of red chlorophyll catabolite reductase, a stroma protein ...fluorescent catabolites (pfccs) during...

Plant Physiol. (1 997) 11 5: 677-682

Partia1 Purification and Characterization of Red Chlorophyll Catabolite Reductase, a Stroma Protein lnvolved in

C h I oro p h y I I B reakdow n'

Simona Rodoni, Fabrizio Vicentini, Maja Schellenberg, Philippe Matile*, and Stefan Hortensteiner

Department of Plant Biology, University of Zürich, CH-8008 Zürich, Switzerland

Red chlorophyll (Chl) catabolite (RCC) reductase, which cata- lyzes the reaction of an intermediary Chl catabolite (RCC) in the two-step cleavage reaction of pheophorbide (Pheide) a into primary fluorescent catabolites (pFCCs) during Chl breakdown, was charac- terized and partially purified. RCC reductase activity was present at all stages of barley leaf development and even in roots. The highest specific activity was found in senescent leaves, which were used to purify RCC reductase 1000-fold. Among the remaining three proteins, RCC reductase activity was most likely associated with a 55-kD protein. RCC reductase exhibited saturation kinetics for RCC, with an apparent Michaelis constant of 0.6 mM. The reaction de- pended on reduced ferredoxin and was sensitive to oxygen. Assays of purified RCC reductase with chemically synthesized RCC as a substrate yielded three different FCCs, two of which could be identified as the stereoisomeric pFCCs from canola (Brassica napus) (pFCC-1) and sweet pepper (Capsicum annuum) (pFCC-2), respectively. In the coupled reaction with Pheide a oxidase and RCC reductase, either pFCC-1 or pFCC-2 was produced, depending on the plant species employed as a source of RCC reductase. Data from 18 species suggest that the stereospecific action of RCC reductase is uniform within a plant family.

~~~~~~~~~~~~~~

Of the enzymes that constitute the pathway of Chl breakdown in senescent leaves (for review, see Matile et al., 1996), PaO deserves special attention for severa1 reasons. It is responsible for the oxygenolytic opening of the porphyrin macrocycle and therefore could be considered to be the center piece of the catabolic system. Indeed, the retention of Chl in senescent leaves of mutant genotypes of Festuca pratensis (Vicentini et al., 1995) and Pisum sativum (Thomas et al., 1996) has been demonstrated to be due to deficient PaO activities. Its key role in Chl breakdown is also em- phasized by the strictly senescence-specific regulation of its activity (Hortensteiner et al., 1995). In contrast, other catabolic enzymes appear to be present in the (potentially) active form at a11 stages of leaf development.

PaO is located in the envelope of senescent chloroplasts (Matile and Schellenberg, 1996) and participates in a reaction in two steps by which Pheide a is converted to a linear tetrapyrrolic product (Fig. 1). As demonstrated in a pre- ceding report (Rodoni et al., 1997), in the first step PaO

' This work was supported in part by the Swiss National Science

* Corresponding author; e-mail phibusQbotinst.unizh.ch; fax Foundation.

41-1-385-42-04. 677

produces an intermediary red bilin (RCC) that is further processed by the action of a stroma protein to a colorless fluorescent catabolite (FCC). Since the structures of a red bilin similar to RCC (Engel et al., 1991) and of an FCC (Miihlecker et al., 1997) were known, the catalytic function of the stroma protein was readily identified as the reduction of the double bond in the 6-methine bridge of RCC. Reduced Fd has been demonstrated to serve as the electron donor of the stroma protein, which is now tentatively called RCC reductase (Rodoni et al., 1997).

Because of the essential contribution of RCC reductase to the cleavage of Pheide a, it seemed worth the effort to purify the enzyme, at least partially, to establish properties that may be relevant for understanding Chl breakdown in senescent leaves.

MATERIALS A N D METHODS

Plant Cul t ivat ion and lnduction of Senescence

Barley (Hordeum vulgare L. cv Express) was grown in a growth chamber and senescence was induced as described elsewhere (Rodoni et al., 1997). Etiolated barley plants were grown in permanent darkness for 10 d. Plants of various species and mature fruits of sweet pepper (Capsi- cum annuum) used for isolation of stroma proteins were either grown in the greenhouse, collected from the field, or purchased in the local market.

Preparation of Gerontoplast Membrane and Stroma Proteins

Senescent leaves were employed for the isolation and fractionation of gerontoplasts (Sitte et al., 1980) into stro- mal proteins (the S1-fraction) and membranes, as outlined by Hortensteiner et al. (1995).

