Planta 9 by Springer-Verlag 1978

Planta 139, 79-83 0978)

Chlorophyllase Activity in Developing Leaves of Phaseolus vulgaris L. W.A.W. Moll, B. deWit, and R. Lutter Department of Plant Physiology,Universityof Amsterdam, IJdijk 26, Amsterdam,The Netherlands

Abstract. In crude extracts of primary leaves from dark grown seedlings of Phaseolus vulgaris L., relatively high hydrolytic activity of chlorophyllase (chlorophyll-chlorophyllido-hydrolase, EC 3.1.1.14) was observed. When plants were exposed to continuous illumination, the enzyme activity in the extracts was doubled within 3 days and both chlorophyll a and b were synthesized. However, when exposed to periodic illumination (1 min light-59min dark) the enzyme activity was doubled within 1 to 2 days and chlorophyll a was synthesized but the formation of chlorophyll b was suppressed. When plants were transferred from periodic illumination to continuous illumination chlorophyll b was synthesized but the activity of chlorophyllase declined. Chloramphenicol blocked the increase in enzyme activity independent of the light regimes, but cycloheximide inhibited the activity more effectively during growth in the light. The presence of chlorophyllase activity in the leaves is discussed in relation to the chlorophyllproteins and the membranes known to be present in chloroplasts. It is suggested that the enzyme is synthesized on plastid ribosomes.

Key words: Chlorophyllase - Chlorophyll-proteins Etiolation - Greening - Phaseolus - Plastid membranes.

Introduction

It is assumed that the chloroplast enzyme chlorophyllase is involved in chlorophyll metabolism in green plants and that the enzyme is intimately concerned with the stability of chlorophyll molecules in photoAbbreviations: CAP=D-threo-chloramphenicol; CHI=cycloheximide; C[-contin~ous ilIumination;PI--periodic illumina~ion

synthetic membranes (Ellsworth et al., 1976; Holden, 1976). Little is known about the site of synthesis of this enzyme. According to Schneider and Beisenherz (1974) in cell cultures of Nicotiana tabacum chlorophyllase is not synthesized within the plastids during growth in darkness and under illumination. Data this subject also were published by Ganoza and McFeeters (1976) working with Chlorella protothecoides. They concluded that the enzyme is synthesized on cytoplasmic ribosomes and that incorporation of the enzyme into the chloroplastmembrane may be promoted by a protein factor synthesized on chloroplast ribosomes. From the work of Terpstra (1974) it can be inferred that chlorophyllase is mainly present in small (stroma) membranes of the chloroplast and that the enzyme is not very active in large (grana) membranes. It was suggested (Terpstra, 1976) that activity of the enzyme is due to the protein part of the chlorophyll-protein complexes (Thornber, 1975; Thornber et al., 1977). If this is true, one would expect the synthesis and the activity of chlorophyllase to be associated with the synthesis and presence of chlorophyll-protein complexes. In this work we followed the activity of chlorophyllase during the development of leaves of Phaseolus vulgaris L. in the presence or absence of antibiotics that inhibit protein synthesis on plastid ribosomes or cytoplasmic ribosomes. Enzyme activity was followed during dark growth and during continuous illumination when the formation of chlorophyll a and b and the chlorophyll-protein complexes were induced. Also, periodic illumination was used. This light regime was first chosen by ArgyroudiAkoyunoglou et al. (1976) and Arntzen et al. (1977) because it retards plastid development by suppressing the formation of grana membranes and the formation of light-harvesting chlorophyll a-b-protein complex without suppressing the formation of chlorophyll a. Thus using periodic illumination we were able to

80

study the activity of chlorophyllase during the synthesis of chlorophyll a while the formation of a part of the chlorophyll-protein complexes in the chloroplast was inhibited.

