Response of magnesium chelatase activity in green pea (Pisum sativum L.) leaves to light, 5-aminolevulinic acid and dipyridyl supply

Joumalof

I:~-KII19~I:IEMIffrRY AND

PI-KIIO/~OIDG~ B:BIOLOGY

ELSEVIER

Journal of Photochemistry and Photobiology B: Biology 36 (1996) 17-22

Response of magnesium chelatase activity in green pea (Pisum sativum L.) leaves to light, 5-aminolevulinic acid and dipyridyl supply N.G. Averina a, E.B. Yaronskaya a, V.V. Rassadina a, G. Walter b,. "Belarussian Academy of Sciences, Institute of Photobiology, Skorina str. 27, 220733 Minsk, Belarus b Humboldt University Berlin, Institute of Biology, Res. Philippstr. 13, Unter den Linden 6 D-10099 Berlin, Germany Received 9 June 1995; accepted 9 October 1995

Abstract

The role of extra-porphyrin production in the magnesium branch of the porphyrin pathway was investigated in green pea (Pisum sa6vum L.) leaves treated with 5-aminolevulinic acid (ALA) and 2,2'-dipyridyl (DP). The magnesium chelatase activity was estimated during protoporphyrin IX, Mg-protoporphyrin IX (monomethyl ester) (MgProto(E)) and protochlorophyllide accumulation in the presence of exogenous ALA and/or DP. The results showed a close correlation only between the enzyme activity and MgProto(E) levels. The inverse dependence of the magnesium chelatase activity on the amount of MgProto(E) accumulated supports the hypothesis that a mechanism is involved which controls the magnesium branch of chlorophyll biosynthesis in vivo through inhibition of the enzymes by their products. Illumination of intact pea leaves predarkened for 17 h resulted in a 1-2 h lag phase of magnesium chelatase activity with consequent strong stimulation. After 3 h of illumination it reached 200% compared with the enzyme activity immediately before illumination. Cycloheximide, if applied during the entire period of dark or light treatment, decreased the magnesium chelatase activity by 19% in the dark and by 39% in light, in comparison with the corresponding control variant on H20. The ADP and especially the ATP content increased in the leaves treated with cycloheximide both in darkness and in light. The photosynthetic activity, measured as 02 evolution by leaf segments, did not change in the presence of cycloheximide. The results are discussed as an additional influence of light on the magnesium chelatase activity not only via photosynthetic supply with ATP but also through light induced synthesis of the enzyme molecules de novo. Keywords: Mg-chelatase; 5-Aminolevulinic acid; 2,2'-Dipyridyl; Cycloheximide; Adenylates; Pea (Pisum sativum L. )

1. Introduction

Magnesium insertion into protoporphyrin IX (Proto) is the first step unique to chlorophyll synthesis. It occurs at the branching point of the two major porphyrin biosynthesis routes in chloroplasts and therefore could be directly involved in the overall coordination of heine and chlorophyll synthesis [1]. Mg-chelatase (MCH) is an ATP-requiring [2-8] enzyme which consists of at least two protein components. In pea chloroplasts membrane-bound and soluble fractions have been separated [9-11 ]. The observations that different levels of endogenous protochlorophyllide (Pchlide) did not affect magnesium chelation [ 12] and that MCH activity in intact cucumber chloroplasts was not inhibited by exogenous Pchlide or chlorophyllide [13] (which had been suggested to regulate the enzyme by feedback inhibition [ 1 ] ), reopen the question of how the magnesium branch of the chlorophyll pathway is regulated. The mechanisms that control the accu* Corresponding author. Tel: (030)2895309; Fax: (030)2895537. 1011-1344/96/$15.00 © 1996 Elsevier Science S.A. All rights reserved SSDI 1011 - 1 344 ( 95 ) 0725 1-9

mulation of excessive amounts of chlorophyll precursors, for example from exogenous 5-aminolevulinic acid (ALA), are still little known [ 14,15 ]. Their study could help us to understand better the structural organization of the chlorophyll pathway in vivo and the relationship between this process and cell porphyrin production. Light affects the MCH in intact plastids [ 16,17 ]. The light stimulation is connected with photosynthetic processes as it is related to the ATP requirement of the reaction [17]. In etiolated leaves and during the early stages of greening when photophosphorylation is absent or low, magnesium chelation could be controlled by mitochondrial ATP [ 4 ]. Nevertheless, the finding that one of the two DNA loci mediating the enzyme protein for the conversion of Proto to magnesium protoporphyrin IX (MgProto) in Chlamydomonas mutants controls the light-dependent step in this process [ 14], and the observation of strong photoactivation of MCH during the early stages of greening of etiolated cucumber seedlings [ 13 ], could indicate additional light-dependent mechanisms involved in this process. The data of Koncz et al. [ 18,19] on

