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Detection of protoporphyrin IX in envelope membranes of pea chloroplasts
BBRC Biochemical and Biophysical Research Communications 299 (2002) 751–754 www.academicpress.com
Detection of protoporphyrin IX in envelope membranes of pea chloroplastsq Anasuya Mohapatra and Baishnab C. Tripathy* School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India Received 22 October 2002
Abstract Envelope membranes were prepared from mature pea chloroplasts. The tetrapyrrole contents of envelope membranes were analysed. The envelope membranes of pea chloroplasts contained substantial amounts of protoporphyrin IX and trace amounts of Mg-protoporphyrin IX and its monoester in addition to protochlorophyllide. The protoporphyrin IX content of envelope membranes was 89.25 pmol (mg protein)1 . Its content in pea envelope membrane was higher than that of protochlorophyllide. The proportion of monovinyl and divinyl forms of protochlorophyllide present in pea chloroplast envelope membrane was 3:7. The significance of the presence of protoporphyrin IX in the envelope membrane is discussed in relation to plastidic Chl biosynthesis. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Chloroplast; Chlorophyll biosynthesis; Envelope membrane; Protochlorophyllide; Protoporphyrin IX
Envelope membranes of chloroplasts are involved in plastid biogenesis [1–3]. Envelope membrane plays a key role in the sorting of plastid proteins that are coded by nuclear genome [4]. Interplay of envelope, stroma, and thylakoids is shown for protoporphyrin IX (Proto IX) synthesis [5]. Envelope also plays a significant role in chlorophyll (Chl) degradation. Chlorophyllase and Mgdechelatase are present in the inner envelope membrane [6,7]. Most of the enzymes of Chl biosynthetic pathway are well characterized [8–10]. Chl is bound to pigment– protein complexes of thylakoid membranes. Chl and its precursors are essential for chloroplast development and nuclear gene expression [1–3]. Envelope membranes play a significant role in Chl biosynthesis. Of the enzymes of Chl biosynthetic pathway, protoporphyrinogen oxidase [11], certain subunits of Mg-chelatase [12], Mg-protoporphyrin IX:S-adenosyl methionine methyl transferase
q Supported by a Grant F. 3-176/2001 (SR-II) from the University Grants Commission. * Corresponding author. Fax: +91-11-6187338. E-mail address: [email protected] (B.C. Tripathy).
[13], and protochlorophyllide oxidoreductase [14] are shown to be present in the envelope membrane. Among Chl biosynthetic intermediates Pchlide and Chlide are present in envelope membranes isolated from green spinach chloroplasts [15]. In the present study it is shown that protoporphyrin IX (Proto IX) is also present in envelope membranes. Envelope membranes of matured pea chloroplasts contain substantial amounts of Proto IX. The Proto IX content of pea chloroplast envelope membranes was much higher than that of protochlorophyllide (Pchlide). The significance of the presence of Proto IX, a chlorophyll biosynthetic intermediate, in the envelope membrane is discussed in relation to plastidic Chl biosynthesis and interorganellar signalling.
Materials and methods Plant source. Pea (Pisum sativum L. cv. bonnie villile) seeds were obtained from Indian Agricultural Research Institute, New Delhi. Plant growth conditions. Pea seeds were grown in vermiculite under cool-white fluorescent light at 25 2 °C for 10 d. Isolation of purified intact pea chloroplasts. Intact chloroplasts were isolated from 60 g batches of tissue over a Percoll gradient [16]. The
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intact chloroplasts which formed a layer at the bottom were collected. The chloroplasts were washed with 4 volumes of isolation buffer and centrifuged at 5000 rpm (2500g) for 5 min to pellet the plastids. Preparation of purified envelope membranes. In order to fractionate the chloroplast into stroma, envelope, and thylakoid fractions, chloroplasts were lysed in ice bucket by suspending them for 15 min in TE buffer consisting of 10 mM Tris–HCl, pH 7.5, and 1 mM EDTA. Lysed chloroplasts were centrifuged in a sucrose step gradient [5]. The sucrose step gradient consisted of 2.5 ml of 1.2 M sucrose at the bottom, 3 ml of 1.0 M sucrose in the middle, and 3 ml of 0.46 M sucrose at the top. Sucrose solutions were prepared in 25 mM MOPS at room temperature pH of 7.5. Lysed chloroplast (1.5 ml) preparation was layered on top of the gradient and