Preparat ion of Soluble Protein Extracts

Soluble protein extracts for the isolation of RCC reductase protein were prepared by homogenization of barley

Abbreviations: Chl, chlorophyll; FCC, fluorescent Chl catabolite; FNR, Fd-NADPH oxidoreductase; FU, fluorescence units; Glc6P, Glc-6-phosphate; Glc6P-DH, Glc6P dehydrogenase; PaO, pheophorbide a oxygenase; Pheide, pheophorbide; RCC, red Chl catabolite.

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678 Rodoni et al. Plant Physiol. Vol. 11 5, 1997

-=I Me

Pheide a oxygenase

reduced Fd; O2 P

Pheophorbide a

Stroma protein (RCC reductasg

1,811.

RCC primary FCC (Bn-FCC-2 =Hv-FCC-4)

Figure 1. Conversion of Pheide a to a primary FCC by the consecutive and possibly channeled action of PaO and RCC reductase. The structural modifications in the respective reactions are outlined.

tissue, centrifugation at 27,50Og, and stepwise fractionation of the supernatant material by ammonium sulfate precipitation. The fraction precipitating between 50 and 80% saturation was utilized as the source of RCC reductase (Rodoni et al., 1997).

Purification of RCC Reductase

The soluble proteins from 200 g of senescent leaf tissue obtained upon precipitation between 50 and 80% of saturation with ammonium sulfate (Rodoni et al., 1997) were loaded on a column (High-Q, Bio-Rad) equilibrated with buffer no. 1 (25 mM Tricine-Tris, pH 8.0, and 10 mM DTT). After washing with buffer no. 1 (five column-volumes), the proteins were eluted stepwise with 150 mM and 300 mM NaC1, respectively, in buffer no. 1 (five column-volumes for each step). Fractions containing activity were pooled (the High-Q pool) and, after desalting on Sephadex G50 (Pharmacia) into buffer no. 2 (10 mM potassium phosphate, pH 7.0, and 10 mM DTT), were applied to a hydroxyapatite column (CHT 11, Bio-Rad) equilibrated in buffer no. 2.

The activity contained in the flow-through (the hydroxyapatite pool) was concentrated by ultrafiltration (Centricon- 50, Amicon). After the sample was loaded on a gel-filtration column (Superdex-200 HiLoad 16 / 60, Pharmacia) equilibrated with buffer no. 3 (50 mM potassium phosphate, pH 7.0, and 10 mM DTT), the column was run with the same buffer and fractions were supplemented with 10% (v/v) glycerol. For M, determination of RCC reductase, an identical run was performed employing blue dextran, catalase, albumin, ovalbumin, chymotrypsinogen A, and RNase A (each at 2 mg mL-l) as standard proteins.

For the final chromatofocusing, a Mono-P column (HR 5/20, Pharmacia) was equilibrated with buffer no. 4 (25 mM methyl piperazine hydrochloride, pH 5.7, and 10 mM DTT). Fractions from the gel-filtration step containing RCC reductase activity (Superdex 200 pool) were desalted on Sephadex-G50 into buffer no. 4 and loaded. The column was developed with a pH gradient employing 40 mL of 10-fold diluted Polybuffer 74 (Pharmacia) adjusted with hydrogen chloride to pH 4.0. Fractions were readjusted to pH 8.0 by the addition of 20% (v/v) of 0.5 M Tricine-Tris, pH 8.0. Fractions containing activity were pooled (the Mono-I’ pool).

Assay of RCC Reductase

The assay mixtures contained an equivalent of 0.5 g fresh weight of gerontoplast membrane protein and soluble proteins. The latter represented either the S1 fraction obtained upon washing of gerontoplast membranes (equivalent to 0.5 g fresh weight) or aliquots (10 pL, equivalent to about 3 4 0 0 pg fresh weight) of fractions obtained in the course of purification. To measure RCC reductase activity after native PAGE, 2-mm gel slices were cut into pieces, mixed with 10 pL of 25 mM Tricine-Tris, pH 8.0, and directly employed. Assay mixtures (40 pL) for measuring the production of FCC from Pheide a were supplemented with 0.5 mM Pheide a, 1.5 mM NADPH, 2.5 mM Glc6P, 10 milliunits of Glc6P-DH, and 10 pg of Fd. After incubation for 30 min in the dark, the reaction was stopped by adding methanol to a final concentration of 70% (v/v).