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N.G. A verina et al. / Journal of Photochemistry and Photobiology B: Biology 36 (1996) 17-22

light activation of expression of the cs(ch-42) gene involved in magnesium chelation in Arabidopsis thaliana and the data of Hudson et al. [ 20] on light depression of oli gene product synthesis which is associated with the magnesium chelation step in chlorophyll biosynthesis in Antirrhinum majus, are contradicting and do not help to solve this problem. One of the purposes of this work was to trace the MCH activity in relation to the levels of excessive Pchlide, MgProto, MgProto-monomethyl ester (MgProtoE), and Proto accumulation in green leaves of pea after their treatment with exogenous ALA, alone or in combination with the chelator 2,2'-dipyridyl (DP), which changes the ratio between porphyrins accumulated from exogenous ALA [21,22]. Along with this, we also aimed at studying the enzyme response to dark-light transition of the plants.

about 5 s each). The homogenate was filtered through two layers of cheesecloth and one layer of nylon (Dederon Filterseide) and centrifuged at 200 g for 1 rain. The pellet was discarded and the chloroplasts in the supernatant were sedimented by centrifugation at 2500 g for 4 min. Chloroplasts were purified by resuspending the pellets in 2 ml of the isolation buffer, layered on 6 ml of 45% (vol/ vol) Percoll (in isolation buffer) and centrifuged at 13 200 g for 7 min. The chloroplast pellets were freed of Percoll contamination by resuspension in 10 ml of the isolation buffer (without BSA) and centrifugation at 2500 g for 4 min. All centrifugations in this preparation were performed on the Centrikon T-124 centrifuge (Germany). The chloroplast intactness, determined by assaying the latent 6-phosphogluconate dehydrogenase activity [23], was 90%.

2.4. Magnesium chelatase assay 2. Materials and methods

2.1. Chemicals ALA, bovine serum albumin (BSA), dithiothreitol (DTT), cycloheximide (CH), kinetin, magnesium-ATP, Percoll, phosphocreatine, creatine phosphokinase, Proto IX, sorbitol, Tricine were obtained from Sigma Chemical Co. All other chemicals were of analytical grade and were obtained from the former Soviet Union chemical firms (Leningrad, plant "Krasnyi khimik"; Olainsk, Chemical plant).

2.2. Plant materials Pea (Pisum sativum L.) seedlings were grown in a growth chamber at 23 °C under cool-white light (10-12 W m -2) with 14 h light-10 h dark cycle for 8-17 days. 14 day old pea leaves were cut and placed in Petri dishes between two gauze layers. They were incubated in the dark for 17 h in solutions containing different concentrations of ALA (pH 3.2), DP or their combinations. Control plants were incubated in H20 adjusted to the same pH. Kinetin (50 mg 1-1) was added to the incubation solutions to prevent the leaves from aging. For investigations of the effect of light on MCH activity, 10-14 day old plants were illuminated after darkness with fluorescent light (28 W m -z) for different time periods. In some experiments plants were placed with roots on the solution of 10 mM CH in H20 for 17 h in darkness before illumination.

2.3. Chloroplast isolation Intact chloroplasts were isolated as described previously [ 10], but with some modifications. Leaves of developing pea seedlings ( 15 g) were excised and ground in 45 ml ice-cold isolation buffer (0.5 M sorbitol+50 mM Tricine + 1 mM Dq"I"+ 1 mM E D T A + 1 mM MgCI2+0.1% BSA, pH 7.8) with an Ultra-Turrax rotor-stator-type tissue disrupter (S 25N-18G probe, speed setting 20 500 min I two times for

The MCH activity was measured as described by Walker and Weinstein [ 10]. Chloroplast incubations were carried out in tubes placed in a heating block set at 30 °C and covered with aluminium foil. In a routine assay, chloroplasts (approximately 1 mg chloroplast protein) were incubated in a total volume of 1 ml incubation buffer (isolation buffer lacking BSA) containing 1.5 × 10 - 6 M Proto (added in dimethyl sulphoxide, 2% (vol/vol) final concentration) and 4.0 mM MgATP in a regenerating system (60 mM phosphocreatine + creatine phosphokinase, 4 units m l - 1) in the dark. In some experiments for investigation of aging and the influence of light on the MCH activity, the Proto was purified as described [ 5 ] ; that resulted in a two-fold increase in the MCH activity. Reactions were started by the addition of plastids and allowed to proceed for 60 min with shaking. Reactions were terminated by adding ice-cold acetone. The MCH activity was described as the amount of MgProto(E) formed from exogenous Proto in the chloroplasts. The activity from different preparations of chloroplasts usually varied no more than 25%. Silicagel thin layer chromatography of the reaction products showed that the main pigment (91%) was MgProto and the remaining 9% belonged to MgProtoE. We did not observe additional formation of Pchlide beyond its endogenous levels during incubation.