centrifuged in Sorvall, Combi plus or Beckman, XL-90 ultracentrifuge at 40,000 rpm in TH 641/SW 41 swinging bucket rotor for 1 h with slow acceleration without brake. After centrifugation envelope membranes appeared as yellow band at the interface of 0.46 and 1.0 M sucrose gradient. The stroma remained at the top of the gradient whereas the thylakoid membranes settled at the bottom. Thylakoid membranes collected from bottom of the tube were washed five times in the buffer consisting of 10 mM Tris (pH 7.5) and 0.4 M sucrose to remove contaminating envelope membranes. The envelope membranes collected from the interface of 0.46 M and 1.0 M sucrose gradient were washed once with 10 ml TE buffer and centrifuged in SW 41 rotor (Beckman) at 40,000 rpm for 30 min to pellet the envelope membranes. The envelope membranes were resuspended in 200 ll TE buffer and purified again by sedimentation on a sucrose step gradient as described before. The yellow membranes which were recovered at the interface of the 0.46 and 1.0 M layers were withdrawn and sedimented after washing with 10 ml of TE buffer and centrifuging in SW 41 rotor (Beckman) at 40,000 rpm for 30 min to pellet the envelope membranes. All the operations were done under diffuse green light to prevent phototransformation of protochlorophyllide to chlorophyllide. Separation of pigments. Pigments from envelope membranes were extracted into 80% acetone and centrifuged at 4 °C in a microfuge for 5 min at 15,000 rpm. Hexane-extracted acetone residue solvent mixture (HEAR) was prepared from 80% acetone extract by adding hexane as described before [17]. HEAR contained Chlide, Pchlide, Proto IX, and other non-esterified porphyrins. In order to take low temperature spectra (77 K) of the pigments present in HEAR, they were transferred to ether phase as follows. To the HEAR, 1/10 volume of saturated NaCl, 1/30 volume of 0.5 M phosphate buffer (pH 7.0), and 1/3 volume of diethyl ether were added, mixed thoroughly in a separating funnel, and allowed to stand and the upper ether layer was collected. The ether phase collected was washed with 0.5 M phosphate buffer (pH 7.0) thrice and used for fluorometric studies. Spectrofluorometry. Fluorescence emission and excitation spectra either at room temperature or at 77 K and fluorometric estimation of pigments were done using SLM Aminco 8000 photon counting spectrofluorometer. The channel A (sample) and channel C (reference) were adjusted to 20,000 counts per second using tetraphenylene butadiene block as standard, excited at 348 nm, and fluorescence emitted was monitored at 422 nm. The samples were excited at appropriate wavelength and emission spectra were recorded in ratio mode (channel A/C) at excitation and emission slit widths of 4 nm. Spectra were corrected for photomultiplier tube response. Excitation spectra were recorded in ratio mode (channel A/C) having slit width of excitation monochromator set at 4 nm and that of emission monochromator set at 8 nm. Using appropriate equations concentrations of Pchlide were quantified [17,18]. Low temperature spectra were taken by freezing the sample at 77 K using liquid nitrogen in a Dewar cuvette. Monovinyl and divinyl forms of Pchlide were quantified from their 77 K fluorescence excitation spectra (F 625) [19,20]. Chlorophyll and protein estimation. Chlorophyll [21] and protein [22] concentrations were measured as described in references.
Results and discussion One mg protein of washed envelope membranes had 3.08 lg Chl(ide). This is in agreement with previous studies [15]. It demonstrates that pea envelope membrane preparations were quite pure. Fluorescence emission and excitation spectra of envelope membranes At room temperature (298 K) the fluorescence emission spectra of washed pea envelope membranes suspended in TE buffer excited at 400 nm (E400 ) had a small hump at 633 nm due to Proto IX and a peak at 681 nm due to pigment–protein complexes associated with trace amounts of thylakoid membrane that marginally contaminated envelope preparation (Fig. 1). When envelope membranes were excited at 440 nm (E440 ) the emission spectrum had a hump at 638 nm due to Pchlide and a peak at 681 nm due to pigment–protein complex. Per chloroplast, the amount of envelope protein is much lower than that of thylakoids. Therefore, small amounts of envelope membrane proteins had to be isolated from a larger number of plastids, i.e., as compared to thylakoid membrane, it was an enrichment process for envelope membrane.