Alternatively, after chromatography on a Mono-I’ column, partially purified RCC reductase (225 ng of protein) was incubated in the presence of 10 pg of RCC, 0.6 mM NADPH, 2 mM Glc6P, 10 milliunits of Glc6P-DH, 12 pg of Fd, and 20 milliunits of FNR. The total volume was 50 pL. Additional supplements were included as outlined in Table 111. For anoxic incubations, the reaction vessels were flooded with nitrogen for 10 min to remove the air (Rodoni et al., 1997). After incubation for 1 h the reaction was terminated by the addition of 40 pL of methanol.

Methanolic extracts of the reaction mixtures were analyzed for FCCs by reverse-phase HPLC (Ginsburg et al., 1994), and detection and characterization of reaction prod- ucts were performed as described elsewhere (Rodoni et al., 1997). The activities of RCC reductase are given as integrated FUs of FCCs produced.

Cel Electrophoresis

For native gel electrophoresis, a discontinuous high-pH system (Hames, 1990) was employed. SDS-PAGE was car- ried out according to the method of Laemmli (1970), and proteins were visualized by silver-staining (Morrisey, 1981).

Protein Determination

ford (1976) using BSA as standard. Protein was measured according to the method of Brad-



Red Chlorophyll Catabolite Reductase in Chlorophyll Breakdown 679

Preparation of Fd, Pheide a, and RCC

Isolation of Fd from spinach leaves and preparation of Pheide a have been detailed elsewhere (Hortensteiner et al., 1995). RCC was chemically synthesized from methyl- Pheide a (Krautler et al., 1997).

RESULTS

Occurrence of RCC Reductase in Barley Seedlings

As shown previously (Ginsburg et al., 1994; Horten- steiner et al., 1995), the stroma protein RCC reductase, which is indispensable for in vitro conversion of Pheide a to Bn-FCC-2, is present in senescent and presenescent canola (Brassica napus) cotyledons. In barley the activity of RCC reductase was also present in senescent and mature green primary leaves (Table I). Since the activity of PaO catalyzing the first step of the conversion of Pheide a to FCC is present only in senescent leaves, the coupled assay had to be performed with washed membranes from senescent leaves. Etiolated primary leaves and, even more sur- prisingly, roots, also yielded preparations of soluble protein containing RCC reductase (Table I). The high specific activities observed in senescent and etiolated leaves, respectively, reflect the low contents of soluble proteins com- pared with green leaves.

Purification of RCC Reductase

Table I1 summarizes the purification of RCC reductase from senescent barley primary leaves. The protein was purified 1000-fold with 24% recovery of the total initial activity in a procedure including fractionated ammonium sulfate precipitation (50-SO%), anion-exchange and hydroxyapatite chromatography, gel filtration, and chromatofocusing. Unfortunately, Fd-Sepharose, which has success- fully and repeatedly been employed for the purification of Fd-dependent proteins in the past (Marquez et al., 1988; Ip et al., 1990; Morigasaki et al., 1990; Rhie and Beale, 1995), for unknown reasons was not suitable for the purification of RCC reductase (data not shown). After chromatography on a Mono-P column, the preparation still yielded about eight protein bands on a 10% polyacrylamide gel (Fig. 2A).

To identify the protein band corresponding to RCC reductase, activity was measured in the Mono-P pool after separation by native gel electrophoresis. Separation on SDS-PAGE of proteins present in gel slices containing activity demonstrated the presence of three proteins with respective molecular masses of 72, 55, and 32 kD (Fig. 2B). RCC reductase is likely identical to the 55-kD protein, since its activity positively correlated with the intensity of this protein band. This conclusion is corroborated by the elution profile of RCC reductase activity in gel-filtration ex- periments indicating an approximate molecular mass of 58 kD (data not shown). As judged from the elution profile on the Mono-P column, RCC reductase has a pI of 4.5 (not shown).

Characterization of Purified RCC Reductase

As shown previously (Rodoni et al., 1997), RCC is converted under anaerobic conditions to three different FCCs when employing an in vitro assay composed of an RCC reductase preparation, Fd, and a Fd-reducing system (NADPH, Glc6P, and Glc6P-DH). These results could be verified by using the highly purified RCC reductase from barley. Activity was strictly dependent on the presence of reduced Fd; however, supplementation of assay mixtures with FNR was necessary because the purification of RCC reductase was associated with the loss of the FNR that had been present at sufficient levels in the crude stroma protein fraction. NADPH was also necessary for FCC formation (Table 111).

