ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

167, 351-365

Chloroplast Biosynthesis and Other

and Accumulation Metalloporphyrins

(1975)

Biogenesis of Mg-Protoporphyrin

by Isolated Chloroplasts’,

CONSTANTIN

Department

Etioplasts ’

A. REBEIZ, BARRY B. SMITH, CAROLE C. REBEIZ. AND DANIEL of Horticulture,

University Received

of Illinois, August

IX Monoester and Developing

JAMES R. MATTHEIS, F. DAYTON

Urbana-Champaign,

Illinois

61801

26,1974

Developing chloroplasts isolated from greening cotyledons and isolated etioplasts were capable of synthesizing and accumulating Mg-protoporphyrin IX monoester as well as longer wavelength metalloporphyrins when incubated in the dark, in the presence of air, &aminolevulinic acid, and cofactors (coenzyme A, glutathione, adenosine triphosphate, nicotinamide adenine dinucleotide, methyl alcohol, magnesium, potassium, and phosphate). The putative metalloporphyrins exhibited distinct fluorescence emission and excitation properties and were detected by spectrofluorometry in situ and after extraction in organic solvents. The cofactors were previously shown to be required for protochlorophyll, and chlorophyll biosynthesis and grana assembly in oitro. The putative long wavelength metalloporphyrins were suggested earlier to represent intermediates between Mg-protoporphyrin IX monomethyl ester and protochlorophyllide. The isolated plastids were similar in this aspect of their biosynthetic activity to etiolated cotyledons greening in distilled H,O. In contrast to greening cotyledons, however, the biosynthetic activity of the isolated plastids depended on the addition of exogenous cofactors and &aminolevulinic acid. This was interpreted as an indication that the isolated plastids were not capable of generating their own &aminolevulinic acid and cofactors under the present incubation conditions. Light was not required for the conversion of added ALA to metalloporphyrins in uitro. The metalloporphyrins synthesized in uitro were more highly fluorescent in situ than those of greening cotyledons. In addition to Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins, isolated etioplasts synthesized and accumulated Zn-protoporphyrin and Zn-protoporphyrin IX monoesterlike compounds.

It was recently reported that excised etiolated cotyledons accumulated Mg-protoporphyrin monester (MPE)3 and other metalloporphyrins during greening in dis-

tilled H,O (1). This biosynthetic activity was observed in the absence of added substrates or inhibitors. MPE and the accompanying metalloporphyrins were detected by spectrofluorometry in situ and after extraction in organic solvents. They were partially segregated by TLC and characterized by their soret fluorescence excitation maxima and their short wave-

‘This work was supported by Research Grant G. B. 40236 from the National Science Foundation and by funds from the Illinois Agricultural Experiment Station. ‘This paper is Paper XIV in a series. Paper XIII appeared in Arch. Biochem. Biophys. 166, 324 (1975). ’ Abbreviations: MPE: Mg-protoporphyrin IX monomethyl ester or monester; Pchlide: protochlorophyllide; Pchl: protochlorophyll; Proto: protoporphyrin IX; ALA: b-aminolevulinic acid; E-X: fluores-

cence emission spectrum elicited by an excitation at Xnm; F-X: fluorescence excitation spectrum recorded with the emission monochromator positioned at Xnm; Zn-Proto: Zn-protoporphyrin IX; Zn-Proto E: Znprotoporphyrin IX monomethyl ester or monoester. 351

Copyright All rights

0 1975 by Academic Press, Inc. of reproduction in any form reserved.

352

REBEIZ

length emission maxima. In general the metalloporphyrins accompanying MPE, exhibited longer wavelength fluorescence emission and excitation maxima than MPE. The chromatographic and spectrofluorometric properties of these metalloporphyrins were compatible with the properties of the intermediates of a P-oxidation sequence between MPE and protochlorophyllide (Pchlide) 3 (1, 2, 3). In our efforts to investigate the stepwise enzymology of protochlorophyll’ (Pchl)’ formation (5) and the assembly of prothylakoid membranes in uitro, we have found that the spectrofluorometric properties of MPE, of the accompanying metalloporphyrins, and of Pchl lend themselves admirably for qualitative and quantitative studies. As a beginning toward the elucidation of the biosynthetic steps between protoporphyrin IX (Proto) and Pchl in vitro, we describe in this paper the net synthesis of MPE and of the longer wavelength metalloporphyrins by isolated etioplasts and developing chloroplasts. The net synthesis of Pchl in uitro will be reported in a separate paper. MATERIALS

AND

METHODS

Cucumber seed, (Cucumis satiuus L. cv. Alpha Green) a gift of the Niagara Chemical Division, FMC Corporation, Modesto, CA were used. The plant material, light pretreatment of excised cotyledons, isolation of etioplasts and developing chloroplasts, extraction of the plastid metalloporphyrins, chromatography, spectrophotometry, spectrofluorometry and the preparation of various metalloporphyrin standards were described in detail elsewhere (1). Determination of the amount of MPE + longer wavelength metalloporphyrins (the MPE-equivalent)’ present in the hexaneextracted acetone fractions was estimated from the integral of the fluorescence emission between 580 and 620 nm, as described earlier (1). Incubation of the isolated plastids. Etioplasts and developing chloroplasts were isolated in a fortified Tris-HCl buffered medium (1) unless otherwise indicated. Etioplasts were prepared from etiolated cotyledons. The latter were exposed to about 50 FW cm-* ‘Protochlorophyll refers to the mixture of protochlorophyllide and protochlorophyllide phytyl ester encountered in etiolated plant tissues (4). 5 The MPE-equivalent refers to the sum of MPE + accompanying longer wavelength metalloporphyrins (1).

