the dark was small (5-7 gg Chl/leaf) compared to that reported for Tradescantia albiflora (up to 60 gg/leaf) (Adamson et al. 1980). Chlorophyll continued to accumulate in Tradescantia for days rather than hours as in barley and was less susceptible to destruction in prolonged darkness. Zostera is another monocotyledon with the capacity for chlorophyll synthesis in darkness 1(Adamson and Hiller 1981). Like Tradescantia it grows well at low light fluxes (Dennison and Alberte 1982) and, as we show in the present paper, is capable of withstanding prolonged darkness and accumulating large amounts of chlorophyll in the absence of light. Since, like barley, Zostera has elongated, strap-like leaves which grow as a result of the elongation of cells produced in an intercalary meristem at the leaf base, it is a very suitable plant in which to study the effect of tissue age on the capacity for dark chlorophyll synthesis. In this paper we describe the changes in levels of chlorophyll a and b and protochlorophyllide in tissue at specified distances from the base of isolated and intact Zostera leaves after prolonged
Abbreviations: Chl = chlorophyll; To = time of transfer to
darkness
I Henceforth, briefly "dark chlorophyll synthesis"
470 d a r k n e s s . C h a n g e s i n c h l o r o p l a s t size, e x t e n t o f thylakoid development and appearance of p r o l a m e l l a r b o d i e s are also d e s c r i b e d . A p r e l i m i n a r y a c c o u n t o f this w o r k w a s p r e s e n t e d b e f o r e ( P a c k e r et al. 1984).
Material and methods Plant material. Zostera capricornii Martens ex Aschers was collected from Cared Bay in Pittwater, north of Sydney, N.S.W., Australia. This site was chosen because it was not polluted. The ability of Zostera from polluted estuaries in industrial locations to withstand prolonged darkness is impaired. Plants which were still covered by water at low tide were collected and transferred within 2-3 h to tanks with aerated seawater in complete darkness at 25 ~ All manipulations of dark-treated plants were performed under a dim green safelight (Wratten filter No. 54; Kodak, Sydney, Australia; 40W incandescent green lamp, Philips, Sydney). The effectiveness of the safelight was evidenced by its failure to convert protochlorophyllide to chlorophyllide in etiolated seedlings used in other experiments. All analyses were carried out on the youngest leaf visible above the sheathing leaf bases of surrounding older leaves.
Experiments with isolated leaves. In these experiments the youngest emergent leaves of 90 plants were isolated and divided at random into three equal groups. The chlorophyll content of pooled successive 10-ram segments measured from the base was determined on one group at the time of transfer of the leaves to darkness (To), and on the other groups of leaves after 22 h and 47 h of darkness, respectively. Any leaf growth during this dark treatment was monitored by determining the mean leaf length of each group at To and at harvest.
Experiments with intact plants. The youngest emergent leaf on 60 intact plants was marked by a V-shaped cut close to the apex. The pigment content was analysed in successive 10-mm segments measured from the base of 30 marked leaves removed and pooled from the whole plants at To, and after 9 d of total darkness in aerated seawater at 25 ~C. Leaf growth was determined on a parallel sample of 120 plants. The youngest emergent leaf was cut at an angle of 45 ~ above the top of the leaf sheath. Sixty plants were chosen at random and the trimmed leaves carefully removed and measured (To). The remaining plants were transferred to darkness and measured after 9 d. The zone of cell division and cell elongation at the base of the youngest emergent leaf was located by carefully removing the leaf from the plant and observing it with a light microscope (450-fold magnification) equipped with a mechanical stage and vernier. Zostera leaves contain elongated air chambers subdivided by transverse septa. The distance between successive septa from the bottom to the top of a single chamber and the number of mesophyll cells in a single file from one septum to the next were determined and mean mesophyll cell length calculated.
Analysis ofporphyrins. Pigments were extracted quantitatively from frozen tissue samples by grinding with acid-washed sand in 85% acetone (10 ml) and approx. 10 mg of magnesium carbonate (Arnon 1949). The absorbance of the acetone extract was recorded at 626, 645 and 663 nm and the chlorophyll concentration calculated using the simultaneous equations of Brouers and Michel-Wolwertz (1983). There was no significant change in the porphyrin composition upon freezing, or after storage of frozen samples over three to four weeks.
