- Email: [email protected]
A Comparison of the Chlorophyll-protein Composition and Chloroplast Ultrastructure in Two Bryophytes and Two Higher Plants
A Comparison of the Chlorophyll-protein Composition and Chloroplast Ultrastructure in Two Bryophytes and Two Higher Plants EVA-MARl
ARO
Department of Biology, University of Turku, SF-20S00 Turku 50, Finland Received April 16, 1982 · Accepted July 14, 1982
Summary The distribution of chlorophyll bet wee n different chlorophyll-protein complexes was compared with the chi al b ratio and chloroplast ultrastructure in two bryo phytes, the protonema of Ceratodon purpureus and thallus of Marchantia polymorpha, and two higher plants, Lemna minor and Cucurbita pepo. The higher plants are sun species, having a chi alb ratio close to 3, but the t wo bryophytes both had a chi alb ratio slighdy above 2, although one of them favours sunny habitats. The bryoph ytes had more of their chloroph yll associated with the LHCP co mplexes and less with the reaction centre complexes than the higher plants. Increase of the quantum flux density from 100 to 500 JLmol photons m- 2s- 1 during growth caused only slight changes in the composition of the chlorophyll-protein complexes and chi alb ratio in Lemna and protone mata of Ceratodon, although the ultrastructure of the chl oroplasts revealed clear red uction in the number of thylakoids per granum. Ceratodon grown in strong light for two to three weeks had grana with only two to three appressed thylakoids, but more t han 60 % of total chlorop hyll was still associated with LHCP, which indicates that the formation of large grana stacks is not necessary for accumulation of large amounts of LHCP in relation to the reaction centre complexes. Cucurbita had big grana stac ks but relatively less LHCP than either Ceratodon or Marchantia.
Key words: Ceratodon purpureus, Cucurbita pepo, Lemna minor, Marchantia polymorpha, bryophytes, chloroplast ult rastructure, chlorophyll alb ratio, chlorophyll·protein complexes.
Introduction Shade plants are typically known to have low chI alb ratios and their chloroplasts are characterized by large grana stacks, which in extreme cases may contain up to 100 th ylakoids (Anderson et aI., 1973). Mosses are also plants with low chI alb ratios (Kallio and Valanne, 1975; Valanne, 1977 a; Arc and Valanne, 1979; Rao et aI. , 1979; Martin, 1980) though not all the species are obligate shade plants. H owever, moss chloroplasts have been observed to be very diverse, some containing large grana stacks and others having only a fe w appressed thylakoids (Kallio and Valanne, 1975; Valanne, Abbreviations: Chi, chlorophyll ; C Pa, ch lorophyll a-protein complex of PSII; CPI, PlOO chloro phyll a-protein complex; FP, free pigment; LHCP, light-harvesting chlorophyll alb-protein co mplex; LHCP"", LHCP':"', LHCP""", oligomers of LHCP w ith increasing molecular weight; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulph ate.
Z. Pjlanzenphysiol. Bd. 108. S. 97-105. 1982.
98
EVA-MARl
ARO
1977 a; Aro and Valanne, 1979). The sun species of higher plants have higher chi alb ratios and their chloroplasts contain smaller grana and larger proportions of stroma thylakoids than the plants of shaded environments (Boardman et al., 1974). The size of the grana can be easily modified by environmental factors like the quantum flux density, especially in the case of the sun species (Ballantine and Forde, 1970; Prioul, 1973; Valanne, 1977 b). Chloroplasts with higher chi alb ratios have less LHCP and more of their chlorophyll is associated with the reaction centre complexes than in chloroplasts with lower chi alb ratios (Anderson 1980). Moreover, lateral heterogeneity has been reported in the distribution of the chlorophyll-protein complexes along the thylakoid membrane system. According to Andersson and Anderson (1980), the P700 chlorophyll a-protein of PSI (CPI) is situated in the stroma thylakoids and only in the end membranes and margins of the grana stacks, while the partitions are almost depleted of PSI reaction centres. These opinions are supported by the studies with PSI mutants (Miller, 1980). The grana partitions, on the other hand, are rich in PSII reaction centre complexes (CPa) and the light-harvesting complexes (LHCP). Only about 10-20 % of the total PSII reaction centre complex and the light-harvesting complex have been calculated to be associated with stroma thylakoids (Andersson and Anderson, 1980). In this study the distribution of chlorophyll between the different chlorophyll-protein complexes was compared with the chi alb ratio and chloroplast ultrastructure in two bryophytes and two higher plants. The effect of the quantum flux density during growth on the chlorophyll-protein complexes, chi alb ratio and chloroplast ultrastructure was also studied in Ceratodon and Lemna. Materials and Methods Cucurbita pepo L. and Marchantia polymorpha L. were grown in the greenhouse under longday conditions with a quantum flux density of 200 "mol photons m- 2 s- 1 for Cucurbita and 100 "mol photons m- 2 s- 1 for Marchantia. Lemna minor L. and protonemata of Ceratodon purpureus (Hedw.) Brid. were grown in MY solution (Waris and Rouhiainen, 1970) at a quantum flux density of 100 and 500 "mol photons m- 2s- 1 (photoperiod 12 h) and 24°C. Lemna was grown for two weeks and Ceratodon for two to four weeks. The sterilization and culture techniques used with Ceratodon were according to Valanne (1966). For isolation of chloroplasts, plant tissue was briefly homogenized in 0.05 M HEPES-KOH, 0.01 M NaCI, 0.5 M sucrose, pH 7.6, filtered through three layers of nylon cloth and centrifuged at 250 x g for 5 min. The chloroplasts were sedimented at 5000 xg for 10 min. The chloroplasts were then broken by washing twice with 1 mM NaEDTA, pH adjusted to 8.0, and the thylakoids were sedimented at 20,000 xg for 20 min. Samples for electrophoresis were prepared by suspending the thylakoids in 0.063 M Tris-HCI, 5 % 2-mercapto-ethanol and 10 % glycerol, pH 6.8, to a chlorophyll concentration of 0.8 mg/ml. Ten per cent SDS (PIERCE) was added to give a final SDS/chl ratio of 6: 1 (w/w) and the thylakoids were gently solubilized at 4°C. SDS-PAGE was carried out according to Laemmli (1970). Slab gels were used with 0.5 cm long, 4 % stacking gel, pH 6.8, and 9 % separation gel, pH 8.8. Samples containing 30 "g of chlorophyll were applied in each well. Electrophoresis was run at 4 °C in the dark for three hours with constant currents of 1 rnA/sample in the stacking gel and 2 rnA/sample in the separation gel. Z. Pjlanzenphysiol. Ed. 108. S. 97-105. 1982.