Fig. 1. Room temperature fluorescence emission spectra of pea chloroplast envelope membranes. Spectra were recorded in a photon counting SLM-AMINCO 8000 spectrofluorometer. Envelope membranes suspended in 10 mM TE buffer were excited at 400 and 440 nm. Emission spectra were recorded at excitation and emission slit widths of 4 nm and were corrected for photomultiplier tube response.
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Fluorescence spectra of solvent extracts of envelope membranes To quantitate the amounts of pigments associated with the envelope membranes, the pigments were extracted into 80% acetone from the envelope membranes and its pigment contents were measured. Esterified pigments were transferred from the acetone layer to the hexane layer and non-esterified pigments, i.e., Proto IX and Pchlide, partitioned to HEAR were measured by spectrofluorometry. As shown in Fig. 2, E400 of HEAR had a peak at 633 nm due to Proto IX and a peak at 674 nm due to Chlide. The E440 fluorescence emission spectrum revealed a peak at 638 nm emanating from Pchlide and a peak at 674 nm due to Chlide. To ascertain the nature of Pchlide present in envelope membrane the pigments from HEAR were transferred to ether and its low temperature (77 K) spectra were recorded as described in Materials and methods. The E440 fluorescence emission spectrum had a peak at 625 nm due to Pchlide and another peak at 673 nm due to Chlide (Fig. 3). The fluorescence excitation spectrum (F625) of the ether extract of HEAR measured at 77 K had peaks at 443 nm and 451 nm due to divinyl (DV) Pchlide [19,20] and a small shoulder at 437 nm due to the presence of small amounts of monovinyl (MV) Pchlide (Fig. 3 inset). The calculation of MV and DV Pchlide [19] revealed that 30% of total pool of Pchlide extracted from enve-
Fig. 2. Room temperature fluorescence emission spectra of HEAR preparation of pea chloroplast envelope membranes. Samples were excited at 400 and 440 nm. Emission spectra were recorded at excitation and emission slit widths of 4 nm and were corrected for photomultiplier tube response.
Fig. 3. Low temperature (77 K) fluorescence emission (E440 ) and excitation (F625) (inset) spectra of ether extract of HEAR preparation of pea envelope membranes. Low temperature spectra were taken by freezing the sample in liquid nitrogen. Emission and excitation spectra were recorded at excitation and emission slit widths of 4 nm and emission spectra were corrected for photomultiplier tube response.
lope membranes was MV Pchlide and 70% was DV Pchlide. Quantitative estimation of pigments Using appropriate equations Proto IX and Pchlide contents were quantified from room temperature fluorescence spectra of E400 and E440 [17]. Pea envelope membranes had only trace amounts of Mg-protoporphyrin IX or its monoester as detected from E420 emission spectrum (data not shown). One mg protein of washed envelope membranes had 89.34 pmol Proto IX, 45.84 pmol Pchlide and 3.08 lg Chl. One mg protein of washed thylakoid membrane had 5 pmol Proto IX, 285.83 pmol Pchlide, and 170 lg Chl. It may be argued that the presence of chlorophyll biosynthetic intermediates in the envelope membrane was due to thylakoid contamination. For calculation purpose it was assumed that all Chl(ide) present in the envelope membrane was due to thylakoid contamination (although envelope membrane contains small amounts of Chlide). In order to correct for Proto IX and Pchlide content of envelope membranes for thylakoid contamination, their content in thylakoid equivalent to 3.08 lg Chl was determined. Thylakoid containing 3.08 lg Chl had 0.09 pmol Proto IX and 5.17 pmol Pchlide. Therefore, envelope membrane had 89:34 0:09 ¼ 89:25 pmol Proto IX (mg protein)1 and 45:844 5:177 ¼ 40:667 pmol Pchlide (mg protein)1 . Thus only a maximum of 0.01% of Proto IX and 11.3% of Pchlide present in the pea envelope membrane could be due to thylakoid contamination. The results demonstrate that Proto IX is present in significant amounts in pea chloroplast envelope membranes. Its concentration in pea envelope membranes