H. Adamson et al.: Dark synthesis of chlorophyll in Zostera Protochlorophyllide was separated from chlorophyll a and b by a modification of the phase partition method of Treffry (1970). The acetone extract was adjusted to a volume of 10 ml. Ammonia (0.2 ml, 16 N) and petroleum ether (40-60 ~ b.p., 3.4 ml) were added and the mixture was shaken vigorously. The upper and lower phases were separated and the lower phase containing the unesterified pigments was re-extracted twice with 2 ml of petroleum ether. The combined upper phase, containing the esterified porphyrins was backwashed twice with 2 ml of ammoniacal acetone (0.02% ammonia in 85% acetone). The concentrations of chlorophyll a and b in the upper layer were calculated from their absorbance values using the equations of Anderson and Boardman (1964). An equal volume of saturated sodium chloride was added to the combined lower phase and the unesterified porphyrins were extracted three times into an equal volume of diethyl ether. The concentration of protochlorophyllide (excitation 436 nm, emission 626 nm) was estimated by low-temperature (77 K) fluorimetry using a fluorescence spectrophotometer (MPF-448; Perkin-Elmer, Norwalk, Conn., USA). Protochlorophyllide extracted from etiolated barley plants was used as reference standard.
Isolation of chloroplasts. Young emergent leaves were removed from intact plants, chilled in ice-cooled seawater, chopped into segments and blotted dry. Approximately 10 g of tissue was homogenised in 100ml of the ice-cold extraction medium (pH 7.6) of Grant and Wright (1980) using a Sorvall Omnimixer (Dupont Instruments, Newtown, Conn., USA) for 2 x 20 s at maximum speed. The ratio of tissue to extracting medium was 1: 10 (w/v). The extract was filtered through three layers of muslin and two layers of Miracloth (Calbiochem. San Diego, Cal., USA) and centrifuged at 2 500 g at 4 ~C for 10 rain. The supernatant was discarded and the pellet washed twice by resuspending it in 20 ml of buffer (pH 7.6) and recentrifuging. The pellet (chloroplast fraction) was finally resuspended in sufficient buffer to give a final volume of 5 ml.
Polyacrylamide gel electrophoresis. Chloroplasts (1 ml) were lysed in 30ml of 0.1M 2-amino-2-(hydroxymethyl)-l,3propanediol(Tris)-acetate buffer (pH 9.2). After centrifuging at 1000 g at 4 ~ for 10 rain to remove starch, the supernatant was centrifuged at 5 000 g for 15 min. The pellet was washed once in the same Tris-acetate buffer and finally resuspended to give a final concentration of 250 ~tg Chl m1-1. The membranes were solubilised by adding 1% sodium dodecyl sulphate (SDS) in 0.1 M Tris-acetate (pH 9.2) to give an SDS:Chl (w/w) ratio of 15. The chlorophyll-protein complexes were separated at 4 ~ on 8% polyacrylamide gels containing 0.15% SDS, as described by Hiller et al. (1977), except that the running buffer was 0.I M Tris-acetate (pH 9.2). The proportions of chlorophyll in each chlorophyll-protein complex were estimated by scanning at 670 and 650 nm (Genge et al. 1974). The complexes were named according to Anderson et al. (1978).
Electron microscopy. Transverse sections of tissue (approx. 0.75 ram) were removed at specified distances from the base of at least six newly-emergent Zostera leaves at To and after 9 d darkness. Tissue was fixed for electron microscopy in a mixture of 5% glutaraldehyde, 1% dimethylsulphoxide, 2% acrolein and 0.4 M Na-eacodylate buffer, pH 7.0, under vacuum for 2 h in darkness. The samples were washed in 0.4 M cacodylate buffer and post-fixed in 2% osmium tetroxide in darkness for 2 h at room temperature. The specimens were dehydrated in acetone and embedded in Spurr's (1969) epoxy resin. The sections were stained sequentially with potassium permanganate, uranyl acetate and lead citrate (Soloff 1973). Chloroplast dimensions were obtained from approx. 20 chloroplasts chosen at
H. Adamson et al.: Dark synthesis of chlorophyll in Zostera
471
random from each leaf position. Thylakoid length/chloroplast section was obtained by measuring between five and ten representative chloroplasts.
160(~
..E
Results
The experiments described in this paper have been repeated three to eight times and although the absolute values of the various parameters measured varied between experiments, the same pattern of response was always observed. Results from typical experiments are described.