Material and Methods Plant Material Seedlings of Phaseolus vulgaris L. cv. Prelude were germinated and grown under conditions described by Sluiters-Scholten et al. (1973). After growing for 9 days in the dark the plants were exposed to periodic illumination (1 rain light-59 min dark) or to continuous illumination. As a light source fluorescent tubes (Philips T1 65W/33) were used and the light intensity was 2000 lx. Leaves were treated with antibiotics on the 4th day and/or on the 9th day after sowing. The inhibitors of protein synthesis, C A P and CHI, were given by wetting the lower side and the upper side of the leaves with about 0.5 ml of a solution of 50 gg/ml C A P or 5 gg/ml C H I under dim green safe light using a fluorescent tube (Philips T L 40W/17).

Preparation of Crude Leaf Extracts All operations were performed under green safe light at 4~ Twelve to sixty pairs of leaves, depending on age were frozen at -30~ for 5 h and freeze dried overnight. The dried leaves were then ground in a mortar and homogenized in a Virtis 45 homogenizer for 5 min in the presence of 30 ml of a cold solution containing 90 % v/v acetone, 2 % v/v glycerol and 8 % v/v destilled water buffered with 0.01mol 1-1 Tris-HCl at pH7.8. The mixture was centrifuged for 15min at 12,000g and the resulting pellet was washed with 100 % cold acetone. To solubilize the chlorophyllase, 6 m l 0.01 tool 1-1 phosphate buffer at p H 7.0 containing 0.2% Triton X-100 and 2 % v/v glycerol was added to the pellet and the suspension was sonicated at 4 ~ for 5 min under gaseous nitrogen. Then the suspension was shaken mechanically for 1 h at 4 ~ and centrifuged for 10min at 30,000g to remove insoluble material. The supernatant was stored in the dark at - 2 0 ~ but could be kept at 10~ for 24 h without loss in chlorophyllase activity.

W.A.W. Moll et al.: Chlorophyllase Activity in Developing Leaves

Results The chlorophyllase activity in extracts of leaves, harvested onwards from the 4th day after sowing is presented in Figure 1. On the 4th day some activity could already be measured and it increased rapidly between the 5th and 7th day of etiolation. During growth in the dark CAP inhibted the enzyme activity for about 70 % and CHI inhibited the activity of the enzyme for about 12 ~o. After the 9th day plants were transferred to continuous illumination and the appropriate leaves treated a second time with the antibiotics 2 h before illumination to maintain the level of these synthesis inhibitors in the leaves. In the absence of antibiotics chlorophyllase activity increased once more until after 3 days the activity was doubled. Treatment with CAP and CHI completely blocked this increase and even reduced activity below the levels found in continuous darkness. Chlorophyllase was also followed in leaves under continuous illumination that were treated only once with the antibiotics (Fig. 2A). Both CAP and CHI were inhibitory after the first 12h, lowering the chlorophyllase activity beneath that of the controls grown further in the dark. In extracts of leaves transferred from the dark to periodic illumination (Fig. 2B) the enzyme activity

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Enzyme Assay The hydrolytic activity of chlorophyllase was measured in the presence of 0.2 % v/v Triton X-100 using purified chlorophyll a as substrate (0.2 mg/ml reaction mixture). The procedure of McFeeters et al. (1971 was followed except that the reactions were performed under nitrogen. Samples of the reaction mixtures were taken after 0, 10, 20 and 60rain. The enzyme activity was expressed as a percentage of the substrate transformed to chlorophyllide per h per 10 pairs of leaves. Chlorophyllcontent and the chlorophyll a/b ratio were determined according to Arnon (1949) in the acetone extracts collected from the centrifnging and washing steps. Chlorophyll a was purified according to the methods of Strain and Svec (1966).

Source of Chemicals CAP, de Watermolen, Zaandam, The Netherlands; CHI, Calbiochem, Los Angeles, Calif. USA; Triton X-100, Merck, Darmstadt, Federal Republic of Germany.