The conversion of RCC to FCCs by purified RCC reductase was sensitive to oxygen. Enzymatic remova1 o€ oxygen from the assay in two different ways (ascorbate/ ascorbate oxidase and Glc/ glucose oxidase / catalase) did not pro- mote FCC production under aerobic conditions. Only when diffusion of oxygen into the assay medium was prevented by overlayering the reaction mixture with silicon oil could RCC reductase activity be partially restored (Table 111).

The conversion of RCC to FCCs by purified RCC reductase proceeded linearly for at least 1 h (data not shown), and the enzyme exhibited saturation kinetics for RCC, with an apparent K , of 0.6 mM (Fig. 3). The enzyme seemed to be specific for RCC, since similar tetrapyrrolic compounds such as biliverdin or bilirubin were not accepted as sub- strates (data not shown).

Table I . RCC reductase activities present in primary barley leaves at different stages of development and in roots

Specific activities refer to protein contents in the preparations of soluble protein employed in the assay (the precipitate between 50 and 80% saturation of ammonium sulfate). The coupled assays were r u n with washed gerontoplast membranes from senescent barley leaves completely devoid of RCC reductase activity.

Tissue Soecific Activities

FU,,,cc.4 min- mg-’ iresh wt pg- protein

Primary leaves, etiolated 1043 133 green 640 78 senescent 606 172

Roots 74 27

FCC Product Specificity 1s a Function of the Source of RCC Reductase

Pheide a is converted to a primary FCC when Pheide a oxygenase and RCC reductase are allowed to act sequen- tially in the in vitro assay. Depending on the source of RCC reductase, two types of primary FCC have so far been observed. Following the nomenclature proposed by Gins- burg and Matile (1993) they have been named Bn-FCC-2 (from B. napus), which is identical to Hv-FCC-4 (from H. vulgare), and Ca-FCC-2 (from C. annuum).

We investigated the stroma fractions from leaves of sev- era1 plant species for their ability to cqnvert Pheide a to a primary FCC (Table IV). Independent of the source of



680 Rodoni et al. Plant Physiol. Vol. 115, 1997

Table II. Partial purification of RCC reductaseActivities of RCC reductase were assessed in a coupled assay with washed membranes from senescent barley leaves as a source of PaO.

12

34

56

Purification Step

Crude extractAmmonium sulfate

precipitateHigh-Q poolHydroxyapatite

poolSuperdex-200 poolMono-P pool

Total Activity

fU^.Kc-4 mi"' '32,181.012,480.0

12,320.012,033.0

9,770.07,580.0

Protein

mg371.0

72.6

3.980.776

0.1490.084

Specific Activity

FUH*-fcc-4 f"in' ' iLg~ '87.0

172.0

3,093.015,507.0

65,568.090,238.0

Yield

%100.039.0

38.037.0

30.024.0

Purification

-fold-

2.0

36.0178.0

754.01,003.0

Pheide a oxygenase used in the coupled assay, one of thetwo FCCs was formed. In all cases the FCCs were identifiedby their retention times on HPLC using Bn-FCC-2 andCfl-FCC-2 as references. Moreover, the typical absorptionspectra with maxima at 320 and 360 nm were checked (datanot shown). Therefore, we propose a specific nomenclaturefor these primary FCCs: pFCC-1 (typified by B«-FCC-2 or

Hp-FCC-4) and pFCC-2 (typified by Ca-FCC-2), in whichthe prefix "p" indicates the primary occurrence of therespective FCC in the catabolic pathway, whereas the num-ber refers to apparent polarity on reverse-phase HPLC.

Although only a small number of plant species have beenexamined, the data suggest that RCC reductases from agiven family have the same product specificity.

B

Figure 2. Illustration of the effectiveness of consecutive purificationsteps for RCC reductase. A, SDS-PAGE protein patterns correspond-ing to purification steps 1 through 6 (see Table II). Each lane con-tained 10 /xg of protein, except lane 5 (5 fi.%) and lane 6 (3 /uig). B,Native PAGE of proteins present in the Mono-P pool (step 6). Slicingof the gel for localization of RCC reductase activity is indicated. C,Profile of RCC reductase activity in the native gel (B) along withpatterns of proteins (SDS-PAGE) present in the slices 3 through 8. Thearrow indicates the putative band of RCC reductase. Gels weresilver-stained.