Chlorophyll-protein complexes and chloroplast ultrastructure
99
The apparent molecular weights of the chlorophyll-protein complexes were determined as described earlier for thylakoid polypeptides (Aro, 1982). The distribution of chlorophyll between different chlorophyll-protein complexes was quantified from densitometer tracings (the height times the width at half height) at 670 nm or by taking the mean value from the tracings at 650 nm and 675 nm. For determination of the chI alb ratios of different complexes, the bands were dissected from the gel slabs, homogenized in 0.05 M HEPES-KOH, pH 7.6, in a glass homogenizer and centrifuged at 10,000 xg for 20 min. Acetone was added up to 80 % to the supernatant, which was centrifuged at 5000 xg for 10 min. T he chI al b ratios of intact plant materials were determined by grinding the material in 80 % acetone in the presence of a small amount of CaC0 3 and centrifuging at 5000 xg for 5 min. Chlorophylls were quantified according to Arnon (1949). For electron microscopy the material was fixed with 3 % glutaraldehyde in 0.1 M Naphosphate buffer, pH 7.0, and postfixed in 1 % osmium tetroxide in the same buffer. After dehydration in an alcohol series the samples were embedded in Epon. The thin sections, cut with an LKB ultramicrotome IV, were stained with uranyl acetate and Reynold's lead citrate, and examined with a Jeol Jem 100 CX electron microscope.
Results Chlorophyll-protein complexes and chI alb ratios There were the same chlorophyll-protein complexes in Ceratodon and Lemna (Fig. 1) (as also in Marchantia and Cucurbita, electrograms not shown). cpr and CPa seemed to be composed of only chi a in Lemna, whereas in Ceratodon some chI b was also present (Table 1). The chI alb ratios of different LHCP complexes were almost the same in Lemna and Ceratodon and the free pigment zones had chI alb ratios close to those observed in the whole plant extracts.
- C P I (lOS) _ LHCP·" (eo) -LMCP • (60) -LHCp · (~6)
---.---LHCP '- c p
Fig. 1: Chlorophyll-protein complexes of Lemna (A) and protonemata of Ceratodon (B) both grown for two weeks at 100 /lmol photons m- 2 s- l . Unstained gels. The apparent molecular we ights of the complexes in kdaltons given in parenthesis.
_ __
~
(38)
(28)
_ _-FP
B
A
Table 1: ChI alb ratios of different chlorophyll-protein complexes and the plant extract of Ceratodon (3 weeks old protonema) and Lemna grown in weak light (100 Itmol photons m- 2s- I). The results are the means of seven independent experiments. Plant
CPT
LHCP""·· LH CP'''' LHCP"'
CPa
Ceratodon Lemna
> 10
chI a
1.04 1.06
1.10
1.13
1.16 1.25
LHCP
FP
Plant extract
>6.0
1.24
chI a
1.39
2.21 2.89
2.07 2.88
Z. Pjlanzenphysiol. Bd. 108. S. 97-105. 1982.
100
EVA-MARl
ARO
The four plants differed, however, in the distribution of chlorophyll between the chlorophyll-protein complexes (Table 2). The bryophytes showed larger amounts of total LHCP than the higher plants and this was consistent with the lower chI alb ratios of the bryophytes. The higher plants contained more of the reaction centre complexes (combined amounts of cpr and CPa). The results obtained differed slightly, depending on the basis for quantification of the chlorophyll-protein complexes (compare tables 2 and 3). rn protonemata of Ceratodon grown in weak light, the two-week-old material was developmentally young and had 19 % of the total chlorophyll associated with cpr and 61 % with total LHCP (Table 3). During further growth there was a gradual decrease in cpr and a concomitant increase in total LHCP. The ratio of LHCP to Table 2: Relative distribution of chlorophyll between different chlorophyll-protein complexes and chI alb ratios of Ceratodon (3 weeks old protonema, weak light), Marchantia, Lemna (weak light) and Cucurbita. Chlorophyll content of different complexes was quantified from densitometer tracings at 670 nm. Results are the mean values of at least three independent experiments and six electrograms. % total chlorophyll in complexes
Plant
CPI
Ceratodon MarciJantia Lonna Cucurbita
16 19 21 29
LHCP': :.,: LHCP':':· LHCP" CPa
21 9
10
9 12 7
10
LHCP FP
total LHCP
CPI+ CPa
total plant LHCPI extract CPI chI alb n5:6
26 38 34 31
57 58 48 47
24 28 33 36
3.6 3.1 2.3 1.6
19 14 19 17
2.07 2.33 2.88 2.87
Table 3: Relative distribution of chlorophyll between different chlorophyll-protein complexes in Ceratodon and Lemna grown under low (LL) (100 Itmol photons m- 2s- 1 and high (HL) (500 Itmol photons m- 2s- l ) quantum flux density. Ceratodon protonemata of different ages were analyzed. Chlorophyll content of the complexes was quantified from densitometer tracings at 650 nm and 675 nm (mean value). Otherwise as in Table 2. % total chlorophyll in complexes Plant