Chlorophyll synthesis in the dark in isolated leaves. When entire young leaves were carefully removed from Zostera plants and floated on seawater in darkness they stopped growing and became distinctly greener. The results of an experiment comparing the distribution of chlorophyll from the base to the apex of Zostera leaves at the time of transfer of the leaves to darkness (To) and after 22 h darkness are shown in Fig. 1. There was a substantial increase in chlorophyll in mature tissue near the apex (approx. 500 ~tg Chl g-1 fresh weight) but little change near the base. Table 1 shows the effect of extending the dark period from 22 to 47 h. Tissue 40-50 mm from the base continued to accumulate chlorophyll; there was no further increase in the chlorophyll content of older tissue near the tip and a net chlorophyll breakdown occurred in immature tissue 10-30 mm from the base. Despite the substantial increase in the chlorophyll content of the 40-50 mm segment over 47 h there was very little change in the chlorophyll a/b ratio. This indicates that chlorophyll a and b were synthesised in approximately the same proportions in light and darkness. These results demonstrate that light is not essential for chlorophyll synthesis in mature isolated leaf tissue of Zostera and that the amount of chlorophyll formed via light-independent routes is substantial. Although immature tissue in isolated leaves failed to accumulate chlorophyll in the dark, we had noticed repeatedly that leaves of intact plants which were growing in the dark did not become noticeably paler at their base. This qualitative observation implied that chlorophyll was accumulating in the newly formed cells at the base of the leaves without exposure to light. Experiments were carried out to determine quantitatively the extent of this greening of intact plants in the dark. Chlorophyll synthesis in the dark in intact plants. Detecting changes in the chlorophyll content of different parts of Zostera leaves when intact plants
|
120{]
800
400
I
i
i
[
I
10
20
30
40
50
Distance
from
Tip
base (mml
Fig. 1. Chlorophyll content (lag g-1FW) at specified distances from the base of isolated Zostera leaves at time of transfer to darkness ( 9 and after 22 h ( 0 )
Table 1. Change in chlorophyll content (Agg g I FW) at specified distances from the base of isolated Zostera leaves after 22 and 47 h continuous darkness, and Chl a/b ratio in 40-50 mm segment at specified times Distance from base (mm)
Ag Chl g-1 FW
Chl a/b
22 h
47 h
To
22 h
47 h
10-20 20-30 30-40 40-50 Tip
-- 60 + 30 +310 + 450 + 530
- 150 -200 + 50 + 800 + 52O
2.47
2.13
2.18
are transferred to darkness is complicated by the continued growth of the leaves from an intercalary meristem at their base. However, the maximum number of mesophyll cells in single file between the transverse septa subdividing the elongated air chambers, and the maximum distance between these septa both occurred about 6 mm, and never more than 10 mm, from the leaf base and remained constant beyond this point. We therefore conclude that cell division and cell elongation are confined to the basal 10 mm of the leaf. This fact enables us to locate tissue further up the leaf and at a specified distance from the base at To, thereafter with great precision, provided the amount of growth which has occurred in the intervening period is known.
H. Adamson et al.: Dark synthesis of chlorophyll in Zostera
472 Table 2. Chlorophyll content at specified distances from the base of Zostera leaves from intact plants at To and after 9 d darkness (T9d). The increase in Chl content is calculated taking into account the 10 mrn growth which occurred at the base of the leaves. The suffixes a, a'; b,b' etc. identify the same tissue at T O and T9d Distance from base (ram)
0-10 10-20 20-30 30-40 40-50 50-60 60-70 a) a'--a, etc.
Chlorophyll content (lag g - l FW)
To
%d
25 a 34 b 238 c 646 a 1 276 e 1974 f 2146g
10 58 a' 346 b' 521 c' 1059 d' 1668 e' 2270 f
Increase in Chl (lagg-I F W ) a
Change in Chl (%)b
33 312 283 413 392 296
132 917 119 64 31 15
b) a'-a • 100, etc. a
At To, the mean length of trimmed, newly emergent leaves was 35.7 :t: 1.2 mm (n = 60). After 9 d dark, the mean length was 45.2:t: 1.3 mm (n = 60). Therefore, approx. 10 mm growth had occurred in leaves of intact plants which had been in the dark for 9 d. The successive leaf segments at To and after 9 d shows very little difference in the distribution of chlorophyll from the base to the apex (Table 2). Chlorophyll synthesis occurred in the dark throughout the leaf. This is obvious when allowance is made for the 10mm growth which occurred. The appropriate comparisons are between tissue at specified distances from the base at To and the same distances + 10 mm after 9 d dark. Chlorophyll accumulation was most noticeable in the region which was 10-20 mm from the base at To. Although tissue in this region was initially very pale green (34 lag Chl g-1 fresh weight) after 9 d darkness it had accumulated almost as much additional chlorophyll (312 gg Chl g-1 fresh weight) as that accumulated by mature tissue. When differences in the extent of photosynthetic membrane development in different parts of the leaf were taken into account by expressing the increase in chlorophyll in darkness as a precentage of chlorophyll at To (Table 2), this tissue appeared to have the greatest capacity for light-independent chlorophyll biosynthesis.