ET

AL.

s-’ of cool white fluorescent light for 15-20 min during harvesting and weighing. During that period the transformable Pchlide was converted into chlorophyllide and chlorophyll a. Developing chloroplasts were prepared from excised greening cotyledons that were previously irradiated for 5 h with 320 rW of cool white fluorescent light. The details of the plastid isolation were described elsewhere (1). Fortified plastids were incubated essentially as described previously (6, 7): Two milliliters of plastid suspensions were incubated with cofactors in cylindrical flat-bottom glass tubes (2 x 10 cm) on a metabolic shaker operated at 10 shakes/min. The incubations were performed at 28°C in the dark for 1 h unless otherwise indicated. Three milliliters of fortified reaction mixture, adjusted to pH 7.7 at room temperature contained: 400 rmol of Tris-HCl, 1 mmol of sucrose, 100 rmol of potassium phosphate, 1 pmol of MgCll, 10 rmol of GSH, 0.6 rmol of CoA, 0.8 mmole of methyl alcohol, 0.8 rmol of ATP, 0.15 rmol of NAD, 0.1 pmol of ALA and 43 mg of plastid proteins. Extmction of the products of incubation. At the beginning or end of incubation the 3.0-ml reaction mixtures were transferred to 50-ml centrifuge tubes with an eye dropper. The reaction was stopped by the addition of 15 ml of acetone: 0.1 N NH,OH (9:l v/v). Preparation of the hexane-extracted acetone fractions containing the metalloporphyrins and chromatography of the latter were as previously described (1). Spectrofluorometry. Metalloporphyrins were characterized by their short wavelength fluorescence emission maxima and their soret excitation maxima in situ and in organic solvents (1). RESULTS

Biosynthesis and accumulation of Mgprotoporphyrin IX monoester and longer wavelength metalloporphyrins by developing chloroplasts during incubation in the dark. Our first objective was to determine

whether isolated developing chloroplasts extracted from Cucumis cotyledons were capable of synthesizing net amounts of MPE and longer wavelength metalloporphyrins as did the greening cotyledons (1). Developing chloroplasts were therefore prepared from greening cotyledons that were preirradiated with 320 PW cmm2 s-’ (250 fc) of cool white fluorescent light for 5 h and the isolated plastids were incubated with and without b-aminolevulinic acid (ALA)’ in the dark. The fluorescence of the plastids was then compared to the fluorescence of greening cotyledons and

Mg-PROTOPORPHYRIN

MONOESTER

to MPE-enriched etioplasts6 before and after incubation. It was reported earlier that greening cotyledons exhibited distinct emission maxima between 580 and 620 nm, that corresponded to MPE and longer wavelegnth metalloporphyrin fluorescence, while MPE-enriched etioplasts constituted a reliable marker of MPE fluorescence in situ (l), Table I. The endogenous metalloporphyrin fluorescence of developing chloroplasts monitored before incubation was as previously described (l), (Table I). Their fluorescence emission spectrum elicited by a 420 nm excitation (E - X = 420 nm)” usually exhibited a weak emission maximum at 598-600 nm that corresponded to the fluorescence of the endogenous MPE (1). In addition it exhibited a weak emission maximum at 607-609 nm and an emission shoulder at 614-61’7 nm that corresponded to the fluorescence of the endogenous longer wavelength metalloporphyrins (1). Such a spectrum is reported in Fig. 1A and is summarized in Table I. After 1 h of dark incubation with ALA, the plastids exhibited an intense increase in the metalloporphyrin fluorescence between 580 and 620 nm, which suggested a net synthesis of metalloporphyrins during incubation (Fig. 1A). The emission spectra of the incubated plastids elicited by a 420 nm excitation (near the soret excitation maximum of MPE) and by a 435 nm excitation (near the soret excitation maxima of the longer wavelength metalloporphyrins) are presented in Fig. 1A and are summarized in Table I. They exhibited enhanced emission maxima or shoulders that corresponded to MPE emission (598-600 nm) and to longer wavelength metalloporphyrin emissions (603-604, 607-609, 614617 nm). In addition an emission shoulder at 590-592 nm was observed. It will be shown later that this emission probably emanated from Zn-protoporphyrin. It was desirable to ascertain that the in6 MPE-enriched etioplasts refer to etioplasts that are highly enriched in Mg-protoporphyrin IX monoester and that contain some Mg-protoporphyrin IX; they are prepared from MPE-enriched etiolated cotyledons 0).

BIOSYNTHESIS

IN VITRO

353

creased metalloporphyrin fluorescence between 580 and 620 nm was due to de novo biosynthesis rather than to incubation-induced enhancement in the fluorescence yield of the endogenous metalloporphyrins. Fortified developing chloroplasts were therefore incubated in the absence of added ALA. After 1 h of incubation, the fluorescence amplitude between 580 and 620 nm decreased below the 0 h level (Fig. 1A). This suggested that no enhancement in the fluorescence yield of the endogenous metalloporphyrins took place during incubation. The foregoing results suggested that isolated developing chloroplasts prepared from greening cotyledons and incubated with ALA and cofactors synthesized and accumulated compounds that exhibited the same fluorescence emission maxima as the metalloporphyrins synthesized by etiolated cotyledons during greening (l), Table I. In order to characterize further the metalloporphyrins synthesized by the developing chloroplasts in vitro, an attempt was made to determine the wavelength of their soret excitation maxima. This was achieved by recording excitation spectra with the emission monochromator positioned at all the metalloporphyrin emission maxima and shoulders reported in Table I for the incubated chloroplasts. The various soret excitation maxima and shoulders thus obtained were matched with the most likely corresponding emission maxima. As reported previously (1) only one soret excitation maximum at about 435 nm, was observed for the metalloporphyrins of greening cotyledons (Table I), and of developing chloroplasts monitored before incubation (Table I, Fig. 1B). The same was true for developing chloroplasts incubated for 1 hr without added ALA (Fig. 1B). However after 1 h of incubation with ALA, the developing chloroplasts exhibited several soret excitation maxima or shoulders (Table I). The matching of these soret excitation bands with the corresponding emission maxima is described below. In an attempt to detect the soret maximum of the MPE synthesized in vitro, an excitation spectrum was recorded at F-600

354

REBEIZ

nm (i.e., with the emission monochromator positioned near the short wavelength emission maximum of MPE in situ). The recorded spectrum failed, however, to exhibit a typical MPE soret excitation maximum at 424-425 nm. Instead it exhibited a novel soret excitation maximum at 430TABLE FLUORESCENCE

431 nm, an MPE shoulder at about 425 nm and a long wavelength shoulder at 434-435 nm (Fig. lB, Table I). This was in contrast to the single soret excitation maximum exhibited by MPE-enriched etioplasts (an MPE marker) at 424-425 nm (Fig. lB, Table I). Since the in situ soret I

EMISSION AND EXCITATION MAXIMA OF METALLOPORPHYRINS’ SYNTHFHZED in AND DEVELOPING CHLOROPLASTS, AND OF METALLOPORPHVRIN STANDARDS

Fraction

Greening

ET AL.