CPI
LHCP"·':':· LHCP':·':· LHCP':·
CPa
LHCP
FP
total LHCP
total plant LHCPI extract CPI chI alb n~6
Ccratodon LL 2 weeks LL 3 weeks LL 4 weeks
19 15
11
4 6 5
HL 2 weeks 18 HL 3 weeks 14
21 22 30
7 8 10
21 19
10
4
29 26 23
15 18 16
61 62 68
3.2 4.1 6.2
2.12 2.07 1.95
32 29
17 21
61 61
3.4 4.4
2.15 2.08
34 35
20 22
52 49
2.6 2.6
2.88 3.01
Lemna
LL HL
20 19
7 2
9 7
Z. Pjlanzenphysiol. Bd. 108. S. 97-105. 1982.
10
C hl orophyll-protein co mplexes and chloroplast ultrastructure
101
CPI increased from 3.2 in two-week-old material to 6.2 in four-week-old material. However, the content of CPa was very stable throughout the study period, whereas the content of LHCP'" clearly increased. A slight decrease in the chi alb ratio took place during ageing of the protonemata. T wo- and three-week-old protonemata of Ceratodon grown in weak light did not differ much in their chlorophyll-protein composition from the protonemata grown in strong light for corresponding periods (Table 3). Lemna was also grown in weak and strong light. O wing to the growth habitat, the culture contains fronds of different ages, which were assumed to be equally distributed in both light conditions. Only slight differences were observed in the chi alb
Figs. 2-4: Chloroplasts of protonemata of Ceratodon grown at 100 I'mol photons m- 2s- I • Fig. 2. 2-week-old protonemata. Fig. 3. 3-week-old protonemata. Fig. 4. 4-week-old protonemata. Bar = 0.5 I'll! . rigs. '}-7: C hloroplasts of prolOne mJta of Ccratodon gro wn at 500 I'mol photons m-zs- I . Fig. 5. 2-week-o ld protonemata. Fig. 6. 3-week-old protonemata. Fig. 7. 4-week-old protone mata. Bar = 0.5 /l11l.
Z. Pjlanzenphysiol. Bd. 108. S. 97-105. 1982.
102
EVA-MARl ARO
ratios and chlorophyll-protein complexes (Table 3). Slightly less chlorophyll was associated with LHCP complexes in strong light, but the ratio of LHCP to cpr remained constant, 2.6 in strong and weak light. Chloroplast ultrastructure
When grown in weak light for two weeks, Ceratodon had chloroplasts with relatively well-developed grana and stroma thylakoids (Fig. 2). However, the thylakoid network was loose, and irregular branched grana structures were typical. When protonemataof Ceratodon were grown under high quantum flux density, the thylakoid network was composed of thin successive grana with 2-3 appressed thylakoids reach-
Fig. 8: Chloroplast of Marchantia. Bar = 0.5 /lm. Fig. 9: Chloroplast of Cucurbita. Bar = 0.5 /lm. Figs. 10-11: Chloroplasts of Lemna. Fig. 10. Grown at 100 /lmol photons m- 2s- l . Fig. 11: Grown at 500 /lmol photons m- 2s- 1. Bar
Z. PJlanzenphysiol. Bd. 108. S. 97-105. 1982.
=
0.5 /lm.
Chlorophyll-protein complexes and chloroplast ultrastructure
103
ing almost from one end of the chloroplast to the other, with reduced amounts of stroma thylakoids (Fig. 5). During ageing of the protonemata, the size of the grana increased in both weak (Figs. 3 and 4) and strong (Figs. 6 and 7) light and the fourweek-old material contained many plastoglobuli independently of whether the quantum flux density during growth was low or high. The chloroplasts of Marchantia were characterized by a dense thylakoid network (Fig. 8) with long grana stacks connected with short stroma thylakoids. The chloroplasts of Cucurbita contained large grana stacks connected with many stroma thylakoids (Fig. 9). However, the thylakoid network was not very dense allowing considerable volume for the stroma in relation to the membrane system. In Lemna grown in weak light, the thylakoid network was typical of higher plants (Fig. 10), with grana usually containing 8-9 appressed thylakoids. The chloroplast ultrastructure was modified by strong light (Fig. 11). The internal membrane system was less well developed than in weak light having longer grana-structures with only 3-4 appressed thylakoids.
Discussion Both higher plants had a chi alb ratio close to 3 and they also contained less total LHCP than the two bryophytes, which had a chi alb ratio slightly above 2 and a relative content of total LHCP corresponding to that of shade-tolerant plants (Anderson, 1980). Marchantia can also be regarded as a shade plant and its chloroplasts are characterized by large grana, but Ceratodon prefers open sunny locations. The low chi alb ratio of Ceratodon is mainly due to a high content of total LHCP and not to lower chi alb ratios of different LHCP complexes than in higher plants, as has been observed in a marine green alga, Caulerpa cactoides (Anderson et aI., 1980). The content of total LHCP in relation to the reaction centre complexes was high in the protonemata of Ceratodon whether the quantum flux density during growth was low or high, but the light conditions clearly influenced the chloroplast ultrastructure. No giant grana existed even in the protonemata grown from two to three weeks in weak light, but both grana stacks and stroma thylakoids were well-defined, whereas in protonemata grown in strong light the inner membrane system of chloroplasts was mainly composed of grana-structures containing only few appressed thylakoids. Accordingly, it seems that the ultrastructure of the thylakoid network can vary, while the chlorophyll-protein composition remains almost the same and that a low chI alb ratio and relatively high content of total LHCP does not necessarily mean that the chloroplasts contain large grana stacks. Strong modification of the chloroplast ultrastructure by light has been observed in a fern, Pteris cretica, although only a slight shift took place in the low chi alb ratio (Hariri and Brangeon, 1977).lt would be interesting to know whether any kind of lateral heterogeinity (Andersson and Anderson, 1980) exists in the distribution of the chlorophyll-protein complexes in Ceratodon-type chloroplasts, especially in those typical of protonemata grown in strong light. The shoots Z. Pjlanzenphysiol. Bd. 108. S. 97-105. 1982.