2.5. Other procedures Pigments were extracted with acetone + 0.1 N NHnOH mixture (9:1, vol/vol). The pigment separation of phytolized, hexane-soluble porphyrins on the one hand, and of unphytolized porphyrins which remained in the water-acetone solution on the other hand, was performed as described by Shlyk et al. [24]. The amounts of Pchlide, MgProto(E) and Proto in hexane-washed water-acetone extracts were measured spectrofluorometrically [24,25] using an F-4500 fluorescence spectrophotometer (Hitachi, Japan). Proteins were assayed according to Bradford [26],

N.G. Averina et al./ Journal of Photochemistry and Photobiology B: Biology 36 (1996) 17-22 Table 1 The MCH activities and amounts of Pchlide in chloroplasts from green pea (Pisum sativum) leaves which were illuminated on the seedlings for 9 h after the last dark period and then continued to be irradiated (L) or were darkened (D) for 17 h Variant

9hL+17hL 9hL+17hD

MCH activity

Pchlide

pmol MgP mg ~ (protein) h - '

%

pmol mg (protein) h -1

%

3895:17 4395:30

100 113±9

1975:18 3395:43

100 172+12

For estimating adenylates, the leaf material was frozen in liquid nitrogen and homogenized in 0.5 N HCIO4 with a mortar. After centrifugation at 20 000 g for 20 min the supernatant was neutralized with 4 N KOH to pH 7.4 and centrifuged once more for 5 min at 1250 g. The samples were adjusted to a final volume of 10 ml with HEPES-KOH buffer (pH 7.75) and stored at - 20 °C. ATP, ADP, and AMP were analysed by the luciferin-luciferase luminescence method [27]. ADP and AMP were determined after enzymatic conversion to ATP [28]. The adenylate energy charge, EC = [ (ATP) + 0.5 (ADP) ] / [ (ATP) + (ADP) + (AMP) ], was calculated to indicate the energy state of the cell.

2.6. 02 evolution assay Pea leaf segments were placed in a laboratory-made vessel with an electrode for measurement of oxygen evolution. Segments were illuminated for 2.5 s with saturating red light flashes with 0.74 s dark intervals. The 02 evolution was measured after 128 flashes and calculated per flash.

3. Results

3.1. Effect of aging, darkening and cutting In the chloroplast preparations from pea leaves which were illuminated for 3-4 h after the last dark period, the MCH activity decreased with aging of plants containing 1654, 1167

19

and 513 pmol MgProto mg -J h -~ for 8, 15 and 17 day old plants respectively. In Table 1 data are presented showing that the MCH activity in leaves of intact plants does not change during 17 h of darkness, compared with the enzyme activity in leaves which continued to be illuminated for the same time period. The amounts of Pchlide increased during the resynthesis period in the dark by 1.7 times, indicating that Pchlide did not control the MCH activity in vivo. The MCH activity in the leaves which were cut and placed in darkness on a water surface for 17 h came to only 66% in comparison with the enzyme activity in leaves of intact plants with roots darkened for the same period (data not shown). Only Pchlide was observed to accumulate under these conditions both in leaves from intact plants with roots and in cut leaves. Approximately equal levels of Pchlide were accumulated.

3.2. ALA and DP treatment Leaves treated with 5 or 10 mM ALA and 1 or 3 mM DP accumulated different amounts of the chlorophyll precursors Proto, MgProto(E) and Pchlide, and showed lower levels of MCH activity in comparison with the control (Table 2 and Fig. 1 ). No correlation was observed between MCH activity and the amounts of accumulated Proto and Pchlide. The enzyme activity changed in a broad interval from 0 to 100% in variants having very similar amounts of Pchlide (see 1 and 3 mM DP variants in relation to the control). However, the variants with Pchlide amounts that differed by 5 times had relatively close mean values of MCH activity (see 1 mM DP and 10 mM ALA variants). The same picture was observed also in the case of Proto accumulation. Similar values of MCH activity were measured in the variants containing 18 and 106 pmol Proto per mg protein (5 and 10 mM ALA variants accordingly). It should also be noted that in four independent experiments in which only DP ( 1 or 3 mM) was applied, no Proto accumulation was observed. Possibly this implies that DP by itself, and in these concentrations, does not influence the MCH activity through chelation of Mg 2÷ ions [ 13 ]. A close correlation between MCH activities and MgProto(E) levels accumulated is evident from Fig. I, in which