Suspension buffer or solvent

Short wavelength emission maxima (nm)

cotyledons

Vitro BY ETIOPLASTS

Soret excitation maxima” (rim)

590-592 596-597 600 603-604' 607-609 614-617

434-436 434-436 434-436 434-436 434-436 434-436

598-600 598-600 607-609 614-617' 590-592’ 598-600

424-425 425,'434-436 434-436 434-436

Reference

~MPE-enriched etioplasts Developing chloroplasts fore incubation Developing chloroplasts 1 h of incubation added ALA

Etioplasts Etioplasts bation

be-

after witk

before incubation after 1 h of incuwith added ALA

Fortified Fortified

Tris-sucrose Tris-sucrose

Fortified

Tris-sucrose

Fortified Fortified

603-604' 607-609 614-617 none 590-592 597 599-600

Trisssucrose Tris-sucrose

603-604' 607-608 614-617' Synthetic Zn-Proto E Synthetic MPE Extract of developing chloroplasts before incubation

Hexane-extracted Hexane-extracted Hexane-extracted

Extract of developing chloro plasm after 1 h of incuba tion with ALAd

Hexane-extracted

aqueous

acetone

Extract of etioplasts incubation

Hexane-extracted

aqueous

acetone

before

aqueous aqueous aqueous

acetone acetone acetone

592 596-597 596-597 599-600 602-604 607-609 613-617' 596-597 600 603 607-608 614-617 none

425,'430-431, 434-435' 424,'430,'433 430,'434 430,'434-436 none 425,'430-431 425,'432, 434436' 425,'434 435 434-436' 417 417-418 418-419 418,c 420 420-421 418,= 421-422 418,’ 422, 428E

none

1

Mg-PROTOPORPHYRIN

MONOESTER TABLE

Fraction

BIOSYNTHESIS

IN VZTRO

I-Continued

Suspension buffer or solvent

Short wavelength emission maxima (rim)

Soret excitation maximab (nm)

Extract of etioplasts after 1 h of incubation with ALA

Hexane-extracted

aqueous acetone

592 597 603-604’ 608’ 614-617’

417 418,421,‘428’ 421,428’

Synthetic Zn-Proto E Synthetic MPE Fast moving band

Methanol:acetone Methanol:acetone Methanol:acetone

(4:l v/v) (4:l v/v) (4:l v/v)

590 596-597 590c 596 602-604c 614-617’ 600 612

415-416 416-417

Slow-moving band

Methanol:acetone

355

(4:l v/v)

Reference

416, 428e 419,427’ 427

“In situ emission spectra were recorded at E-420 or E-435 nm; spectra in organic solvents were recorded at E-420 or E-425 nm. All emission spectra were recorded at an excitation bandwidth of 6 nm and an emission bandwidth of 3 nm. Excitation spectra were recorded at an excitation bandwidth of 3 nm and an emission bandwidth of 6 nm. Both emission and excitation spectra were corrected for photomultiplier and monochromator responses as well as for variation in the output of the light source. Additional information is given in the text. *The excitation maxima were recorded at or near the emission maxima or shoulders reported in the corresponding emission column. e Refers to a fluorescence emission or excitation shoulder. d The fluorescence amplitudes at the emission maxima or shoulder were higher after incubation than before.

excitation maximum of putative MPE may be obscured by the excitation bands of the metalloporphyrins emitting at longer wavelength, an attempt was made to minimize the latter interference. This was achieved by recording the excitation spectrum at an emission wavelength further removed from the interference of the emissions of the longer wavelength metalloporphyrins. Such a wavelength was found to be conveniently located at 595 nm. Although the excitation spectrum recorded at F-595 nm exhibited an enhanced MPE soret excitation shoulder at 425 nm, the main soret excitation maximum was nevertheless observed at 430-431 nm. A longer wavelength soret excitation shoulder was also detected at 434-436 nm (Fig. 1B). In an attempt to detect the soret excitations of the longer wavelength metalloporphyrins, excitation spectra were recorded with the emission monochromator positioned at longer emission wavelength, further removed from the MPE emission

maximum. It was observed that the soret excitation maximum shifted gradually to longer wavelength as the emission monochromator was moved from shorter to longer wavelength. For example at F-600, 603, 607, and 617 nm, the soret excitation maxima were observed at 430-431, 433, 434, and 434-436 nm, respectively (Fig. lB, Table I). This in turn suggested that the longer wavelength soret excitations corresponded to longer wavelength emission maxima. The assignment of soret excitations to the various emission maxima is presented in Table I. The foregoing fluorescence excitation results suggested that developing chloroplasts incubated with exogenous ALA synthesized and accumulated compounds that exhibited the in. situ short wavelength emission and soret excitation maxima of MPE. In addition the longer wavelength metalloporphyrins synthesized in vitro, exhibited distinct soret excitation maxima between 424 and 436 nm in situ (Fig. lB, Table I).

REBEIZ ET AL.

356

E-420

I 0

I

550 A

I 600

(nm)

A (nm)

FIG. 1. In situ fluorescence emission and excitation spectra of the metalloporphyrins synthesized by developing chloroplasts in uitro, A: Emission spectra elicited by the excitations (E-X) indicated. Developing chloroplasts before incubation, l/10 x oridnate (a, e); developing chloroplasts after 1 h of incubation without ALA, l/10 x ordinate (b, f); developing chloroplasts after 1 h of incubation with ALA, l/10 x ordinate (c, g); MPE-enriched etioplasts, 33.3 x ordinate (d), 10 x ordinate (h). B: Fluorescence excitation spectra recorded at the emission wavelength (F-X) indicated; scale, l/3.5 x ordinate. Developing chloroplasts before incubation, (a); developing chloroplasts after 1 h of incubation without ALA, (b); developing chloroplasts incubated for 1 h with ALA (c, e-i); MPE-enriched etioplasts, 33.3 x ordinate (d). Single headed arrows point to wavelengths of interest; double headed arrows indicate noise level; absence of double headed arrows denotes negligible noise. For the convenience of presentation the vertical arrangement of the spectra was arbitrary. Emission spectra a-c and f-g were normalized at 500 nm. Excitation spectra a-c were normalized at 380 nm. All emission spectra were recorded at an excitation bandwidth of 6 nm and an emission bandwidth of 3 nm. Excitation spectra were recorded at an excitation bandwidth of 3 nm and an emission bandwidth of 6 nm.