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was higher than that of Pchlide. The presence of Pchlide and Chlide has already been shown before [15,16]. Unpublished observations in the authorÕs laboratory demonstrate the presence of only trace amounts of Proto IX and MP(E) in the envelope membranes of cucumber etioplast and spinach chloroplast. However, Pchlide was present in significant amounts in both cucumber and spinach. These demonstrate that tetrapyrrole content of chloroplast envelope membrane varies from species to species and may change during different developmental stages [23]. Envelope membrane has significant amount of protoporphyrinogen oxidase [11]. Proto IX is synthesized from its substrate protoporphyrinogen IX in the envelope membrane [5] and may play an important role in chlorophyll biosynthesis and chloroplast biogenesis. It is proposed that Chl biosynthetic intermediates may act as signalling molecules that regulate the cross-talk between plastid and nucleus [2,24]. Protoporphyrinogen IX, the precursor of Proto IX, is a water soluble tetrapyrrole that could diffuse from its site of action to the cytoplasm through chloroplast envelope membrane and subsequently migrate to the nucleus and regulate the expression of nuclear gene involved in chloroplast biogenesis. References [1] L.A. Eichacker, J. Soll, P. Lauterbach, W. Rudiger, R.R. Klein, J.E. Mullet, In vitro synthesis of chlorophyll a in the dark triggers accumulation of chlorophyll a apoproteins in barley etioplasts, J. Biol. Chem. 265 (1990) 13566–13571. [2] J. Kropat, U. Oster, W. R€ udiger, C.F. Beck, Chloroplast signalling in the light induction of nuclear HSP70 genes requires the accumulation of chlorophyll precursors and their accessibility to cytoplasm/nucleus, Plant J. 24 (2000) 523–531. [3] J.K. Hoober, L.L. Eggink, Assembly of light harvesting complex II and biogenesis of thylakoid membranes in chloroplasts, Photosynth. Res. 61 (1999) 197–215. [4] K. Keegstra, K. Cline, Protein import and routing systems of chloroplasts, Plant Cell 11 (1999) 557–570. [5] M.S. Manohara, B.C. Tripathy, Regulation of protoporphyrin IX biosynthesis by intraplastidic compartmentalization and adenosine triphosphate, Planta 212 (2000) 52–59. [6] P. Matile, S. H€ ortensteiner, H. Thomas, B. Kr€autler, Chlorophyll breakdown in senescent leaves, Plant Physiol. 112 (1996) 1403– 1409. [7] P. Matile, S. H€ ortensteiner, H. Thomas, Chlorophyll degradation, Annu. Rev. Plant Physiol. Plant Mol. Biol. 50 (1999) 67–95. [8] D. von Wettstein, S. Gough, C.G. Kannangara, Chlorophyll biosynthesis, Plant Cell 7 (1995) 1039–1057. [9] J. Papenbrock, B. Grimm, Regulatory network of tetrapyrrole biosynthesis-studies of intracellular signaling involved in metabolic and developmental control of plastids, Planta 213 (2001) 667–681. [10] C.A. Rebeiz, Analysis of Intermediates and end products of the chlorophyll biosynthetic pathway, in: A.G. Smith, M. Witty
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(Eds.), Heme, Chlorophyll, and Bilins: Methods and Protocols, Humana Press, USA, 2001, pp. 111–155. M. Matringe, J.M. Camadro, M.A. Block, J. Joyard, R. Scalla, P. Labbe, R. Douce, Localization within chloroplasts of protoporphyrinogen oxidase, the target enzyme for diphenylether like herbicides, J. Biol. Chem. 267 (1992) 4646–4651. M. Nakayama, T. Masuda, T. Bando, H. Yamagata, H. Ohta, K. Takamiya, Cloning and expression of the soybean chl H gene encoding a subunit of Mg-chelatase and localization of the Mg2þ concentration-dependent Chl H protein within the chloroplast, Plant Cell Physiol. 39 (1998) 275–284. M.A. Block, A.K. Tewari, C. Albrieux, E. Marechal, J. Joyard, The plant S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase is located in both envelope and thylakoid chloroplast membranes, Eur. J. Biochem. 269 (2002) 240–248. J. Joyard,, M. Block, B. Pineau, C. Albrieux, R. Douce, Envelope membranes from mature spinach chloroplasts contain a NADPH:protochlorophyllide reductase on the cytosolic side of the outer membrane, J. Biol. Chem. 265 (1990) 21820–21827. B. Pineau, G. Dubertret, J. Joyard, R. Douce, Fluorescence properties of the envelope membranes from spinach chloroplasts; Detection of protochlorophyllide, J. Biol. Chem. 261 (1986) 9210– 9215. N. Chakraborty, B.C. Tripathy, Involvement of singlet oxygen in 5-aminolevulinic acid-induced photodynamic damage of cucumber (Cucumis sativus L.) chloroplasts, Plant Physiol. 