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5 6 7 8 9 10 11 1213 (days) Time after start of germination Fig. 1. Chlorophyllase activity in extracts of leaves of Phaseolus vulgaris L., grown in the dark and under continuous illumination. Antibiotics were added on the 4th day and 2 h before illumination. a, 9 9 dark control; b, o o light control; c, 9 9 activity in the dark, C H I (5 gg/ml) added on the 4th day; c~ [] activity under continuous illumination, CHI (5 gg/ml) added a second time 2 h before illumination; d, 9 9 activity in the dark, C A P (50gg/ml) added on the 4th day; zx zx activity under continuous illumination, C A P (50 gg/ml) added a second time 2 h before illumination. CI, continuous illumination

W.A.W. Moll et al.: Chlorophyllase Activity in Developing Leaves 30

Table 1. Pigments in leaves of Phaseolus vulgaris L. exposed to continuous or periodic illumination after an etiolation period of 9 days

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Total chlorophyll a (mg/10 leaf pairs)

Chlorophyll a/b"

C

CAP

CHI

C

CAP

CHI

9 D + 1 2 h C1 b 24 h 36 h 48 h 72 h 96 h

0,20 0,30 1.21 3,50 7.40 9.30

0.15 0,26 0.42 0.71 1.50 2.16

0,15 0,20 0,30 0.36 0.41 0.47

3.3 3.0 2.8 2.6 2.6 2.6

4.2 3.1 3.0 2.9 2.7 2.6

5.0 4.1 3.9 3.7 3.6 3.6

9 D + 1 2 h PI b 24 h 36 h 48 h 35.1 72 h 72 h PI + 2 4 h CI

0.10 0.20 0.49 1.10 2.35 5.80

0.10 0.15 0.25 0.36 0,58 0.96

0.07 0.10 0.13 0.20 0.24 0.29

>40 >40 >40 > 40 35,1 3.5

>40 >40 >40 > 40 38.5 10.2

>40 >40 >40 > 40 >40 38.0

Illumination conditions

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Time offer s t a r t of first illuminolion Fig. 2A and B. Chlorophyllase activity in extracts of leaves of Phaseolus vulgaris L., grown under continuous illumination A or periodic illumination followed by continuous illumination for 24 h B. The activity was followed in plants that were germinated and grown in darkness for 9 days, and then at 0 h exposed to the light. Antibiotics were added 2 h before illumination, a, 9 9 dark control; b, o o light control; c, [] [] CHI added (5 gg/ml); d, z~ ~ C A P added (50 gg/ml). CI, continuous illumination; PI, periodic illumination

was doubled within 1 to 2 days, faster even than under continuous illumination. CAP-treatment again inhibited the development of the enzyme activity but CHI-treatment was slightly less effective. When after 72 h periodic illumination, the plants were transferred to continuous illumination, the chlorophyllase activity in extracts of leaves that were not treated with the antibiotics, declined sharply. In Table 1 the chlorophyll content and the chlorophyll a/b ratio in extracts of leaves are presented. The chlorophyll content of the leaves grown under continuous illumination increased sharply after 24 h. CAP- and CHI-treatment inhibited chlorophyll synthesis for about 75 % and 95 % respectively after 72 h, and from the slightly higher a/b ratios compared with the controls, it seemed that chlorophyll b synthesis was the most sensitive to inhibition. Under periodic illumination the formation of chlorophyll was retarded, relative to under continuous illumination. Little if any chlorophyll b was synthesized, which indicates the absence of light-harvesting chlorophyll a-b-protein complex and the absence of grana membranes. However, when after 72 h the plants were transferred to contiinuous illumination and the chlorophyllase activity had declined (Fig. 2B), chlorophyll and from the drop in a/b ratios we can

" C = c o n t r o l experiment; C A P = a f t e r treatment with 501ag/ml C A P ; C H I = a f t e r treatment with 5 g g / m l CHI. The antibiotics were added 2 h before the onset of illumination. The chlorophyllase activity extracted from these leaves is presented in Figure 2 A and B b CI = c o n t i n u o u s illumination; PI =periodic illumination

say particularly chlorophyll b, continued to be synthesized.