Determination of the amount of MPE and longer wavelength metalloporphyrins synthesized by developing chloroplasts in the dark. Further experiments were aimed at confirming that the increase in the metalloporphyrin fluorescence observed in situ after 1 h of incubation with ALA, was due to de novo synthesis. This was accomplished by extracting the putative metalloporphyrins in aqueous acetone and by determining their amount before and after incubation, with and without added ALA. The metalloporphyrins were extracted with acetone: 0.1 N NH,OH (9:l v/v) as described earlier; fluorescence emission and excitation spectra were recorded on the hexane-extracted acetone fractions (1). It

was shown earlier (1) that the extract of developing chloroplasts prepared from greening cotyledons contained MPE with an emission maximum or shoulder at 596597 nm and longer wavelength metalloporphyrins; the latter exhibited emission maxima or shoulders at 599-600, 602-604, 607-609, and 613-617 nm (Table I). It was considered that a net synthesis of any one of those metalloporphyrins is likely to be reflected by an increased fluorescence amplitude at the corresponding emission maximum. It was assumed that energy transfer between metalloporphyrins was negligible. Indeed the concentration of the metalloporphyrins in the hexane-extracted acetone fractions was so low as to preclude

Mg-PROTOPORPHYRIN

MONOESTER

any significant energy transfer between them (Table II). The emission spectra of the extracts from various treatments are presented in Fig. 2 and are summarized in Table I. They were elicited by a 420 nm excitation (near the soret excitation of MPE in hexane-extracted acetone) and by a 425 nm excitation (near the soret excitations of the longer wavelength metalloporphyrins). The emission spectra of the extracts of developing chloroplasts prepared before incubation, and after incubation in the absence of added ALA, were qualitatively very similar (Fig. 2). They exhibited emission maxima and shoulders that corresponded to MPE and longer wavelength metalloporphyrins. However the extract of the plastids incubated for 1 hr without added ALA exhibited a distinct reduction in the fluorescence amplitudes at 596 nm (MPE emission) and at 600, 603, 607, and 614 nm (longer wavelength metalloporphyrin emissions) (Fig. 2). The reduction in the fluorescence integral between 580 and 620 nm as calculated from the emission spectrum recorded at E-425 nm TABLE

II

BIOSYNTHESIS AND ACCUMULATION OF MgPROTOPORPHYRIN MONOESTER AND LONGER WAVELENGTH METALLOPORPHYRINS BY DEVELOPING DURING FIVE CONSECUTIVE CHLOROPLASTS” EXPERIMENTS Experiment

A B

C D E

MPE-equivalentb pmol/lOO mg protein Before incubation

After incubation

417 429 516 532 501

735 702 837 647 966

% increase

76 64 62 22 93

a Developing chloroplasts were prepared in a fortitied buffered medium from greening cotyledons which had been irradiated with 320 rW cm-l SC’ of cool white fluorescent light for 5 h. The plastids were incubated with 1D.l rmol of ALA for 1 h in the dark. Additional details are given in Materials and Methods. “MPE-equivalent refers to the sum of MPE + longer wavelength metalloporphyrins.

BIOSYNTHESIS

0 500

IN

E-420 550

VITRO

357

JLI

I 600

0

I

A (nm)

FIG. 2. Fluorescence emission spectra in hexaneextracted acetone of the metalloporphyrins synthesized by developing chloroplasts in uitro. The spectra were recorded at the E-X indicated; scale, l/3.5 x ordinate unless otherwise indicated. Synthetic MPE, 12 x ordinate (a) and 11 x ordinate (e); extract of developing chloroplasts before incubation (b, fl; extract after 1 h of incubation without ALA (c, g); extract after 1 h of incubation with ALA (d, h). Other symbols are as described in Fig. 1. Spectra b-d and f-h were normalized at 500 nm.

amounted to a decrease in the MPE + longer wavelength metalloporphyrins (the MPE-equivalent) of about 18%. After 1 h of incubation with ALA the fluorescence integral between 580 and 620 nm of the hexane-extracted acetone fraction increased significantly (Fig. 2). The emission spectrum recorded at E-420 nm and E-425 nm exhibited enhanced emission maxima and shoulders at 596-597 nm (MPE fluorescence), and at 600, 603, 607608, and 614-617 nm (longer wavelength metalloporphyrins) (Fig. 2). In the experiment reported in Fig. 2, the increase in the MPE-equivalent calculated from the emission spectrum recorded at E-425 nm amounted to 70%. In five consecutive experiments, the increase in MPE-equivalent after 1 h of incubation with ALA ranged from 22 to 93%. The mean increase

358

REBEIZ

amounted to 63% with a standard deviation of 23% (Table II). The foregoing results confirmed the in situ fluorescence data derived from developing chloroplasts incubated for 1 h with and without ALA. They indicated that developing chloroplasts, during incubation with ALA in the dark, synthesized net amounts of a compound that exhibited the short wavelength fluorescence emission characteristics, in aqueous acetone, of synthetic MPE. The results also indicated that longer wavelength metalloporphyrins similar to those accumulated by greening cotyledons were also synthesized in vitro. No attempts were made to carry any further the purification and characterization of the metalloporphyrins synthesized by the developing chloroplasts in vitro. Indeed, the presence of endogenous MPE and longer wavelength metalloporphyrins coupled to uncertain recoveries after TLC segregation (1) was likely to obscure qualitative and quantitative differences between treatments. Biosynthesis of MPE and longer wavelength metalloporphyrins by developing chloroplasts in the light or in the absence of cofactors. The previous experiments indicated that isolated developing chloroplasts were capable of synthesizing MPE and longer wavelength metalloporphyrins in the dark. It was pertinent to determine whether isolated developing chloroplasts were equally capable of such a biochemical activity in the light. Developing chloroplasts were therefore prepared from excised greening cotyledons that were previously irradiated for 5 h with 320 PW of cool white fluorescent light. They were incubated for 1 h with 0.033 InM ALA in the dark or under 13 PW cm-’ SK’ (about 10 fc) of cool white fluorescent light. The metalloporphyrins were extracted in aqueous acetone and the MPE-equivalent was determined. The amount of MPE-equivalent formed during the light incubations did not exceed that formed during the dark incubations. In some preparations it was significantly lower. The results of a representative experiment are reported in Table III.