Table 2 Porphyrin contents and MCH activity in chloroplasts from isolated green pea (Pisum sativum) leaves, treated with ALA and DP of different concentrations for 17 h in the dark Variant

ALA (5 mM) ALA ( 10 mM) DP ( 1 raM) DP (3 raM) DP (3 raM) + A L A (5 mM)

Proto pmol m g (protein)

18 106 0 0 30

PChlide

MCH activity

pmol mg ~ (protein)

% to control on H20

pmol MgP mg ~ (protein) h ~

% to control on H20

1006 1180 413 270 860

518 498 116 120 443

101 74 128 0 0

66 61 48 10 0

The data of five representative experiments (preparations) axe shown; all parameters in each preparation, determined in duplicate assays, rarely varied by more than 5%.

N.G. Aver±ha et al. / Journal of Photochemistry and Photobiology B: Biology 36 (1996) 17-22

20

250

lO0 80

U ill

10

40

[]

control

0

ALA (5 raM)

• A

ALA(10 raM) DP (1 mM)

[]

DP

~¢

ALA (5 raM)

200

=" 150

(3raM)

g

+ DP (3 raM)

o

-r-

100

20 50

0 0

500

1000

1500 0

MgProto(E) [pmol'mg-lprotein]

i

,

,

i

2

4

6

8

10

Fig. 1. Dependence of the Mg chelatase activity (relative to the corresponding control variants on HzO = 100%) on the amount of endogenous MgP in isolated green pea (Pisum sati~urn) leaves placed for 17 h in darkness on ALA, DP and ALA + DP solutions of different concentrations. The data represent 15 independent preparations.

Illumination, h Fig. 2. Influence of light on the Mg chelatase activity (relative to the corresponding control variants on H20 = 100%) in green pea (Pisum sat±rum) leaves from seedlings predarkened for 17 h before continuous illumination for 10h.

the data of 15 individual experiments are represented. It is obvious that the MCH activity depends inversely on the amount of MgProto (E). ALA on its own at a concentration of 10/.tM in the reaction mixture increased slightly the MCH activity during incubation of the chloroplasts with Proto. In the chloroplasts incubated for 1 h at 30 °C in the presence of ALA, 49 + 3 pmol MgProto per mg protein was formed from Proto, and 38 + 0 pmol MgProto was found in control samples incubated without ALA. No MgProto was formed in chloroplast samples boiled for 2 min before incubation, showing that there exists no non-enzymatic introduction of Mg into Proto.

amounted to 115 (100%) and 675 (100%), 71 (62%) and 739 (109%), 254 (221%) and 1003 (149%) respectively. Illumination for 5 and 10 h led to a gradual decrease in MCH activity, reaching after 10 h the level which was observed in predarkened leaves immediately before illumination. If the plants were placed with their roots on a solution of CH (10 mg 1- ~) during 17 h of darkness, the MCH activity at the end of this period decreased to 81% compared with control leaves (Table 3). The illumination of plants treated with CH did not result in enzyme photostimulation but rather strengthened the inhibition of its activity up to 41% at 3 h of illumination. The contents of ATP, ADP and AMP in leaves of intact plants exposed to dark-light transitions are represented in Table 3. The action of CH during 17 h of darkening resulted in an increase in total adenylates (130%) and especially the ATP content (143%) compared with control plants. The illumination of leaves for 3 h increased the levels of ADP and ATP in both control and CH treated plants. The action of CH resulted in a two-fold increase in the ATP content compared with control variant. Nevertheless the energy charge (EC) of the plants changed little in the presence of CH (Table 3). In parallel, the photosynthetic activities were determined as the 02 evolution of leaf segments from control and CH treated green pea leaves in two independent experiments. The

3.3. Effect of dark-light transition and CH treatment When intact plants after 17 h of predarkening were illuminated, the MCH activity showed a lag phase of 1-2 h, followed by strong simulation (Fig. 2). There was threefour-fold photostimulation in some individual experiments. The photostimulation was observed in chloroplast preparations showing both low and high MCH activities (when 10 or 14 day old plants and unpurified or purified Proto were used as substrate). For example, in two experiments the values of MCH activities for 0, 1 and 3 h of illumination

Table 3 Adenylate contents and MCH activity in intact green pea (Pisum sat±rum) leaves placed for 17 h in darkness on water or on CH solution with following illumination for 3 h Variant