Discussion

A marked increase in chlorophyllase activity was found in etiolated leaves of Phaseolus vulgaris L., 5-7 days after germination. This is correlated with the formation of the prolamellar bodies (Klein and Schiff, 1972) but it seems doubtful whether the hydrolytic activity that we assay for the enzymes presence has any physiological significance at this time. Perhaps the enzyme has a synthetic role to play, or perhaps it is merely a passive component of the membrane depot which the etioplast represents. Nonetheless, the synthesis of chlorophyllase in the absence of chlorophyll illustrates that chlorophyllase activity is not necessarily associated with the chlorophyll-protein complexes (Thornber, 1975; Thornber et al., t977). Leaves transferred to continuous light exhibit a further marked rise in chlorophyllase activity, which is associated with chlorophyll synthesis, but leaves transferred to intermittent illumination exhibit an even more dramatic rise in activity while chlorophyll synthesis, and particularly chlorophyll b synthesis, is

82 appreciably less than in continuous light. Thus synthesis of the enzyme preceeds the formation of chlorophyll rather than paralleling it (Bogorad, 1976). There is no apparent correlation between chlorophyllase activity and the formation of chlorophyll-protein complexes. We conclude that the protein part of the chlorophyll-complexes are not responsible for the chlorophyllase activity or at least that it does not require to be complexed with chlorophyll to be active, It might be that chlorophyllase will become associated with proteins that during plastid development under illumination are complexed with chlorophyll a. However a more definite identification between chlorophyll a-protein complexes and chlorophyllase activity is needed. Chlorophyllase is a membrane protein which Terpstra (1974) gas shown to be mainly associated with the stroma membranes of the chloroplast as opposed to the grana membranes. Our results support this contention because during the rapid increase in activity in leaves under periodic illumination chloroplast development is incomplete and only the stroma membrane precursors, the primary thylakoids are present (Argyroudi-Akoyunoglou et al., 1976; Arntzen et al., 1977). We therefore assume that this newly synthesized chlorophyllase is associated with these stroma membrane precursors and not with the as yet absent grana membranes. Evidence is presented that CAP, the chloroplast protein synthesis inhibitor, is very effective in inhibiting the synthesis of chlorophyllase during the development of the primary leaves of Phaseolus vulgaris L. However this effect of C A P was not observed by others. During transformation of proplastids to chloroplasts in cell culture of Nicotiana tabacum (Schneider and Beisenherz, 1974) and during regeneration of chloroplasts in greening cell cultures of ChIorella Protothecoides (Ganoza and McFeeters, 1976) C A P is less effective in inhibiting the synthesis of chlorophyllase. According to these authors the influence of C A P can be ascribed to inhibition of the synthesis of subsidiary proteins necessary for transport, localisation or incorporation of chlorophyllase in the chloroplast membranes. According to Ganoza and McFeeters (1976) these protein(s) protect the enzyme from rapid degradation. The presence of high chlorophyllase activity in triton extractions from etiolated leaves of Phaseolus vulgaris L., suggests that subsidiary proteins already can be synthesized during etiolation and that the enzyme molecules turn over very slowly even in the absence of the chloroplast structure. G a n o z a and McFeeters (1976) observed that in greening cells C H I inhibited the increase in chlorophyllase activity and they concluded that the

W.A.W. Moll et al.: Chlorophyllase Activity in Developing Leaves enzyme is synthesized on cytoplasmic ribosomes. Our results do not support that conclusion. If we firstly consider the inhibition of chlorophyllase synthesis in developing leaves of Phaseolus vulgaris L., in the dark, then C A P is clearly more inhibitive than C H I and the simplest interpretation would be that the enzyme is synthesized within the etioplast. This is in agreement with the hypothesis of Siddell and Ellis (1977) that a number of proteins are synthesized on plastid ribosomes during the very early stages of etioplast development. If we consider synthesis in the light, then we must bear in mind that in light cytoplasmic ribosomes are supposed to be very active in synthesizing many (chloroplast) components (Siddell and Ellis, 1977). Thus, inhibition by C H I in the light is not a good criterion for cytoplasmic protein synthesis. Nevertheless, with respect to chlorophyllase, although C H I was more effective in the light, it was not more effective than C A P and in periodic light, appreciably less effective. We suggest therefore that chlorophyllase is synthesized in the chloroplast.