104
EVA-MARl ARO
of Ceratodon growing in natural habitats also have chloroplasts with a low chI alb ratio and with grana-structures containing only few appressed thylakoids (Valanne, 1977 a; Aro and Valanne, 1979). During ageing of the protonemata there was a gradual decrease in CPI and a concomitant increase in total LHCP, which has usually been observed during senescence (Wolindska, 1976; Jenkins et aI., 1981). In particular, the content LHCP':increased and this may be connected with the observed increase in the size of the grana. The chloroplasts of four-week-old protonemata grown under both low and high quantum flux densities showed many large plastoglobuli, which is also a typical phenomenon of senescence (Vala~ne et aI., 1979; Hudak, 1981). In Lemna also, the chloroplast ultrastructure was modified by the quantum flux density, although the LHCP/CPI ratio remained constant. The lower number of appressed thylakoids per granum in strong light was probably compensated by the greater length of the grana in relation to the stroma thylakoids. The chloroplasts of Cucurbita almost resembled those described for shade plants in respect to the size of the grana (Melis and Harvey, 1981), although the content of total LHCP was relatively small. In other respects, however, they resembled typical chloroplasts of sun species in having long stroma thylakoids and a large volume of stroma in relation to the membrane system. It seems that the ultrastructure of the chloroplast is more flexible than the composition of the chlorophyll-protein complexes. Probably the chlorophyll content of the chloroplast has more effect on the differentiation of the internal membranes into grana and stroma thylakoids than the distribution of chlorophyll between different chlorophyll-protein complexes. Acknowledgements
I express my sincere thanks to Mrs. Ulla-Maija Suo rant a for technical assistance in electron microscopy. This work was supported by The Academy of Finland.
References ANDERSON, ]. M.: Chlorophyll-protein complexes of higher plant thylakoids: Distribution, stoichiometry and organization in the photosynthetic unit. FEBS Lett. 117, 327-331 (1980). ANDERSON, J. M., D. ]. GOODCHILD, and N. K. BOARDMAN: Composition of the photosystems and chloroplast structure in extreme shade plants. Biochim. Biophys. Acta 325, 573-585
(1973).
ANDERSON, J. M., J. c. WALDRON, and S. W. THORNE: Chlorophyll-protein complexes of a marine green alga, Cau/elpa cactoides. Plant Sci. Lett. 17, 149-157 (1980). ANDERSSON, B. and]. M. ANDERSON: Lateral heterogeneity in the distribution of chlorophyllprotein complexes of the thylakoid membranes of spinach chloroplasts. Biochim. Biophys. Acta 593,427-440 (1980). ARNON, D. 1.: Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Eetavl1lgaris. Plant Physiol. 24,1-15 (1949). ARO, E.-M.: Polypeptide patterns of the thylakoid membranes of bryophytes. Plant Sci. Lett.
24,335-345 (1982).
Z. Pjlanzenphysiol. Ed. 108. S. 97-105. 1982.
Chlorophyll-protein complexes and chloroplast ultrastructure
105
ARO, E.-M. and N. VALANNE: Effect of continuous light on CO 2 fixation and chloroplast structure of the mosses Plcurozium schreberi and Ceratodon purpureus. Physiol. Plant. 45, 460-466 (1979). BALLANTINE, J. E. M. and B. J. FORDE: The effect of light intensity and temperature on plant growth and chloroplast ultrastructure in soybean. Amer.]. Bot. 57,1150-1159 (1970). BOARDMAN, N. K., J. M. ANDERSON, O. BJORKMAN, D. ]. GOODCHILD, L. H. GRIMME, and S. W. THORNE: Chloroplast differentiation in sun and shade plants: Relationship between chlorophyll content, grana formation, photochemical activity and fractionation of the photosystems. Port. Acta BioI. Ser. A 14, 213-236 (1974). HARIRI, M. and J. BRANGEON: Light-induced adaptive responces under greenhouse and controlled conditions in the fern Pteris cretica var. ouvrardii 1. Structural and infrastructural features. Physiol. Plant. 41, 280-288 (1977). HUDAK, J.: Plastid senescence 1. Changes of chloroplast structure during natural senescence in cotyledons of Sinapis alba L. Photosynthetica 15,174-178 (1981). JENKINS, G. 1., N. R. BAKER, and H. W. WOOLHOUSE: Changes in chlorophyll content and organization during senescence of the primary leaves of Phascolus vulgaris L. in rela,t,ion to photosynthetic electron transport. J. Exp. Bot. 32, 1009-1020 (1981). KALLIO, P. and N. V ALANNE: On the effect of continuous light on photosynthesis in mosses. In: WIELGOLASKI, f. E. (ed.): Ecological studies. Analysis and synthesis, vol. 16. Fennoscandian Tundra Ecosystems, Part 1,149-162. Springer-Verlag, Berlin, Heidelberg, New York, 1975. LAEMMLI, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 (1970). MARTIN, C. E.: Chlorophyll alb ratios of eleven North Carolina mosses. The Bryologist 83, 84-87 (1980). MELIs, A. and G. W. HARVEY: Regulation of photosystem stoichiometry, chlorophyll a and chlorophyll b content and relation to chloroplast ultrastructure. Biochim. Biophys. Acta 631, 138-145 (1981). MILLER, K. R.: A chloroplast membrane lacking photosystem 1. Changes in unstacked membrane region. Biochim. Biophys. Acta 592, 143-152 (1980). PRIOUL, J. L.: Eclairement de croissance et infrastructure des chloroplastes de Lolium multi[ forum Lam. Relation avec les resistances au transfert de CO 2 . Photosynthetica 7, 373-381 (1973). RAo, K. R., N. R. KUMAR, and A. N. REDDY: Studies of photosynthesis in some liverworts. The Bryologist 82, 286-289 (1979). VALANNE, N.: The germination phases of moss spores and their control by light. Ann. Bot. Fenn.3, 1-60 (1966). - Effect of continuous light on CO 2 fixation, chlorophyll content, growth and chloroplast structure in Ceratodon purpureus. Z. Pflanzenphysiol. 81, 347-357 (1977 a). - The combined effects of light intensity and continuous light on the CO 2 fixation, chlorophy II content and chloroplast structure of the protonema of Ceratodon purpureus. Z. Pflanzenphysiol. 83, 275-283 (1977 b). VALANNE, N., A. PENNANEN, and E. VAPAAVUORI: Comparison between the preservation of chloroplast structure in the dark and the turnover rate of chlorophyll-protein complexes in a moss and two varieties of pea. Plant & Cell Physiol. 20,1511-1522 (1979). W ARIS, H. and 1. ROUHIAINEN: Permanent and temporary morphological changes in Mierasterias induced by gamma rays. Ann. Acad. Sci. Fenn. A, IV, 167,1-13 (1970). WOLINDSKA, D.: Functional and structural changes in chloroplasts of senescent tobacco leaves. Acta Soc.Bul. Pol. 45, 341-351 (1976).