ET AL.

When developing chloroplasts were isolated in unfortified Tris-sucrose, i.e., lacking cofactors, and were incubated with ALA in the absence of added cofactors no net synthesis of MPE equivalent was observed (Table III). Biosynthesis and accumulation of Mgmonoester and longer protoporphyrin wavelength metalloporphyrins by isolated etioplasts. Etiolated cotyledons and isolated etioplasts do not contain detectable amounts of endogenous MPE or other metalloporphyrins before incubation (1). Metalloporphyrins detected after incubation are therefore synthesized de novo. In order to test the metalloporphyrin biosynthetic capacity of isolated etioplasts, their fluorescence was compared to that of MPEenriched etioplasts (an MPE marker) and developing chloroplasts, after incubation in the dark with and without added ALA. The emission spectra of etioplasts, elicTABLE

III

BIOSYNTHESIS OF MPE AND LONGER WAVELENGTH METALLOPORPHYRINS BY DEVELOPING CHLOROPLASIS IN THE LIGHT OR IN THE AESENCE OF ADDED COFACTORS MPE-equivalent

pmol/lOO

mg proteins

Experiment

A

Experiment

B

’ Developing chloroplasts were isolated in a fortified buffered medium and incubated for 1 h with 0.1 rmol of ALA either in the dark or under 13 rW cm-* SK’ of cool white fluorescent light. ’ Greening cotyledons were ground in 0.5 M sucrose, 0.2 M Tris-HCl adjusted to pH 8.0 at room temperature. The centrifuged plastids were suspended in the same medium adjusted to pH 7.7 at room temperature. The unfortified plastids were incubated for 1 h with 0.1 pmol of ALA in the dark, but without added cofactors. The volume of the reaction mixture was 3 ml.

Mg-PROTOPORPHYRIN

MONOESTER

ited by 420 and 435 nm excitations, before incubation, and after 1 h of incubation without added ALA, exhibited no detectable metalloporphyrin fluorescence in the 580-620 nm wavelength region. After 1 h of incubation with ALA in the dark, the etioplasts exhibited an intense metalloporphyrin fluorescence in the 580-620 nm wavelength region indicating the net synthesis of metalloporphyrins (Fig. 3A). The emission spectra of the incubated etioplasts elicited by a 420 nm excitation and by a 435 nm excitation are presented in Fig. 3A and are summarized in Table I. They were similar to the spectra of develoninn chloronlasts incubated with ALA. They exhibited-emission maxima or shoulders that corresponded to MPE emission (599-600 nrn) and to longer wavelength emissions (603-604, metalloporphyrin

BIOSYNTHESIS

VITRO

IN

607-608, and 614-617 nm) (Fig. 3A, Table Il. However in contrast to the spectra of developing chloroplasts incubated with ALA, the emission at 590-592 nm (probably Zn-Proto) was more pronounced. In addition the main emission maximum in the spectrum recorded at E-420 nm was at 597 nm instead of at 598-600 nm (Fig. lA, 3A). The foregoing results indicated that etioplasts incubated with ALA synthesized and accumulated compounds with in situ emission properties very similar to the metalloporphyrins synthesized by etiolated cotyledons greening in distilled Hz0 (1) and by developing chloroplasts incubated with ALA (Table I). Attempts were also made to determine the wavelength of the soret excitation maxima of the metalloporphyrins synthe-

E-420

400 A (nml

359

450 A (nm)

FIG. 3. In situ fluorescence emission and excitation spectra of the metalloporphyrins synthesized by isolated etioplasts. A. Emission spectra elicited by the excftations (E-X) indicated; scale, l/8.5 x ordinate unless otherwise indicated. Etioplasts before incubation (a, e); etioplasts after 1 h of incubation without ALA (b, t); etioplasts after 1 h of incubation with ALA (c, g); MPE-enriched etioplasts, 33.3 x ordinate (d), 10 x ordinate (h). B. Fluorescence excitation spectra recorded at the emission wavelength (F-h) indicated, scale l/7 x ordinate unless otherwise indicated. Etioplasts before incubation l/3.5 x ordinate (a); etioplasts after 1 h of incubation without ALA l/3.5 x ordinate (b); etioplasts after 1 h of incubation with ALA (c, d-h). Other symbols are as described in Fig. 1. Emission spectra a-c and f-g were normalized at 500 nm. Excitation spectra a-c were normalized at 380 nm.

360

REBEIZ ET AZ,.

sized by isolated etioplasts, and to compare them with those of developing chloroplasts incubated with ALA. Excitation spectra were therefore recorded at all the emission maxima and shoulders just described as well as at other wavelengths. The various soret excitation maxima and shoulders thus obtained were matched with the most likely corresponding emission maxima (Table I). Excitation spectra recorded before incubation, and after 1 h of incubation without added ALA exhibited only a scatter excitation band with a maximum at about 444 nm; there was no evidence of any metalloporphyrin soret excitation bands (Fig. 3B). After 1 hr of incubation with ALA and in an attempt to detect the soret excitation of MPE an excitation spectrum was recorded at F-600 nm (the short wavelength emission maximum of MPE in situ). The excitation spectrum exhibited an MPEexcitation shoulder at 425 nm and a longer wavelength excitation shoulder at about 434-436 nm; however the main excitation maximum was found at about 432 nm (Fig. 3B) as was observed with developing chloroplasts incubated with ALA in the dark (Fig. lB, 3B). The similarity between the excitation spectra of etioplasts and developing chloroplasts after incubation with ALA was also observed in the wavelength position of the soret excitations of the longer wavelength metalloporphyrins. At F-595, 600, 604, and 608 nm soret excitation maxima that corresponded to longer wavelength emissions were observed at 430-431, 432, 434, and 435 nm, respectively (Fig. 3B). At F-595, 600, and 604 nm, MPE excitation shoulders were observed at 425 nm (Fig. 3B). At F-616 nm, the soret excitation maximum was reduced to a broad shoulder between 434 and 436 nm, on the short wavelength tail of the scatter excitation band (Fig. 3B). The assignment of soret excitations to the various emission maxima is presented in Table I. In addition to confirming the impression conveyed by the in situ emission data, the foregoing results indicated that etioplasts that were incubated with ALA, synthesized