98 (1992) 7– 11. P. Hukmani, B.C. Tripathy, Spectrofluorometric estimation of intermediates of chlorophyll biosynthesis: protoporphyrin IX, Mg-protoporphyrin and protochlorophyllide, Anal. Biochem. 206 (1992) 125–130. C.A. Rebeiz, J.R. Matheis, B.B. Smith, C.C. Rebeiz, D.F. Dayton, Chloroplast biogenesis: biosynthesis and accumulation of Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins by greening cotyledons, Arch. Biochem. Biophys. 166 (1975) 446–465. B.C. Tripathy, C.A. Rebeiz, Chloroplast biogenesis: quantitative determination of monovinyl and divinyl Mg-protoporphyrins and protochlorophyllides by spectrofluorometry, Anal. Biochem. 149 (1985) 43–61. B.C. Tripathy, C.A. Rebeiz, Chloroplast biogenesis: demonstration of monovinyl and divinyl monocarboxylic routes of chlorophyll biosynthesis in higher plants, J. Biol. Chem. 261 (1986) 13556–13564. R.J. Porra, W.A. Thompson, P.E. Kriedemann, Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy, Biochim. Biophys. Acta 975 (1989) 384–394. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275. X. Barthelemy, G. Bouvier, A. Radunz, S. Docquier, G.H. Schimd, F. Franck, Localization of NADPH-protochlorophyllide reductase in plastids of barley at different greening stages, Photosynth. Res. 64 (2000) 63–76. N. Mochizuki, J.A. Brusslan, R. Larkin, A. Nagatani, J. Chory, Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction, Proc. Natl. Acad. Sci. USA 98 (2001) 2053– 2058.
Detection of protoporphyrin IX in envelope membranes of pea chloroplastsq Anasuya Mohapatra and Baishnab C. Tripathy* School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India Received 22 October 2002
Abstract Envelope membranes were prepared from mature pea chloroplasts. The tetrapyrrole contents of envelope membranes were analysed. The envelope membranes of pea chloroplasts contained substantial amounts of protoporphyrin IX and trace amounts of Mg-protoporphyrin IX and its monoester in addition to protochlorophyllide. The protoporphyrin IX content of envelope membranes was 89.25 pmol (mg protein)1 . Its content in pea envelope membrane was higher than that of protochlorophyllide. The proportion of monovinyl and divinyl forms of protochlorophyllide present in pea chloroplast envelope membrane was 3:7. The significance of the presence of protoporphyrin IX in the envelope membrane is discussed in relation to plastidic Chl biosynthesis. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Chloroplast; Chlorophyll biosynthesis; Envelope membrane; Protochlorophyllide; Protoporphyrin IX
Envelope membranes of chloroplasts are involved in plastid biogenesis [1–3]. Envelope membrane plays a key role in the sorting of plastid proteins that are coded by nuclear genome [4]. Interplay of envelope, stroma, and thylakoids is shown for protoporphyrin IX (Proto IX) synthesis [5]. Envelope also plays a significant role in chlorophyll (Chl) degradation. Chlorophyllase and Mgdechelatase are present in the inner envelope membrane [6,7]. Most of the enzymes of Chl biosynthetic pathway are well characterized [8–10]. Chl is bound to pigment– protein complexes of thylakoid membranes. Chl and its precursors are essential for chloroplast development and nuclear gene expression [1–3]. Envelope membranes play a significant role in Chl biosynthesis. Of the enzymes of Chl biosynthetic pathway, protoporphyrinogen oxidase [11], certain subunits of Mg-chelatase [12], Mg-protoporphyrin IX:S-adenosyl methionine methyl transferase
q Supported by a Grant F. 3-176/2001 (SR-II) from the University Grants Commission. * Corresponding author. Fax: +91-11-6187338. E-mail address: [email protected] (B.C. Tripathy).
[13], and protochlorophyllide oxidoreductase [14] are shown to be present in the envelope membrane. Among Chl biosynthetic intermediates Pchlide and Chlide are present in envelope membranes isolated from green spinach chloroplasts [15]. In the present study it is shown that protoporphyrin IX (Proto IX) is also present in envelope membranes. Envelope membranes of matured pea chloroplasts contain substantial amounts of Proto IX. The Proto IX content of pea chloroplast envelope membranes was much higher than that of protochlorophyllide (Pchlide). The significance of the presence of Proto IX, a chlorophyll biosynthetic intermediate, in the envelope membrane is discussed in relation to plastidic Chl biosynthesis and interorganellar signalling.