Z. Pjlanzenphysiol. Bd. 108. S. 97-105. 1982.
ARO
Department of Biology, University of Turku, SF-20S00 Turku 50, Finland Received April 16, 1982 · Accepted July 14, 1982
Summary The distribution of chlorophyll bet wee n different chlorophyll-protein complexes was compared with the chi al b ratio and chloroplast ultrastructure in two bryo phytes, the protonema of Ceratodon purpureus and thallus of Marchantia polymorpha, and two higher plants, Lemna minor and Cucurbita pepo. The higher plants are sun species, having a chi alb ratio close to 3, but the t wo bryophytes both had a chi alb ratio slighdy above 2, although one of them favours sunny habitats. The bryoph ytes had more of their chloroph yll associated with the LHCP co mplexes and less with the reaction centre complexes than the higher plants. Increase of the quantum flux density from 100 to 500 JLmol photons m- 2s- 1 during growth caused only slight changes in the composition of the chlorophyll-protein complexes and chi alb ratio in Lemna and protone mata of Ceratodon, although the ultrastructure of the chl oroplasts revealed clear red uction in the number of thylakoids per granum. Ceratodon grown in strong light for two to three weeks had grana with only two to three appressed thylakoids, but more t han 60 % of total chlorop hyll was still associated with LHCP, which indicates that the formation of large grana stacks is not necessary for accumulation of large amounts of LHCP in relation to the reaction centre complexes. Cucurbita had big grana stac ks but relatively less LHCP than either Ceratodon or Marchantia.
Key words: Ceratodon purpureus, Cucurbita pepo, Lemna minor, Marchantia polymorpha, bryophytes, chloroplast ult rastructure, chlorophyll alb ratio, chlorophyll·protein complexes.
Introduction Shade plants are typically known to have low chI alb ratios and their chloroplasts are characterized by large grana stacks, which in extreme cases may contain up to 100 th ylakoids (Anderson et aI., 1973). Mosses are also plants with low chI alb ratios (Kallio and Valanne, 1975; Valanne, 1977 a; Arc and Valanne, 1979; Rao et aI. , 1979; Martin, 1980) though not all the species are obligate shade plants. H owever, moss chloroplasts have been observed to be very diverse, some containing large grana stacks and others having only a fe w appressed thylakoids (Kallio and Valanne, 1975; Valanne, Abbreviations: Chi, chlorophyll ; C Pa, ch lorophyll a-protein complex of PSII; CPI, PlOO chloro phyll a-protein complex; FP, free pigment; LHCP, light-harvesting chlorophyll alb-protein co mplex; LHCP"", LHCP':"', LHCP""", oligomers of LHCP w ith increasing molecular weight; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulph ate.
Z. Pjlanzenphysiol. Bd. 108. S. 97-105. 1982.
98
EVA-MARl
ARO
1977 a; Aro and Valanne, 1979). The sun species of higher plants have higher chi alb ratios and their chloroplasts contain smaller grana and larger proportions of stroma thylakoids than the plants of shaded environments (Boardman et al., 1974). The size of the grana can be easily modified by environmental factors like the quantum flux density, especially in the case of the sun species (Ballantine and Forde, 1970; Prioul, 1973; Valanne, 1977 b). Chloroplasts with higher chi alb ratios have less LHCP and more of their chlorophyll is associated with the reaction centre complexes than in chloroplasts with lower chi alb ratios (Anderson 1980). Moreover, lateral heterogeneity has been reported in the distribution of the chlorophyll-protein complexes along the thylakoid membrane system. According to Andersson and Anderson (1980), the P700 chlorophyll a-protein of PSI (CPI) is situated in the stroma thylakoids and only in the end membranes and margins of the grana stacks, while the partitions are almost depleted of PSI reaction centres. These opinions are supported by the studies with PSI mutants (Miller, 1980). The grana partitions, on the other hand, are rich in PSII reaction centre complexes (CPa) and the light-harvesting complexes (LHCP). Only about 10-20 % of the total PSII reaction centre complex and the light-harvesting complex have been calculated to be associated with stroma thylakoids (Andersson and Anderson, 1980). In this study the distribution of chlorophyll between the different chlorophyll-protein complexes was compared with the chi alb ratio and chloroplast ultrastructure in two bryophytes and two higher plants. The effect of the quantum flux density during growth on the chlorophyll-protein complexes, chi alb ratio and chloroplast ultrastructure was also studied in Ceratodon and Lemna. Materials and Methods Cucurbita pepo L. and Marchantia polymorpha L. were grown in the greenhouse under longday conditions with a quantum flux density of 200 "mol photons m- 2 s- 1 for Cucurbita and 100 "mol photons m- 2 s- 1 for Marchantia. Lemna minor L. and protonemata of Ceratodon purpureus (Hedw.) Brid. were grown in MY solution (Waris and Rouhiainen, 1970) at a quantum flux density of 100 and 500 "mol photons m- 2s- 1 (photoperiod 12 h) and 24°C. Lemna was grown for two weeks and Ceratodon for two to four weeks. The sterilization and culture techniques used with Ceratodon were according to Valanne (1966). For isolation of chloroplasts, plant tissue was briefly homogenized in 0.05 M HEPES-KOH, 0.01 M NaCI, 0.5 M sucrose, pH 7.6, filtered through three layers of nylon cloth and centrifuged at 250 x g for 5 min. The chloroplasts were sedimented at 5000 xg for 10 min. The chloroplasts were then broken by washing twice with 1 mM NaEDTA, pH adjusted to 8.0, and the thylakoids were sedimented at 20,000 xg for 20 min. Samples for electrophoresis were prepared by suspending the thylakoids in 0.063 M Tris-HCI, 5 % 2-mercapto-ethanol and 10 % glycerol, pH 6.8, to a chlorophyll concentration of 0.8 mg/ml. Ten per cent SDS (PIERCE) was added to give a final SDS/chl ratio of 6: 1 (w/w) and the thylakoids were gently solubilized at 4°C. SDS-PAGE was carried out according to Laemmli (1970). Slab gels were used with 0.5 cm long, 4 % stacking gel, pH 6.8, and 9 % separation gel, pH 8.8. Samples containing 30 "g of chlorophyll were applied in each well. Electrophoresis was run at 4 °C in the dark for three hours with constant currents of 1 rnA/sample in the stacking gel and 2 rnA/sample in the separation gel. Z. Pjlanzenphysiol. Ed. 108. S. 97-105. 1982.