PIastid development. We have two lines of evidence that the chlorophyll which is formed in the dark in intact Zostera plants is incorporated into thylakoids in the normal manner. (1) There was no significant difference in the proportions of the main chlorophyll-protein com-
Table 3. Percentage of total Chl in various chlorophyll-protein complexes in newly ermergent leaves of Zostera at time of transfer to darkness (To) and after 4 d darkness (T4d) Complex
% of total chlorophyll To
T4~
CPla CP1 CPa
24.5• 5.0 a 7.84-2.5 18.4 • 2.3
17.9• 14.7• 22.7 4- 3.4
Total CP1
50.6 ~ 4.8
56.3 + 4.8
LHCP 1 LHCP 3
38.8 -4-2.2 5.8 • 2.2
31.6 • 3.4 8.7 + 0.05
Total LHCP
44.5 • 4.4
40.2 9 2.9
Free pigment
4.6 • 0.3
6.0 -t=0.05
a Mean values -4- SE ( n = 3, where n is a separate experiment)
plexes separated by sodium dodecyl sulphatepolyacrylamide gel electrophoresis from chloroplasts isolated from normal plants and from plants which had been kept in darkness for 4 d (Table 3). Since the percentage of free chlorophyll associated with the chloroplasts in dark-treated plants was the same as that observed under normal growth conditions it would appear that chlorophyll which accumulates in the dark is complexed with chloroplast proteins in the usual way. (2) Chloroplasts continued to develop in the absence of light. Different stages in the normal development of chloroplasts are illustrated on the left-hand side of Fig. 2; immature plastids, at the base of the leaf, are at the bottom of the figure and mature plastids towards the apex are at the top. Proplastids visible in the region 0-10 mm from the base develop into the etioplasts with single lamellae and prolamellar bodies found 10-20 mm from the base. Expansion of the etioplast is accompanied by synthesis and stacking of thylakoids (20-30 mm). Finally, the prolamellar bodies disappear and a mature chloroplast is formed (30 mm from leaf base). Each micrograph on the right-hand side of the figure illustrates the changes which occurred in these plastids after 9 d without light. Proplastids at the base of the leaf developed into etioplasts. Etioplasts increased enormously in size and lamellar content. Mature chloroplasts remained virtually unchanged. Prolonged darkness did not induce the formation of prolamellar bodies in mature chloroplasts; however it did enhance their development in etioplasts. The changes in plastid dimensions and thylakoid development illustrated in Fig. 2 are quantified in Figs. 3 and 4.