and accumulated compounds with distinct metalloporphyrin emission and excitation properties in situ. The fluorescence of these metalloporphyrins was similar to the in situ fluorescence of the metalloporphyrins synthesized by isolated developing chloroplasts after incubation with ALA. Fluorescence of the extracted metalloporphyrins synthesized by isolated etioplasts. Further comparison of the metallo-

porphyrins synthesized by isolated etioplasts with those formed by greening cotyledons and developing chloroplasts was achieved after extraction of the putative metalloporphyrins in aqueous acetone. The metalloporphyrins of incubated etioplasts were therefore extracted in acetone: 0.1 N NH,OH (9:l v/v) and the fluorescence emission and excitation spectra of the hexane-extracted acetone fractions were recorded. The extracts of etioplasts processed before incubation, and after incubation without added ALA, exhibited no metalloporphyrin fluorescence between 580 and 620 nm (Table I). These results confirmed the in situ emission data as to the absence of metalloporphyrin biosynthesis in uitro, when no ALA is added to the incubated etioplasts. After 1 h of dark incubation of the etioplasts with ALA, the emission spectrum of the hexane-extracted acetone fraction exhibited a pronounced metalloporphyrin fluorescence between 580 and 620 nm, which indicated the synthesis and accumulation of metalloporphyrins during incubation. However the emission spectrum elicited by a 420 nm excitation exhibited a pronounced emission maximum at 592 nm instead of an MPE emission maximum at 596-597 nm. The emission maximum at 592 nm corresponded to the short wavelength emission maximum of synthetic Zn-Proto E (Table I). In addition, the emission spectrum exhibited a pronounced MPE shoulder at 597 nm and broad emission shoulders at 603608 and 614-617 nm (Table I). The latter corresponded to emissions of some of the longer wavelength metalloporphyrins observed in extracts of developing chloroplasts (Table I). In order to minimize the contribution of the Zn-Proto-like com-

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pound and enhance the contribution of the longer wavelength metalloporphyrins to the fluorescence integral between 580 and 620 nm, emmission spectra were also elicited by 425 and 430 nm excitations. At E-425 nm the Zn-Proto-like emission maximum at 592 nm was reduced to a shoulder while the MPE shoulder at 597 nm became the dominant emission peak in the spectrum. The broad shoulder between 603 and 608 nm was split into two emission shoulders with maxima at about 604 and 608 nm. The relative fluorescence amplitude of the 614-617 nm shoulder increased with respect to the amplitude of the MPE emission maximum at 597 nm. In the spectrum recorded at E430 nm, the Zn-Proto shoulder at 592 nm disappeared. However, the overall decrease in fluorescence amplitude made detection of spectral details difficult, except for the MPE emission maximum at 597 nm and a broad shoulder between 603 and 608 nm. A distinct, emission maximum or shoulder at 599 to 600 nm was not resolved. The foregoing emission results are summarized in Table I. They confirmed the in situ emission data which suggested the biosynthesis of metalloporphyrins by isolated etioplasts incubated with ALA. The putative metalloporphyrins exhibited the same emission maxima in aqueous acetone as MPE and some of the longer wavelength metalloporphyrins synthesized by greening cotyledons and by isolated developing chloroplasts. In addition, a compound with the short wavelength emission maximum of standard Zn-Proto E was also synthesized. We have observed, however, that both etioplasts and developing chloroplasts accumulated massive amounts of a Zn-Protolike compound, in addition to MPE, under certain incubation conditions. This was especially true when the incubation was performed in the presence of large amounts of added ALA (2-50 pmollassay), after prolonged incubation in vitro (3-18 h), in the absence of added cofactors, after excessive homogenization of the plastid preparations, after prolonged contact of the plastids with the cell sap, or after exposure to low pH values. Under these conditions the predominant emission maxima were observed at 590-592 nm in situ, and after extraction

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in organic solvents. Additional information about the acetone extracted metalloporphyrins of incubated etioplasts was derived from their excitation spectra. The latter were recorded at all emission maxima and shoulders just described; they were then compared to those reported earlier for developing chloroplasts prepared from greening cotyledons and extracted before incubation (Table I). Excitation spectra recorded before incubation, and after incubation without ALA, showed no metalloporphyrin excitation bands between 380 and 440 nm (Table I). This in turn confirmed the in situ excitation and emission data which suggested the absence of metalloporphyrin biosynthesis in vitro when no ALA was added to the incubated etioplasts (Fig. 3). After 1 h of dark incubation of the etioplasts in the presence of added ALA, the excitation spectrum recorded at F-592 nm (the short wavelength emission maximum of Zn-Proto E) exhibited a soret excitation maximum at about 417 nm identical to the soret excitation maximum of synthetic ZnProto E (Table I). At F-597 nm (the short wavelength emission maximum of MPE) the excitation spectrum exhibited a maximum at 418 nm that corresponded to the soret excitation maximum of synthetic MPE; longer wavelength excitation shoulders were also observed at about 421 and 428 nm (Table I). At F-604 nm (the short wavelength emission maximum of one of the long wavelength metalloporphyrins) the soret excitation maximum was observed at about 421 nm; an excitation shoulder was also observed at 428 nm. As reported previously (1) an identical soret excitation maximum was observed at 421 nm for one of the long wavelength metalloporphyrins present in the extract of developing chloroplasts monitored at F-604 nm before incubation (Table I). The fluorescence amplitudes at the 608 and 614-617 nm emission shoulders observed in the etioplast extract were too low to allow the recording of meaningful soret excitation data. The foregoing results are summarized in Table I. They indicated that isolated etioplasts synthesized and accumulated compounds that exhibited the short wavelength