Chlorophyll-protein complexes and chloroplast ultrastructure
99
The apparent molecular weights of the chlorophyll-protein complexes were determined as described earlier for thylakoid polypeptides (Aro, 1982). The distribution of chlorophyll between different chlorophyll-protein complexes was quantified from densitometer tracings (the height times the width at half height) at 670 nm or by taking the mean value from the tracings at 650 nm and 675 nm. For determination of the chI alb ratios of different complexes, the bands were dissected from the gel slabs, homogenized in 0.05 M HEPES-KOH, pH 7.6, in a glass homogenizer and centrifuged at 10,000 xg for 20 min. Acetone was added up to 80 % to the supernatant, which was centrifuged at 5000 xg for 10 min. T he chI al b ratios of intact plant materials were determined by grinding the material in 80 % acetone in the presence of a small amount of CaC0 3 and centrifuging at 5000 xg for 5 min. Chlorophylls were quantified according to Arnon (1949). For electron microscopy the material was fixed with 3 % glutaraldehyde in 0.1 M Naphosphate buffer, pH 7.0, and postfixed in 1 % osmium tetroxide in the same buffer. After dehydration in an alcohol series the samples were embedded in Epon. The thin sections, cut with an LKB ultramicrotome IV, were stained with uranyl acetate and Reynold's lead citrate, and examined with a Jeol Jem 100 CX electron microscope.
Results Chlorophyll-protein complexes and chI alb ratios There were the same chlorophyll-protein complexes in Ceratodon and Lemna (Fig. 1) (as also in Marchantia and Cucurbita, electrograms not shown). cpr and CPa seemed to be composed of only chi a in Lemna, whereas in Ceratodon some chI b was also present (Table 1). The chI alb ratios of different LHCP complexes were almost the same in Lemna and Ceratodon and the free pigment zones had chI alb ratios close to those observed in the whole plant extracts.
- C P I (lOS) _ LHCP·" (eo) -LMCP • (60) -LHCp · (~6)
---.---LHCP '- c p
Fig. 1: Chlorophyll-protein complexes of Lemna (A) and protonemata of Ceratodon (B) both grown for two weeks at 100 /lmol photons m- 2 s- l . Unstained gels. The apparent molecular we ights of the complexes in kdaltons given in parenthesis.
_ __
~
(38)
(28)
_ _-FP
B
A
Table 1: ChI alb ratios of different chlorophyll-protein complexes and the plant extract of Ceratodon (3 weeks old protonema) and Lemna grown in weak light (100 Itmol photons m- 2s- I). The results are the means of seven independent experiments. Plant
CPT
LHCP""·· LH CP'''' LHCP"'
CPa
Ceratodon Lemna
> 10
chI a
1.04 1.06
1.10
1.13
1.16 1.25
LHCP
FP
Plant extract
>6.0
1.24
chI a
1.39
2.21 2.89
2.07 2.88
Z. Pjlanzenphysiol. Bd. 108. S. 97-105. 1982.
100
EVA-MARl
ARO
The four plants differed, however, in the distribution of chlorophyll between the chlorophyll-protein complexes (Table 2). The bryophytes showed larger amounts of total LHCP than the higher plants and this was consistent with the lower chI alb ratios of the bryophytes. The higher plants contained more of the reaction centre complexes (combined amounts of cpr and CPa). The results obtained differed slightly, depending on the basis for quantification of the chlorophyll-protein complexes (compare tables 2 and 3). rn protonemata of Ceratodon grown in weak light, the two-week-old material was developmentally young and had 19 % of the total chlorophyll associated with cpr and 61 % with total LHCP (Table 3). During further growth there was a gradual decrease in cpr and a concomitant increase in total LHCP. The ratio of LHCP to Table 2: Relative distribution of chlorophyll between different chlorophyll-protein complexes and chI alb ratios of Ceratodon (3 weeks old protonema, weak light), Marchantia, Lemna (weak light) and Cucurbita. Chlorophyll content of different complexes was quantified from densitometer tracings at 670 nm. Results are the mean values of at least three independent experiments and six electrograms. % total chlorophyll in complexes
Plant
CPI
Ceratodon MarciJantia Lonna Cucurbita
16 19 21 29
LHCP': :.,: LHCP':':· LHCP" CPa
21 9
10
9 12 7
10
LHCP FP
total LHCP
CPI+ CPa
total plant LHCPI extract CPI chI alb n5:6
26 38 34 31
57 58 48 47
24 28 33 36
3.6 3.1 2.3 1.6
19 14 19 17
2.07 2.33 2.88 2.87
Table 3: Relative distribution of chlorophyll between different chlorophyll-protein complexes in Ceratodon and Lemna grown under low (LL) (100 Itmol photons m- 2s- 1 and high (HL) (500 Itmol photons m- 2s- l ) quantum flux density. Ceratodon protonemata of different ages were analyzed. Chlorophyll content of the complexes was quantified from densitometer tracings at 650 nm and 675 nm (mean value). Otherwise as in Table 2. % total chlorophyll in complexes Plant