H. Adamson et al.: Dark synthesis of chlorophyll in Zostera
473
Fig. 2. Stages in the normal development of Zostera plastids and changes occuring in darkness. Plastids at specified distances up from the base of Zostera leaves from intact plants at To (left-hand side of the figure) are compared with plastids from the same distance + 10 m m after 9 d growth in the dark (right-hand side). 1 = 0-10 m i n x 10.000; 2 = 10-20 m m x 10.000; 3 = 20-30 m m • 10.000; 4 = > 3 0 m i n x 10.000
474
H. Adamson et al. : Dark synthesis of chlorophyll in Zostera
5
E ~L 4
rlr
160
r 0
140
O) e"
I
m 3
o
o
_o
120
2 la m e~ O h. O
e-
1
0
I
0
10
I 20
I 30
mm
I 40
I 50
I 6O
f r o m base
e~
100
80
e"
eo
E f-
+
O
ro
40
>. e-
J~
20
o w0 e-
I tO
[
20 mm
I 30 from
I 40
I SO
I SO
0 ~
0
10
20
30
40
50
60
m m from base
base
Fig. 3. Mean length and breadth of chloroplast sections obtained at specified distances from the base of Zostera leaves at time of transfer to darkness ( 9 and after 9 d ( e ) . Error bars = • S E ( n = 20)
Fig. 4. Mean thylakoid length per chloroplast section for chloroplasts at specified distances from the base of Zostera leaves at the time of transfer to darkness ( 9 and after 9 d darkness ( e ) . Error bars = ~: SE (n = 5-10)
Immature chloroplasts in the first 30 mm of the leaf increased significantly in length in the dark. The breadth of the chloroplast sections also increased but overall the effect was not significant (Fig. 3). Chloroplast expansion in the dark was accompanied by a substantial increase in mean thylakoid length per chloroplast section (Fig. 4). Thylakoids formed in the dark became appressed in the usual manner. There was no difference in the percentage of thylakoid per chloroplast section in grana in chloroplasts in the regioin 20-30 mm from the base at To (60 + 4) and in the same chloroplasts after 9 d darkness (70 • 6) even though total thylakoid length per chloroplast section had almost doubled. Prolamellar-body formation did not depend on the accumulation of either protochlorophyllide or protochlorophyll. Protochlorophyllide was present in trace amounts 5-10 mm from the base of Zostera leaves under normal diurnal conditions but prolamellar bodies were observed 20 mm from the base in the absence of any detectable protochlorophyllide (Table 4). Chlorophyll synthesis in the dark was accompanied by the accumulation of very small amounts ofprotochlorophyllide in tissue
Table
4. Protochlorophyllide (Pchlide) content and presence or absence ( + or - ) of prolamellar bodies (PLB) in plastids at specified distances from the base of Zostera at times of transfer to darkness (To) and after 9 d darkness (T9d) Distance from base (mm)
0-10 10-20
20-30 3040
To
~9d
Pchlide PLB (ggg ]FW)
Pchlide PLB (~tgg-l FW)
9" 10 .3 nd a nd nd
-}-
1.6
+
+
0.4
+
-
nd nd
+
a Not detectable by fluorescence spectroscopy
up to 20 mm from the base but again prolamellar bodies were observed in plastids further up the leaf. Using low-temperature fluorimetry we could not detect any protochlorophyll among the esterified pigments extracted from either normal or darktreated plants. Discussion
Protochlorophyllide reduction to chlorophyllide is an essential step in chlorophyll formation. Light is
H. Adamson et al.: Dark synthesis of chlorophyll in Zostera
required for this reduction in dark-grown angiosperm seedlings but not in some gymnosperm seedlings (see review by Castelfranco and Beale 1983). This difference has led to widespread acceptance of the view that light is essential for chlorophyll synthesis in angiosperms (Kirk and Tilney-Bassett 1978, pp. 627, 722). Our results indicate that Zostera capricornii is an exception to this general rule. When intact Zostera plants were transferred from daylight to darkness, substantial amounts of chlorophyll accumulated in both immature and mature tissue (up to 400 gg Chl g-~ fresh weight). Chlorophyll also accumulated in isolated leaves deprived of light, but only in mature tissue. The reason for this difference is not clear. Possibly chlorophyll synthesis in immature isolated leaves was inhibited by a shortage of nutrients and-or plant growth-regulating substances normally supplied by the rhizome and older leaves. This could also account for the failure of the isolated leaves to grow, although the possibility that the basal meristern was damaged when the leaves were excised cannot be excluded. In growing leaves the capacity of immature chloroplasts in young tissue for dark chlorophyll synthesis exceeded that of mature chloroplasts in older tissue. Young tissue also accumulated small amounts of protochlorophyllide in the dark. Protochlorophyllide accumulation and prolamellar-body formation, however, were