Materials and methods Plant source. Pea (Pisum sativum L. cv. bonnie villile) seeds were obtained from Indian Agricultural Research Institute, New Delhi. Plant growth conditions. Pea seeds were grown in vermiculite under cool-white fluorescent light at 25 2 °C for 10 d. Isolation of purified intact pea chloroplasts. Intact chloroplasts were isolated from 60 g batches of tissue over a Percoll gradient [16]. The
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A. Mohapatra, B.C. Tripathy / Biochemical and Biophysical Research Communications 299 (2002) 751–754
intact chloroplasts which formed a layer at the bottom were collected. The chloroplasts were washed with 4 volumes of isolation buffer and centrifuged at 5000 rpm (2500g) for 5 min to pellet the plastids. Preparation of purified envelope membranes. In order to fractionate the chloroplast into stroma, envelope, and thylakoid fractions, chloroplasts were lysed in ice bucket by suspending them for 15 min in TE buffer consisting of 10 mM Tris–HCl, pH 7.5, and 1 mM EDTA. Lysed chloroplasts were centrifuged in a sucrose step gradient [5]. The sucrose step gradient consisted of 2.5 ml of 1.2 M sucrose at the bottom, 3 ml of 1.0 M sucrose in the middle, and 3 ml of 0.46 M sucrose at the top. Sucrose solutions were prepared in 25 mM MOPS at room temperature pH of 7.5. Lysed chloroplast (1.5 ml) preparation was layered on top of the gradient and centrifuged in Sorvall, Combi plus or Beckman, XL-90 ultracentrifuge at 40,000 rpm in TH 641/SW 41 swinging bucket rotor for 1 h with slow acceleration without brake. After centrifugation envelope membranes appeared as yellow band at the interface of 0.46 and 1.0 M sucrose gradient. The stroma remained at the top of the gradient whereas the thylakoid membranes settled at the bottom. Thylakoid membranes collected from bottom of the tube were washed five times in the buffer consisting of 10 mM Tris (pH 7.5) and 0.4 M sucrose to remove contaminating envelope membranes. The envelope membranes collected from the interface of 0.46 M and 1.0 M sucrose gradient were washed once with 10 ml TE buffer and centrifuged in SW 41 rotor (Beckman) at 40,000 rpm for 30 min to pellet the envelope membranes. The envelope membranes were resuspended in 200 ll TE buffer and purified again by sedimentation on a sucrose step gradient as described before. The yellow membranes which were recovered at the interface of the 0.46 and 1.0 M layers were withdrawn and sedimented after washing with 10 ml of TE buffer and centrifuging in SW 41 rotor (Beckman) at 40,000 rpm for 30 min to pellet the envelope membranes. All the operations were done under diffuse green light to prevent phototransformation of protochlorophyllide to chlorophyllide. Separation of pigments. Pigments from envelope membranes were extracted into 80% acetone and centrifuged at 4 °C in a microfuge for 5 min at 15,000 rpm. Hexane-extracted acetone residue solvent mixture (HEAR) was prepared from 80% acetone extract by adding hexane as described before [17]. HEAR contained Chlide, Pchlide, Proto IX, and other non-esterified porphyrins. In order to take low temperature spectra (77 K) of the pigments present in HEAR, they were transferred to ether phase as follows. To the HEAR, 1/10 volume of saturated NaCl, 1/30 volume of 0.5 M phosphate buffer (pH 7.0), and 1/3 volume of diethyl ether were added, mixed thoroughly in a separating funnel, and allowed to stand and the upper ether layer was collected. The ether phase collected was washed with 0.5 M phosphate buffer (pH 7.0) thrice and used for fluorometric studies. Spectrofluorometry. Fluorescence emission and excitation spectra either at room temperature or at 77 K and fluorometric estimation of pigments were done using SLM Aminco 8000 photon counting spectrofluorometer. The channel A (sample) and channel C (reference) were adjusted to 20,000 counts per second using tetraphenylene butadiene block as standard, excited at 348 nm, and fluorescence emitted was monitored at 422 nm. The samples were excited at appropriate wavelength and emission spectra were recorded in ratio mode (channel A/C) at excitation and emission slit widths of 4 nm. Spectra were corrected for photomultiplier tube response. Excitation spectra were recorded in ratio mode (channel A/C) having slit width of excitation monochromator set at 4 nm and that of emission monochromator set at 8 nm. Using appropriate equations concentrations of Pchlide were quantified [17,18]. Low temperature spectra were taken by freezing the sample at 77 K using liquid nitrogen in a Dewar cuvette. Monovinyl and divinyl forms of Pchlide were quantified from their 77 K fluorescence excitation spectra (F 625) [19,20]. Chlorophyll and protein estimation. Chlorophyll [21] and protein [22] concentrations were measured as described in references.