CPI
LHCP"·':':· LHCP':·':· LHCP':·
CPa
LHCP
FP
total LHCP
total plant LHCPI extract CPI chI alb n~6
Ccratodon LL 2 weeks LL 3 weeks LL 4 weeks
19 15
11
4 6 5
HL 2 weeks 18 HL 3 weeks 14
21 22 30
7 8 10
21 19
10
4
29 26 23
15 18 16
61 62 68
3.2 4.1 6.2
2.12 2.07 1.95
32 29
17 21
61 61
3.4 4.4
2.15 2.08
34 35
20 22
52 49
2.6 2.6
2.88 3.01
Lemna
LL HL
20 19
7 2
9 7
Z. Pjlanzenphysiol. Bd. 108. S. 97-105. 1982.
10
C hl orophyll-protein co mplexes and chloroplast ultrastructure
101
CPI increased from 3.2 in two-week-old material to 6.2 in four-week-old material. However, the content of CPa was very stable throughout the study period, whereas the content of LHCP'" clearly increased. A slight decrease in the chi alb ratio took place during ageing of the protonemata. T wo- and three-week-old protonemata of Ceratodon grown in weak light did not differ much in their chlorophyll-protein composition from the protonemata grown in strong light for corresponding periods (Table 3). Lemna was also grown in weak and strong light. O wing to the growth habitat, the culture contains fronds of different ages, which were assumed to be equally distributed in both light conditions. Only slight differences were observed in the chi alb
Figs. 2-4: Chloroplasts of protonemata of Ceratodon grown at 100 I'mol photons m- 2s- I • Fig. 2. 2-week-old protonemata. Fig. 3. 3-week-old protonemata. Fig. 4. 4-week-old protonemata. Bar = 0.5 I'll! . rigs. '}-7: C hloroplasts of prolOne mJta of Ccratodon gro wn at 500 I'mol photons m-zs- I . Fig. 5. 2-week-o ld protonemata. Fig. 6. 3-week-old protonemata. Fig. 7. 4-week-old protone mata. Bar = 0.5 /l11l.
Z. Pjlanzenphysiol. Bd. 108. S. 97-105. 1982.
102
EVA-MARl ARO
ratios and chlorophyll-protein complexes (Table 3). Slightly less chlorophyll was associated with LHCP complexes in strong light, but the ratio of LHCP to cpr remained constant, 2.6 in strong and weak light. Chloroplast ultrastructure
When grown in weak light for two weeks, Ceratodon had chloroplasts with relatively well-developed grana and stroma thylakoids (Fig. 2). However, the thylakoid network was loose, and irregular branched grana structures were typical. When protonemataof Ceratodon were grown under high quantum flux density, the thylakoid network was composed of thin successive grana with 2-3 appressed thylakoids reach-
Fig. 8: Chloroplast of Marchantia. Bar = 0.5 /lm. Fig. 9: Chloroplast of Cucurbita. Bar = 0.5 /lm. Figs. 10-11: Chloroplasts of Lemna. Fig. 10. Grown at 100 /lmol photons m- 2s- l . Fig. 11: Grown at 500 /lmol photons m- 2s- 1. Bar
Z. PJlanzenphysiol. Bd. 108. S. 97-105. 1982.
=
0.5 /lm.
Chlorophyll-protein complexes and chloroplast ultrastructure
103
ing almost from one end of the chloroplast to the other, with reduced amounts of stroma thylakoids (Fig. 5). During ageing of the protonemata, the size of the grana increased in both weak (Figs. 3 and 4) and strong (Figs. 6 and 7) light and the fourweek-old material contained many plastoglobuli independently of whether the quantum flux density during growth was low or high. The chloroplasts of Marchantia were characterized by a dense thylakoid network (Fig. 8) with long grana stacks connected with short stroma thylakoids. The chloroplasts of Cucurbita contained large grana stacks connected with many stroma thylakoids (Fig. 9). However, the thylakoid network was not very dense allowing considerable volume for the stroma in relation to the membrane system. In Lemna grown in weak light, the thylakoid network was typical of higher plants (Fig. 10), with grana usually containing 8-9 appressed thylakoids. The chloroplast ultrastructure was modified by strong light (Fig. 11). The internal membrane system was less well developed than in weak light having longer grana-structures with only 3-4 appressed thylakoids.
Discussion Both higher plants had a chi alb ratio close to 3 and they also contained less total LHCP than the two bryophytes, which had a chi alb ratio slightly above 2 and a relative content of total LHCP corresponding to that of shade-tolerant plants (Anderson, 1980). Marchantia can also be regarded as a shade plant and its chloroplasts are characterized by large grana, but Ceratodon prefers open sunny locations. The low chi alb ratio of Ceratodon is mainly due to a high content of total LHCP and not to lower chi alb ratios of different LHCP complexes than in higher plants, as has been observed in a marine green alga, Caulerpa cactoides (Anderson et aI., 1980). The content of total LHCP in relation to the reaction centre complexes was high in the protonemata of Ceratodon whether the quantum flux density during growth was low or high, but the light conditions clearly influenced the chloroplast ultrastructure. No giant grana existed even in the protonemata grown from two to three weeks in weak light, but both grana stacks and stroma thylakoids were well-defined, whereas in protonemata grown in strong light the inner membrane system of chloroplasts was mainly composed of grana-structures containing only few appressed thylakoids. Accordingly, it seems that the ultrastructure of the thylakoid network can vary, while the chlorophyll-protein composition remains almost the same and that a low chI alb ratio and relatively high content of total LHCP does not necessarily mean that the chloroplasts contain large grana stacks. Strong modification of the chloroplast ultrastructure by light has been observed in a fern, Pteris cretica, although only a slight shift took place in the low chi alb ratio (Hariri and Brangeon, 1977).lt would be interesting to know whether any kind of lateral heterogeinity (Andersson and Anderson, 1980) exists in the distribution of the chlorophyll-protein complexes in Ceratodon-type chloroplasts, especially in those typical of protonemata grown in strong light. The shoots Z. Pjlanzenphysiol. Bd. 108. S. 97-105. 1982.
104
EVA-MARl ARO
of Ceratodon growing in natural habitats also have chloroplasts with a low chI alb ratio and with grana-structures containing only few appressed thylakoids (Valanne, 1977 a; Aro and Valanne, 1979). During ageing of the protonemata there was a gradual decrease in CPI and a concomitant increase in total LHCP, which has usually been observed during senescence (Wolindska, 1976; Jenkins et aI., 1981). In particular, the content LHCP':increased and this may be connected with the observed increase in the size of the grana. The chloroplasts of four-week-old protonemata grown under both low and high quantum flux densities showed many large plastoglobuli, which is also a typical phenomenon of senescence (Vala~ne et aI., 1979; Hudak, 1981). In Lemna also, the chloroplast ultrastructure was modified by the quantum flux density, although the LHCP/CPI ratio remained constant. The lower number of appressed thylakoids per granum in strong light was probably compensated by the greater length of the grana in relation to the stroma thylakoids. The chloroplasts of Cucurbita almost resembled those described for shade plants in respect to the size of the grana (Melis and Harvey, 1981), although the content of total LHCP was relatively small. In other respects, however, they resembled typical chloroplasts of sun species in having long stroma thylakoids and a large volume of stroma in relation to the membrane system. It seems that the ultrastructure of the chloroplast is more flexible than the composition of the chlorophyll-protein complexes. Probably the chlorophyll content of the chloroplast has more effect on the differentiation of the internal membranes into grana and stroma thylakoids than the distribution of chlorophyll between different chlorophyll-protein complexes. Acknowledgements
I express my sincere thanks to Mrs. Ulla-Maija Suo rant a for technical assistance in electron microscopy. This work was supported by The Academy of Finland.