not always associated. Michel-Wolwertz and Bronchart (1974) also noted that there was no correlation between the formation of prolamellar bodies in gymnosperms greening in the dark and the accumulation of protochlorophyllide. In general, the pattern of chlorophyll synthesis and chloroplast development established in Zostera under normal growth conditions was maintained in darkness. Chlorophyll a and b were synthesised in the dark and distributed among the various chlorophyll-protein complexes in essentially the same proportions as in the light. This indicates, and our electron micrographs confirm, that chlorophyll synthesised in the dark is incorporated into thylakoids in the usual manner. These findings with Zostera are remarkably similar to our earlier findings with Tradescantia (Adamson etal. 1980). In both species, dark chlorophyll synthesis is a major process which enables normal chloroplast development to continue in the absence of light. Although small prolamellar bodies sometimes form when darkness is prolonged, in each case this is only occasionally accompanied by the accumulation of protochlorophyllide. Unlike many gymnosperms,both Zostera and
475
Tradescantia require prior exposure to light before their dark chlorophyll biosynthetic pathways become operational. In both species, as in all angiosperms, leaves which have been initiated in the dark remain devoid of chlorophyll until they are exposed to light. We do not know how light induces the capacity for dark chlorophyll synthesis and have not investigated which wavelengths are effective. Phytochrome may be involved but since short periods of exposure to daylight (minutes to a few hours) are not sufficient to develop the capacity for light-independent chlorophyll synthesis in etiotated seedlings (Adamson 1982a), other factors must also be operating. Light-independent chlorophyll synthesis has also been reported in other monocotyledons. When Maitra and Mukherji (1983) exposed etiolated rice seedlings to light for different lengths of time and then returned them to darkness they observed an increase of 30 gg Chl g-1 fresh weight during a 24h dark period. Their findings are reminiscent of those of Popov and Dilova (1969) who observed increases of 50 gg Chl g-~ fresh weight following the return of partially greened, etiolated barley seedlings to darkness. Adamson (1982a,b) confirmed the findings of Popov and Dilova (1969) and extended them to barley seedlings grown under glasshouse conditions. Apel et al. (1984), however, were not able to confirm these findings. When they exposed etiolated barley seedlings to light and then returned isolated leaf pieces to darkness in the presence of radioactive amino levulinic acid (ALA) they observed that only protochlorophyllide became highly labelled. Since they did not present data on the chlorophyll content of their tissue at the beginning and end of the dark period it is not clear whether their failure to incorporate labelled ALA into chlorophyll was because chlorophyll was not being synthesised in the leaf pieces they used or because exogenous ALA was not readily incorporated into the light-independent pathway. In addition to the monocotyledonous plants already discussed, there is some evidence for lightindependent pathways of chlorophyll biosynthesis in dicotyledons. We have observed an increase in the chlorophyll content of young expanding pea leaves when glasshouse-grown seedlings are transferred to darkness (Adamson and Packer 1984). The amount of chlorophyll which accumulates in the dark varies with leaf age and rate of growth but is similar to that formed in barley and rice. Ikegami etal. (1984) have also reported very small (0.0037gg g-1 fresh weight) increases in the chlorophyll content of tobacco tissue cultured on agar in the dark and interpreted as these evidence
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that tobacco is genetically capable of reducing protochlorophyllide in darkness. The published evidence for dark chlorophyll synthesis in other light-grown angiosperms is sparse and requires further confirmation, and the contribution of the dark pathway, except in Zostera and Tradescantia is small. However, these observations when taken together, imply that lightindependent pathway(s) of chlorophyll biosynthesis have not been entirely lost in the course of evolution of the angiosperms. This is an important idea because it raises the possibility that the failure of some plants or tissues to form chlorophyll in the dark might be the consequence of repression rather than absence of the necessary genetic information. This project was funded by the Australian Research Grants Scheme and Macquarie University.