HzO (17 h D) CH ( 1 7 h D ) H20 (17 h D + 3 h L ) CH ( 1 7 h D + 3 h L )

ATP (nmol g - ' (fresh weight) )

98.65:13.0 140.65:24.3 166.5±18.3 317.35:52.4

ADP (nmol g - ~ (fresh weight) )

86,1 5:10.4 101.6+ 16.1 107.3+14.2 134.8±14.5

AMP (nmol g - ~ (fresh weight) )

2.305:6.0 0.39±0.67 1,78±3.48 0.765:1.96

ATP (relative)

ATP/ADP

EC

Mg-chelatase activity pmol rag- 1 (protein) h -

100 143 169 322

1.17+0.26 1.42±0.36 1.57±0.25 2.395:0.52

0.76-t-0.02 0.795:0.03 0.805:0.02 0.855:0.02

121 + 15 95+11 1785:31 69±9

relative l

100 81+7 151 + 22 59:[:6

N.G. A verina et al. / Journal of Photochemistry and Photobiology B: Biology 36 (1996) 17-22

O2 evolution per leaf area and flash was actually equal in control and CH variants, both in leaves predarkened for 17 h and in leaves irradiated for 3 h (data not shown).

4. Discussion In our previous paper [ 15 ] we showed a gradual inhibition of the S-adenosyl-L-methionine: MgProto methyltransferase (MT) activity during MgProto(E) and Pchlide accumulation from exogenous ALA. We proposed that a mechanism exists in plants which controls the accumulation of excessive amounts of chlorophyll precursors. The limitation in membrane sites available for membrane-bound porphyrine molecules possibly inhibits transportation of enzyme products from their place of origin, thus inhibiting enzyme-product complex dissociation and thereby obstructing the activities of the enzymes involved. In the present study we have obtained results supporting this idea. A tight correlation was observed between the MCH activity and MgProto(E) levels accumulated from exogenous ALA during 17 h of darkness. The enzyme activity decreased gradually during MgProto(E) accumulation and diminished to nearly zero when high levels of MgProto (E) accumulated. Gradual inhibition of MCH activity, as well as MT activity [ 15], during porphyrin accumulation were observed in the presence of kinetin which prevents seedlings from aging. We assume that the inhibition of MCH activity in vivo in the presence of exogenous ALA is the result of accumulation of the MgProto molecules (which cannot transform into Pchlide because of a limitation in membrane sites) and the inability of MgProto to dissociate from the MCH complex; hence, the enzyme is inhibited by its product. In cases of ALA and DP use, when a transformation of MgProtoE into Pchlide is inhibited by the chelator, we observe the accumulation of surprisingly high levels of MgProto(E) with very low MCH activity. The possibility cannot be excluded that the chelator changes the membrane conformation in vivo [29], so that exogenous Proto can no longer associate with MCH in isolated chloroplasts. Therefore in the presence of DP we observed hardly any enzyme activity in our incubation medium. A detailed investigation of the mechanisms involved in the control of extra porphyrin production should be a subject of future work. The light dependence of the MCH is conditioned by ATP demands of the reaction which can utilize ATP from different cell sources such as mitochondria and chloroplasts at different stages of plant development [4,17]. The decrease in MCH activity in darkened isolated leaves compared with freshly harvested leaves could be due to degradation of some enzyme molecules under these conditions. The CH data which showed inhibition of MCH activity not only in the light but also in darkness (Table 3) may support this explanation. Our data (Fig. 2) show a 1-2 h lag-period before two-fold light stimulation in MCH activity, though ATP does not seem to be limited during this period. This points to additional

21

mechanisms of light action which could be connected with photocontrol of the MCH activity through synthesis of new enzyme molecules. The effects of CH in our experiments support this explanation. Synthesis of MCH de novo and its active state may be favoured by enhanced contents of ATP and ADP (typically, the cellular EC changes only very little in balanced metabolic processes). CH application led to an accumulation of adenylates, because adenylate-dependent anabolic processes are blocked by CH in light as well as in the dark (Table 3). Under such conditions there is no correlation between adenylate content and MCH activity which is very small in leaves with application of CH. Our results underline the influence of light on MCH not only by photosynthetic supply of the Mg chelation with ATP but also through light-induced synthesis of the enzyme molecules de novo. This supports the data on light activation of the cs(ch42) gene transcription, the product of which is involved in the Mg chelation step of chlorophyll biosynthesis [ 18,19].

Acknowledgements This research was supported by the Volkswagen-Stiftung, grant 1/68 478. We thank Erika Helmer and Camillo Kitzmann for skilful estimation of the adenylate contents and Vladimir Domanskii for oxygen evolution assay.

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