References ANDERSON, ]. M.: Chlorophyll-protein complexes of higher plant thylakoids: Distribution, stoichiometry and organization in the photosynthetic unit. FEBS Lett. 117, 327-331 (1980). ANDERSON, J. M., D. ]. GOODCHILD, and N. K. BOARDMAN: Composition of the photosystems and chloroplast structure in extreme shade plants. Biochim. Biophys. Acta 325, 573-585
(1973).
ANDERSON, J. M., J. c. WALDRON, and S. W. THORNE: Chlorophyll-protein complexes of a marine green alga, Cau/elpa cactoides. Plant Sci. Lett. 17, 149-157 (1980). ANDERSSON, B. and]. M. ANDERSON: Lateral heterogeneity in the distribution of chlorophyllprotein complexes of the thylakoid membranes of spinach chloroplasts. Biochim. Biophys. Acta 593,427-440 (1980). ARNON, D. 1.: Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Eetavl1lgaris. Plant Physiol. 24,1-15 (1949). ARO, E.-M.: Polypeptide patterns of the thylakoid membranes of bryophytes. Plant Sci. Lett.
24,335-345 (1982).
Z. Pjlanzenphysiol. Ed. 108. S. 97-105. 1982.
Chlorophyll-protein complexes and chloroplast ultrastructure
105
ARO, E.-M. and N. VALANNE: Effect of continuous light on CO 2 fixation and chloroplast structure of the mosses Plcurozium schreberi and Ceratodon purpureus. Physiol. Plant. 45, 460-466 (1979). BALLANTINE, J. E. M. and B. J. FORDE: The effect of light intensity and temperature on plant growth and chloroplast ultrastructure in soybean. Amer.]. Bot. 57,1150-1159 (1970). BOARDMAN, N. K., J. M. ANDERSON, O. BJORKMAN, D. ]. GOODCHILD, L. H. GRIMME, and S. W. THORNE: Chloroplast differentiation in sun and shade plants: Relationship between chlorophyll content, grana formation, photochemical activity and fractionation of the photosystems. Port. Acta BioI. Ser. A 14, 213-236 (1974). HARIRI, M. and J. BRANGEON: Light-induced adaptive responces under greenhouse and controlled conditions in the fern Pteris cretica var. ouvrardii 1. Structural and infrastructural features. Physiol. Plant. 41, 280-288 (1977). HUDAK, J.: Plastid senescence 1. Changes of chloroplast structure during natural senescence in cotyledons of Sinapis alba L. Photosynthetica 15,174-178 (1981). JENKINS, G. 1., N. R. BAKER, and H. W. WOOLHOUSE: Changes in chlorophyll content and organization during senescence of the primary leaves of Phascolus vulgaris L. in rela,t,ion to photosynthetic electron transport. J. Exp. Bot. 32, 1009-1020 (1981). KALLIO, P. and N. V ALANNE: On the effect of continuous light on photosynthesis in mosses. In: WIELGOLASKI, f. E. (ed.): Ecological studies. Analysis and synthesis, vol. 16. Fennoscandian Tundra Ecosystems, Part 1,149-162. Springer-Verlag, Berlin, Heidelberg, New York, 1975. LAEMMLI, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 (1970). MARTIN, C. E.: Chlorophyll alb ratios of eleven North Carolina mosses. The Bryologist 83, 84-87 (1980). MELIs, A. and G. W. HARVEY: Regulation of photosystem stoichiometry, chlorophyll a and chlorophyll b content and relation to chloroplast ultrastructure. Biochim. Biophys. Acta 631, 138-145 (1981). MILLER, K. R.: A chloroplast membrane lacking photosystem 1. Changes in unstacked membrane region. Biochim. Biophys. Acta 592, 143-152 (1980). PRIOUL, J. L.: Eclairement de croissance et infrastructure des chloroplastes de Lolium multi[ forum Lam. Relation avec les resistances au transfert de CO 2 . Photosynthetica 7, 373-381 (1973). RAo, K. R., N. R. KUMAR, and A. N. REDDY: Studies of photosynthesis in some liverworts. The Bryologist 82, 286-289 (1979). VALANNE, N.: The germination phases of moss spores and their control by light. Ann. Bot. Fenn.3, 1-60 (1966). - Effect of continuous light on CO 2 fixation, chlorophyll content, growth and chloroplast structure in Ceratodon purpureus. Z. Pflanzenphysiol. 81, 347-357 (1977 a). - The combined effects of light intensity and continuous light on the CO 2 fixation, chlorophy II content and chloroplast structure of the protonema of Ceratodon purpureus. Z. Pflanzenphysiol. 83, 275-283 (1977 b). VALANNE, N., A. PENNANEN, and E. VAPAAVUORI: Comparison between the preservation of chloroplast structure in the dark and the turnover rate of chlorophyll-protein complexes in a moss and two varieties of pea. Plant & Cell Physiol. 20,1511-1522 (1979). W ARIS, H. and 1. ROUHIAINEN: Permanent and temporary morphological changes in Mierasterias induced by gamma rays. Ann. Acad. Sci. Fenn. A, IV, 167,1-13 (1970). WOLINDSKA, D.: Functional and structural changes in chloroplasts of senescent tobacco leaves. Acta Soc.Bul. Pol. 45, 341-351 (1976).
Z. Pjlanzenphysiol. Bd. 108. S. 97-105. 1982.