References Adamson, H. (1982a) Evidence for a light-independent protochlorophyllide reductase in green barley leaves. In: Progress in clinical and biological research, vol. 102B: Cell function and differentiation, pp. 33-41, Akoyunoglou, G., Evangelopoulos, A.E. Georgatsos, J., Palaiologos, G., Trakatellis, A., Tsiganos, C.P., eds. Alan R. Liss, New York Adamson, H. (1982b) Chloroplast development in green barley leaves transferred to darkness. In: Progress in clinical and biological research, vol. 102B: Cell function and differentiation, pp. 189-199, Akoyunoglou, G., Evangelopoulos, A.E., Georgatsos, J., Palaiologos, G., Trakatellis, A., Tsiganos, C.P., eds. Alan R. Liss, New York Adamson, H., Hiller, R.G. (1981) Chlorophyll synthesis in the dark in angiosperms. Proc. Vth Int. Congr. on Photosynthesis, vol. 5: Chloroplast development, pp. 213-221, Akoyunoglou, G., ed. Balaban Services, Philadelphia, Pa., USA Adamson, H., Hiller, R.G., Vesk, M. (1980) Chloroplast development and the synthesis of chlorophyll a and b and chlorophyll-protein complexes I and II in the dark in Tradescantia albiflora (Kunth). Planta 150, 269-274 Adamson, H., Packer, N. (1984) Dark synthesis of chlorophyll in vivo and dark reduction of protochlorophyllide in vitro by pea chloroplasts. In: Protochlorophyllide reduction and greening, pp. 353-363, Sironval, C., Brouers,M., eds. Martinus Nijhoff/Dr. W. Junk, The Hague Anderson, J.M., Boardman, N,K. (1964) Studies on the greening of dark-grown bean plants. II Development of photochemical activity, Aust. J. Biol. Sci. 17, 93-101 Anderson, J.M., Waldron, J.C., Thorne, S.W. (1978) Chlorophyll-protein complexes of spinach and barley thylakoids. FEBS Lett. 92, 222233 Apel, K., Motzkus, M., Dehesh, K. (1984) The biosynthesis of chlorophyll in greening barley (Hordeum vulgare). Is there a light-independent protochlorophyllide reductase? Planta 161, 550-554
H. Adamson et al.: Dark synthesis of chlorophyll in Zostera Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1-15 Brouers, M., Michel-Wolwertz, M. (1983) Estimation of protochlorophyll(ide) contents in plant extracts; reevaluation of the molar absortption coefficient of protochlorophyll(ide). Photosynth. Res. 4, 265 270 Castelfranco, P.A., Beale, S.I. (1983) Chlorophyll biosynthesis: recent advances and areas of current interest. Annu. Rev. Plant Physiol. 34, 241 278 Dennison, W.C., Alberte, R.S. (1982) Photosynthetic responses of Zostera marina L. (eelgrass) to in situ manipulations of light intensity. Oecologia 55, 137-144 Genge, S., Pilger, D., Hiller, R.G. (1974) The relationship between chlorophyll b and pigment-protein complex II. Biochim. Biophys. Acta 347, 22-30 Godnev, T.N., Shlyk, A.A., Rotfarb, R.M. (1959) The synthesis of chlorophyll in the dark in angiosperms. Faziol. Rast. 6, 33-37 Grant, B.R., Wright, S.W. (1980) Purity of chloroplasts prepared from the siphonous green alga, Caulerpa simpliuscula, as determined by their ultrastructure and enzymic content. Plant Physiol. 66, 130-138 Hiller, R.G., Pilger, T.B.G., Genge, S. (1977) Effect of lincomycin on the chlorophyll-protein complex I content and photosystem I activity of greening leaves. Biochim. Biophys. Acta 460, 431M44 Ikegami, I., Kamiya, A., Hase, E. (1984) Dark formation of chlorophyll in cultured tobacco cells. Plant Cell Physiol. 25, 343-348 Kirk, J.T.O., Tilney-Bassett, R.A.E. (1978) The plastids. Elsevier, North Holland Biomedical Press, Amsterdam New York Oxford Maitra, P., Mukherji, S. (1983) Dark conversion of protochlorophyllide to chlorophyllide following brief illumination and temperature dependence of photo-induced carotenoid synthesis in rice (Oryza sativa L.) seedlings. Biochem. Physiol. Pflanzen 178, 207 211 Michel-Wolwertz, M.R., Bronchart, R. (1974) Formation of prolamellar bodies without correlative accumulation of protochlorophyllide in pine cotyledons. Plant Sci. Lett. 2, 45-54 Packer, N., Adamson, H., Gregory, J. (1984) Chloroplast development and associated changes in protochlorophyllide and chlorophyll levels in seagrasses transferred to darkness. In: Advances in photosynthesis research, vol. 4: pp. 745-748, Sybesma, C., ed. Martinus Nijhoff/Dr. W. Junk, The Hague Boston Lancaster Popov, K., Dilova, S. (1969) On the dark synthesis and stabilisation of chlorophyll. In: Progress in photosynthesis research, vol. 2, pp. 606-609, Metzner, H., ed. Int. Union Biol. Sci., Tfibingen, FRG Soloff, B.L. (1973) Buffered potassium permanganate-uranyl acetate-lead citrate staining sequence for ultrathin sections. Stain Technol. 48, 159-165 Spurr, A.R. (1969) A low viscosity Epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43 Treffry, T. (1970) Phytylation of chlorophyllide and prolamellar body transformation in etiolated peas. Planta 91, 279-284 Received 27 February 1984; accepted 24 February 1985