References Argyroudi-Akoyunoglou, J.H., Kondylaki, S., Akoyunoglou, G.: Growth of grana form "primary" thylakoids in Phaseolus vulgaris. Plant & Cell Physiol. 17, 939-954 (1976) Arnon, D.I.: Copper enzymes in isolated chloroplasts. Plant Physiol. 24, 1 15 (1949) Arntzen, C.J., Armond, P.A., Briantais, J.M., Burke, J.J., Novitzky, W.P.: Dynamic interactions among structural components of the chloroplast membrane. In: Chlorophyll-proteins, reaction centres and photosynthetic membranes,: Brookhaven symposia in biology, vol. 28, pp. 316-337. Olson, J.M., Hind, G., eds. New York: Upton 1977 Bogorad, L.: Chlorophyll biosynthesis. In: Chemistry and biochemistry of plant pigments, vol.2, pp. 118 120, Goodwin, T., ed. London-New York-San Francisco: Academic Press 1976 Ellsworth, R.K., Tsuk, R.M., St. Pierre, L.A.: Studies on chlorophyllase IV. Attribution of hydrolytic and esterifying "chlorophyllase" activities observed in vitro to two enzymes. Photosynthetica 10 (3), 312-323 (1976) Ganoza, V.G., McFeeters, R.F.: Chlorophyllase activity during pigmentation changes in Chlorella protothecoides. Photosynthetica 10, 1 6 (1976) Holden, M.: Chlorophylls. In: Chemistry and biochemistry of plant pigments, vol. 2, pp. 28-30, Goodwin, T., ed. LondonNew York-San Francisco: Academic Press 1976 Klein, S., Schiff, J.A.: The correlated appearance of prolamellar bodies, protochlorophyl(lide) species and the Shibata shift during development of bean etioplasts in the dark. Plant Physiol. 49, 619-626 {1972) McFeeters, R.F., Chichester, C.O., Whitaker, J.R.: Purification and properties of chlorophyllase from Ailanthus altissima (tree of heaven). Plant Physiol. 47, 609-618 (1971) Siddell, S.G., Ellis, F.R. : Protein synthesis by etioplasts. Biochem. Soc. Tra.ans.ns.5, 98-102 (1977)

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Sluiters-Scholten, C.M.Th., Berg, F.M., van den Stegwee, D.: Aminoleavulinate dehydratase in greening leaves of Phaseolus vulgaris L. Z. Pflanzenphysiol. 69, 217-227 (1973) Schneider, Hj.A.W., Beisenherz, W.W.: Determination of the sites of synthesis of chlorophyll synthesizing enzymes in cell cultures of Nicotiana rabacum. Biochem. Biophys. Res. Commun. 48, 907-913 (1972) Strain, H.H., Svec, W.A.: Extraction, separation, estimation and isolation of the chlorophylls. In: The chlorophylls, pp. 54-57, Vernon, P., Seely, G.R., eds. New York: Academic Press 1966 Terpstra, W.: Properties of chloroplasts and chloroplasts fragments as deduced from internal chlorophyll --+ chlorophyllide conversion. Z. Pflanzenphysiol. 71, 129-143 (1974) Terpstra, W.: Chlorophyllase and lamellar structure in Phaeodac-

tylum tricornutum. III Situation of chlorophyllase in pigment membranes. Z. Pflanzenphysiol. 80, 177-188 (1976) Thornber, J.P.: Chlorophyll-proteins: Light-harvesting and reaction centre components of plants. In: Ann Rev. Plant Physiol. 26, 127-158 (1975) Thornber, J.P., Alberte, R.S., Hunter, F.A., Shiozawa, J.A., Kan, K.S.: The organization of chlorophyll in the plant photosynthetic unit. In: Chlorophyll-proteins, reaction centres and photosynthestic membranes, Brookhaven Symposia in biology 28, 132-147. Olson, J.M., Hing, G., eds. New York: Upton 1977

Received 27 October; accepted 7 November 1977

Chlorophyllase activity in developing leaves of Phaseolus vulgaris L.

In crude extracts of primary leaves from dark grown seedlings of Phaseolus vulgaris L., relatively high hydrolytic activity of chlorophyllase (chlorop...
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