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emission and soret excitation maxima of synthetic MPE and Zn-Proto E in hexaneextracted aqueous acetone. In addition, compounds that exhibited longer wavelength emission and excitation maxima, similar to some of the metalloporphyrins observed in extracts of developing chloroplasts, were also formed. Partial segregation of the metalloporphyrins synthesized by isolated etioplasts. Further characterization of the metalloporphyrins synthesized by etioplasts in vitro was achieved by spectrofluorometry after segregation by TLC. Fortified etioplast suspensions three times more concentrated than usual and containing about 20 mg of protein per assay were prepared. Two reaction mixtures were incubated with 0.1 prnol of ALA for 1 h in the dark. in situ fluorescence spectra exhibited the usual MPE fluorescence emission maximum at 599 nm as well as the longer wavelength emission maxima and shoulders depicted in Fig. 3A. However, the emission spectrum recorded at E-420 nm exhibited a more pronounced emission maximum at 592 nm (most probably a ZnProto emission) than depicted in Fig. 3A. At the end of incubation the reaction mixtures were combined and the metalloporphyrins were extracted in acet0ne:O.l N NH,OH (9:l v/v) and were transferred to ether. The ether extract (13 ml) was concentrated under N, to 0.35 ml was streaked on two thin layer plates of silica gel H, 5 x 20 cm and 500 pm in thickness. The plates were developed in benzene:ethylacetate: ethanol (8:2:2 v/v) in the dark at 4°C. The developed chromatograms revealed the presence of two red fluorescent bands when viewed under uv light. In some extracts that exhibited a very pronounced Zn-Proto emission maximum at 592 nm, two more red fluorescent bands were detected close to the origin. One of these bands exhibited the mobility and the fluorescence emission and excitation properties of synthetic Zn-Proto. These bands were not eluted or investigated any further. The Rf of the slow moving band (band 1) varied from 0.3 to 0.4; it was highly enriched in endogenous chlorophyllide and contained trace amounts of protochloro-

ET AL.

phyllide. A similar slow moving band was previously observed in extracts of developing chloroplasts prepared from greening cotyledons (1). The fast moving band (band 2) cochromatographed with standard MPE and ZnProto E. Its R, values varied from 0.44 to 0.51, and it was enriched in both protochlorophyllide and chlorophyllide. Putative metalloporphyrins of the fastmoving band. The fast moving band was eluted in methanol:acetone (4:l v/v) and its fluorescence emission and excitation spectra were elicited by 420 and 425 nm excitations. As usual, recoveries after chromatography were very low. The main metalloporphyrin of band 2 consisted of MPE with an emission maximum at 596 nm, and a soret excitation maximum at 416 nm (Table I). In addition it contained small amounts of Zn-Proto E as evidenced by an emission shoulder at 590 nm and very small amounts of longer wavelength metalloporphyrins that appeared as emission shoulders at 602-604 nm and 614-617 nm (Table I). Putative metalloporphyrins of the slowmoving band. The slow moving band that segregated with endogenous chlorophyllide was eluted in methanol:acetone (4:l v/v) and its fluorescence emission and excitation spectra were recorded. The fluorescence emission spectra elicited by 420 and 425 nm excitations were of low fluorescence amplitude due to poor recoveries. They exhibited a broad emission band with maxima at 600 and 612 nm (Table I). The soret excitation maxima corresponding to the 600 and 612 nm emissions were found at 419 and 427 nm, respectively (Table I). Similar long wavelength metalloporphyrins were detected in the slow moving band of extracts prepared from developing chloroplasts (l), (Table I). These metalloporphyrins were designated earlier as MP (E418, F600) and MP(E427, F612) (1). Estimation of the amounts of MPEequivalent synthesized by isolated etioplasts incubated in the dark. The amount of MPE-equivalent synthesized by isolated etioplasts, after 1 hr of incubation in the dark with ALA and cofactors, ranged from

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about 200 to 300 pmol/lOO mg of protein. These values were not corrected for the presence of minor amounts of Zn-Proto + Zn-Proto E-like compounds. Etioplasts incubated in the dark but in the absence of added ALA synthesized no MPE or other metalloporphyrins.

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biosynthetic activity (Table III). The need of exogenous cofactors was not surprising as these same cofactors were previously shown to be essential for the biosynthesis of Pchl, chlorophyll, the maintenance of the plastid membranes and the assembly of grana in vitro (2, 5-8). The individual effect of the above cofactors on the incorporation DISCUSSION of a-amino [4-‘4C]levulinic acid into MPE Developing chloroplasts and etioplasts and Pchl was reported earlier (2). The need incubated with added ALA, and the cofac- of exogenous cofactors suggested at least a tors previously shown to be required for partial dependence of the plastids on other the biosynthesis of protochlorophyll and parts of the cell for all or some of the rechlorophyll from ALA in vitro (2, 7,8), syn- quired cofactors. The requirement for exthesized and accumulated several metalogenous ALA indicated that under the loporphyrins. One metalloporphyrin ex- present incubation conditions, the isolated hibited the chromatographic mobility and plastids lacked the potential of generating fluorescence emission and excitation prop- their own ALA. It is not clear however erties of synthetic MPE (Table I). We whether this was motivated by the absence conclude that this putative compound is of the enzymes required for ALA biosynmost probabl,y MPE. thesis in the plastids or by inactivation of In addition to MPE, other compounds the enzymes during isolation of the organthat exhibited the fluorescence emission elles. The absence of a light requirement and excitation properties of the longer for the biosynthesis of metalloporphyrins in wavelength metalloporphyrins that were vitro is compatible with the assumption previously detected in greening cotyledons, that the effect of light on metalloporphyrin were also synthesized in vitro. These me- biosynthesis, that was observed in greening talloporphyrins were detected by their cotyledons (1) may be indirect. Light may short wavelength emission and soret exci- be involved in the maintenance of a contintation maxima in situ (Table I, Figs. 1, 3). uous supply of ALA and some of the reThey were also detected by their short quired cofactors in the greening cotylewavelength emission maxima and in some dons. cases by their soret excitation maxima In addition to MPE and the longer waveafter extraction in acetone (Table I, Fig. 2). length metalloporphyrins, etioplasts were However segregation by TLC of the vari- also capable of synthesizing a compound ous metalloporphyrins synthesized from with the emission and excitation characterALA by isolated etioplasts was partially istics in organic solvents of Zn-Proto (Table successful (Table I). This was due to a low I). The Zn-Proto fluorescence became very concentration of the putative long wave- noticeable only after extraction in acetone. length metalloporphyrins in the incubated It then dominated the emission spectrum of acetone fraction reetioplasts and to poor recoveries from the hexane-extracted TLC. It is concluded that isolated devel- corded at E-420 nm. In contrast, the in situ oping chloroplasts and etioplasts are simi- emission spectra of the etioplasts incubated with ALA exhibited only a Zn-Proto lar in some aspects of their metalloporphyrin biosynthetic activity to etiolated coty- emission shoulder at 590-592 nm and were ledons, greening in distilled H,O (1). dominated by the emissions of MPE and In contrast to etiolated cotyledons green- the longer wavelength metalloporphyrins (Fig. 3A). This behavior is highly suggesing in distilled HzO, the isolated plastids tive of energy transfer in situ between the required cofactors (GSH, CoASH, ATP, NAD, Mg’+, K+, Pi, methyl alcohol) and putative Zn derivative, MPE, some of the This exogenous ALA to perform their biosyn- longer wavelength metalloporphyrins. in turn suggested that the sites of synthesis thetic activity (Table III). However, light was not essential for the expression of this of these compounds were close enough to