Results and discussion One mg protein of washed envelope membranes had 3.08 lg Chl(ide). This is in agreement with previous studies [15]. It demonstrates that pea envelope membrane preparations were quite pure. Fluorescence emission and excitation spectra of envelope membranes At room temperature (298 K) the fluorescence emission spectra of washed pea envelope membranes suspended in TE buffer excited at 400 nm (E400 ) had a small hump at 633 nm due to Proto IX and a peak at 681 nm due to pigment–protein complexes associated with trace amounts of thylakoid membrane that marginally contaminated envelope preparation (Fig. 1). When envelope membranes were excited at 440 nm (E440 ) the emission spectrum had a hump at 638 nm due to Pchlide and a peak at 681 nm due to pigment–protein complex. Per chloroplast, the amount of envelope protein is much lower than that of thylakoids. Therefore, small amounts of envelope membrane proteins had to be isolated from a larger number of plastids, i.e., as compared to thylakoid membrane, it was an enrichment process for envelope membrane.
Fig. 1. Room temperature fluorescence emission spectra of pea chloroplast envelope membranes. Spectra were recorded in a photon counting SLM-AMINCO 8000 spectrofluorometer. Envelope membranes suspended in 10 mM TE buffer were excited at 400 and 440 nm. Emission spectra were recorded at excitation and emission slit widths of 4 nm and were corrected for photomultiplier tube response.
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Fluorescence spectra of solvent extracts of envelope membranes To quantitate the amounts of pigments associated with the envelope membranes, the pigments were extracted into 80% acetone from the envelope membranes and its pigment contents were measured. Esterified pigments were transferred from the acetone layer to the hexane layer and non-esterified pigments, i.e., Proto IX and Pchlide, partitioned to HEAR were measured by spectrofluorometry. As shown in Fig. 2, E400 of HEAR had a peak at 633 nm due to Proto IX and a peak at 674 nm due to Chlide. The E440 fluorescence emission spectrum revealed a peak at 638 nm emanating from Pchlide and a peak at 674 nm due to Chlide. To ascertain the nature of Pchlide present in envelope membrane the pigments from HEAR were transferred to ether and its low temperature (77 K) spectra were recorded as described in Materials and methods. The E440 fluorescence emission spectrum had a peak at 625 nm due to Pchlide and another peak at 673 nm due to Chlide (Fig. 3). The fluorescence excitation spectrum (F625) of the ether extract of HEAR measured at 77 K had peaks at 443 nm and 451 nm due to divinyl (DV) Pchlide [19,20] and a small shoulder at 437 nm due to the presence of small amounts of monovinyl (MV) Pchlide (Fig. 3 inset). The calculation of MV and DV Pchlide [19] revealed that 30% of total pool of Pchlide extracted from enve-
Fig. 2. Room temperature fluorescence emission spectra of HEAR preparation of pea chloroplast envelope membranes. Samples were excited at 400 and 440 nm. Emission spectra were recorded at excitation and emission slit widths of 4 nm and were corrected for photomultiplier tube response.
Fig. 3. Low temperature (77 K) fluorescence emission (E440 ) and excitation (F625) (inset) spectra of ether extract of HEAR preparation of pea envelope membranes. Low temperature spectra were taken by freezing the sample in liquid nitrogen. Emission and excitation spectra were recorded at excitation and emission slit widths of 4 nm and emission spectra were corrected for photomultiplier tube response.