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allow energy transfer between them in situ. It is not clear however whether the ZnProto compound and the longer wavelength metalloporphyrins were synthesized on the same or on adjacent sites. Thin layer chromatography provided additional information about the Zn-Protolike compounds synthesized in uitro. After chromatography on silica gel H, the MPE that was synthesized de novo and that was eluted with the fast moving band was slightly contaminated by a compound that exhibited the same fluorescent properties as Zn-Proto E (Table I). This was evident as an emission shoulder at 590 nm in the emission spectrum of the fast moving band recorded at E-420 nm (Table I). Since this contaminant moved with the mobility of synthetic MPE and Zn-Proto E it is suggested that it is very likely Zn-Proto E. This in turn indicated that the bulk of the compound that was present in the hexaneextracted acetone fraction and that exhibited Zn-Proto-like emission and excitation maxima did not cochromatograph with MPE. Preliminary results suggested that it may be made up to Zn-Proto of much slower chromatographic mobility than the esterified derivative. This is compatible with the observation that the enzymatic esterification of Zn-Proto is much slower than the esterification of Mg-Proto (9). Under the incubation conditions described here, developing chloroplasts were less prone to form Zn-Proto-like compounds than etioplasts. We have observed, however, that both etioplasts and developaccumulated massive hs chloroplasts amounts of Zn-Proto and Zn-Proto E-like compounds, in addition to MPE, under certain incubation conditions. The biological role of the Zn-Proto derivatives is not well understood. Neither is their relationship to the biosynthesis of Mg-Proto derivatives. They may, however, play a certain role in the chlorophyll biosynthetic chain. It has been observed consistently that in situ, the relative fluorescence yield of MPE and of the longer wavelength metalloporphyrins, that were accumulated by developing chloroplasts during incubation, was much higher than that of the metalloporphyrins that were present in the plas-

ET

AL.

tids before incubation. For example a 20fold increase in. the fluorescence integral between 580 and 620 nm was observed in situ after incubation of the developing chloroplasts with ALA (Fig. 1A). However, this increase amounted to only 70% when the actual MPE-equivalent was determined after extraction of the metalloporphyrins in acetone: 0.1 N NH,OH (9:l v/v) (Fig. 2). The reason for this discrepancy is not clearly understood. It was suggested earlier that the reactions between Proto and Pchl were membrane bound (5, 10). If such were the case, the high relative fluorescence yield of the metalloporphyrins formed in vitro may be attributed to an incomplete incorporation of the putative metalloporphyrins into the plastid membranes. That the enhanced fluorescence yield observed in situ was not due to solubilization of the endogenous metalloporphyrins during incubation, is evidenced by the low fluorescence yield observed after 1 h of incubation in the absence of added ALA (Figs. lA, 2). In spite of the high relative fluorescence yield of the long wavelength metalloporphyrins synthesized in vitro, it was not possible to correlate with any degree of certainty the emission maxima observed in situ to those observed after extraction in acetone. The multiplicity of the emission maxima observed in situ and in organic solvents as well as the lack of specific information about the solvent induced shifts of the various emitting compounds rendered such an attempt highly speculative. Finally the in situ soret excitation maximum of putative MPE in developing chloroplasts and etioplasts that were incubated with ALA, was observed at 430-432 nm; in MPE-enriched etioplasts prepared from MPE-enriched cotyledons, it was observed at 424-425 nm (Table I). It is uncertain, at this stage, whether this discrepancy is due to interference by longer wavelength metalloporphyrins in the incubated etioplasts and developing chloroplasts, or to a different MPE-protein association in the incubated plastids. REFERENCES 1. REBEIZ, C. A., MATTHEIS, J. R., SMITH, B. B., REBEIZ, C. C., AND DAYTON, D. F. (1975) Arch. Biochem. Biophys. 166,446-465.

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2. REBEIZ, C. A., AND CASTELFRANCO, P. A. (1971) Plant Physiol. 47, 24-32. 3. ELLSWORTH, R. K., AND ARONOFF, S. (1969) Arch. Biochem. Biophys. 130, 374-383. 4. KIRK, J. T. O., AND TILNEY-BASSET, R. A. E. (1967) The Plastids, p. 402, Freeman, San Francisco. 5. REBEIZ, C. A.., AND CASTELFRANCO, P. (1973) Annu. Rev. Plant Physiol. 24, 129-172. 6. REBEIZ, C. A., LARSON, S., WEIFX, T. E., AND

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7. 8. 9. 10.

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CASTELFRANCO, P. A. (1973) Plant Physiol. 51, 651-659. REBEIZ, C. A., CRANE, J. C., NISHIJIMA C., AND REBEIZ, C. C. (1973) Plant Physiol. 51,660-666. REBEIZ, C. A., AND CASTELFRANCO, P. A. (1971) Plant Physiol. 47, 33-37. RADMER, R. J., AND BOGORAD, L. (1967) Plant Physiol. 42, 463-465. REBEIZ, C. A., CRANE, J. C., AND NISHIJIMA, C. (1972) Plant Physiol. 50, 185-186.