lope membranes was MV Pchlide and 70% was DV Pchlide. Quantitative estimation of pigments Using appropriate equations Proto IX and Pchlide contents were quantified from room temperature fluorescence spectra of E400 and E440 [17]. Pea envelope membranes had only trace amounts of Mg-protoporphyrin IX or its monoester as detected from E420 emission spectrum (data not shown). One mg protein of washed envelope membranes had 89.34 pmol Proto IX, 45.84 pmol Pchlide and 3.08 lg Chl. One mg protein of washed thylakoid membrane had 5 pmol Proto IX, 285.83 pmol Pchlide, and 170 lg Chl. It may be argued that the presence of chlorophyll biosynthetic intermediates in the envelope membrane was due to thylakoid contamination. For calculation purpose it was assumed that all Chl(ide) present in the envelope membrane was due to thylakoid contamination (although envelope membrane contains small amounts of Chlide). In order to correct for Proto IX and Pchlide content of envelope membranes for thylakoid contamination, their content in thylakoid equivalent to 3.08 lg Chl was determined. Thylakoid containing 3.08 lg Chl had 0.09 pmol Proto IX and 5.17 pmol Pchlide. Therefore, envelope membrane had 89:34 0:09 ¼ 89:25 pmol Proto IX (mg protein)1 and 45:844 5:177 ¼ 40:667 pmol Pchlide (mg protein)1 . Thus only a maximum of 0.01% of Proto IX and 11.3% of Pchlide present in the pea envelope membrane could be due to thylakoid contamination. The results demonstrate that Proto IX is present in significant amounts in pea chloroplast envelope membranes. Its concentration in pea envelope membranes
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was higher than that of Pchlide. The presence of Pchlide and Chlide has already been shown before [15,16]. Unpublished observations in the authorÕs laboratory demonstrate the presence of only trace amounts of Proto IX and MP(E) in the envelope membranes of cucumber etioplast and spinach chloroplast. However, Pchlide was present in significant amounts in both cucumber and spinach. These demonstrate that tetrapyrrole content of chloroplast envelope membrane varies from species to species and may change during different developmental stages [23]. Envelope membrane has significant amount of protoporphyrinogen oxidase [11]. Proto IX is synthesized from its substrate protoporphyrinogen IX in the envelope membrane [5] and may play an important role in chlorophyll biosynthesis and chloroplast biogenesis. It is proposed that Chl biosynthetic intermediates may act as signalling molecules that regulate the cross-talk between plastid and nucleus [2,24]. Protoporphyrinogen IX, the precursor of Proto IX, is a water soluble tetrapyrrole that could diffuse from its site of action to the cytoplasm through chloroplast envelope membrane and subsequently migrate to the nucleus and regulate the expression of nuclear gene involved in chloroplast biogenesis. References [1] L.A. Eichacker, J. Soll, P. Lauterbach, W. Rudiger, R.R. Klein, J.E. Mullet, In vitro synthesis of chlorophyll a in the dark triggers accumulation of chlorophyll a apoproteins in barley etioplasts, J. Biol. Chem. 265 (1990) 13566–13571. [2] J. Kropat, U. Oster, W. R€ udiger, C.F. Beck, Chloroplast signalling in the light induction of nuclear HSP70 genes requires the accumulation of chlorophyll precursors and their accessibility to cytoplasm/nucleus, Plant J. 24 (2000) 523–531. [3] J.K. Hoober, L.L. Eggink, Assembly of light harvesting complex II and biogenesis of thylakoid membranes in chloroplasts, Photosynth. Res. 61 (1999) 197–215. [4] K. Keegstra, K. Cline, Protein import and routing systems of chloroplasts, Plant Cell 11 (1999) 557–570. [5] M.S. Manohara, B.C. Tripathy, Regulation of protoporphyrin IX biosynthesis by intraplastidic compartmentalization and adenosine triphosphate, Planta 212 (2000) 52–59. [6] P. Matile, S. H€ ortensteiner, H. Thomas, B. Kr€autler, Chlorophyll breakdown in senescent leaves, Plant Physiol. 112 (1996) 1403– 1409. [7] P. Matile, S. H€ ortensteiner, H. Thomas, Chlorophyll degradation, Annu. Rev. Plant Physiol. Plant Mol. Biol. 50 (1999) 67–95. [8] D. von Wettstein, S. Gough, C.G. Kannangara, Chlorophyll biosynthesis, Plant Cell 7 (1995) 1039–1057. [9] J. Papenbrock, B. Grimm, Regulatory network of tetrapyrrole biosynthesis-studies of intracellular signaling involved in metabolic and developmental control of plastids, Planta 213 (2001) 667–681. [10] C.A. Rebeiz, Analysis of Intermediates and end products of the chlorophyll biosynthetic pathway, in: A.G. Smith, M. Witty
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