DISCUSSION

It is interesting that the two enzymes that jointly catab-olize Pheide a to a primary FCC, PaO and RCC reductase,are regulated in different ways. Whereas the activity ofPaO has so far been detected exclusively in connection withChl degradation in either developing gerontoplasts orchromoplasts, RCC reductase appears to represent a con-stitutive component of plastids, even in nongreen organssuch as etiolated leaves or roots. Therefore, etioplasts andleucoplasts appear to contain the enzyme, as well as ma-ture green chloroplasts. The occurrence in nongreen tissuesraises the question of whether RCC reductase has catalyticfunctions other than Chl breakdown. Such a function wasconsidered to be the reduction of an analog of RCC, bi-liverdin, representing the first intermediate of phytochro-mobilin synthesis from heme (Weller et al., 1996). How-ever, this possibility was ruled out because RCC reductasedid not recognize biliverdin as a substrate.

RCC reductase from barley has been purified 1000-foldto near homogeneity. After four chromatographic stepseight protein bands still were present on SDS-PAGE. Na-tive gel electrophoresis reduced this to only three majorprotein bands. The possibility that purified RCC reductasecould be composed of three different subunits, comprisingdifferent protein bands under denaturing conditions, couldbe ruled out by two observations. First, gel-filtration ex-periments showed the molecular mass of the native proteinto be 58 kD, and after native PAGE, activity was mainlypresent in a gel slice that contained the protein of 55 kD inSDS gels. The nature of the other two bands is unknown.

It was shown previously that in a 50 to 80% ammoniumsulfate protein extract from barley, RCC reduction to FCCrequired reduced Fd (Rodoni et al., 1997). Reduction of Fdwas accomplished by the addition of NADPH and anNADPH-regenerating system (Glc6P and Glc6P-DH) to thein vitro assay. Although Fd was demonstrated to be essen-tial, FCCs were also synthesized in the absence of exoge-



Red Chlorophyl l Catabolite Reductase in Chlorophyll Breakdown 681

Table 111. Demonstration of the requirement of RCC reductase for reduced Fd and anoxic conditions Complete systems contained purified RCC reductase, RCC as substrate, and a blend of cofactors consisting of NADPH, Glc6P, GIc~P-DH, Fd,

and FNR. Anoxic conditions were established by flushing sealed glass vials with nitrogen. Removal of oxygen was also attempted enzymatically using ascorbate (Asc; 2 mM)/Asc-oxidase (0.2 unit) or Clc (12 mM)/Glc-oxidase (1 pg)/catalase (1 milliunit), but was partially effective only when the assay mixture was covered with silicon oil.

Assay Mixture

Complete -Fd,-FNR -NADPH, -GIc6P,-GlcGF -NADPH Complete +Asc, +Asc-oxidase +Clc, +Clc-oxidase, + +Asc, +Asc-oxidase +Glc, +Glc-oxidase, +

'-DH

catalase

catalase

Product Formation lncubation Conditions Peak 1 pFCC-1 pFCC-2

FU min- pg- protein Anoxic 4118 3235 1838 Anoxic O O O Anoxic O O O Anoxic O O O Air O O O Air O O O Air O O O Air, silicon oil 735 809 147 Air, silicon oil 1471 1029 368

nous Fd, since the enzyme preparation used contained considerable amounts of both Fd and FNR (Rodoni et al., 1997). With purified RCC reductase, the requirement of reduced Fd for the supply of electrons could be confirmed. Fd and FNR were both removed by the purification procedure, so the supplementation with these cofactors was indispensable for keeping Fd in the reduced state.

Of the three FCCs that were produced when RCC reductase was incubated in the presence of RCC (Rodoni et al., 1997), only two have been identified so far as the naturally occurring primary catabolites Bn-FCC-2 ( B . napus, pFCC-1) and Ca-FCC-2 (C. annuum, pFCC-2). Whether the third FCC represents a hitherto undiscovered natural FCC or an arti- fact is unknown. The structure of pFCC-1 (Mühlecker et al., 1997) is depicted in Figure 1. In the meantime, pFCC-2 has also been purified by employing the Pheide a-cleaving

system from C. annuum chromoplasts (Moser and Matile, 1997). The structural analysis has revealed pFCC-2 to be the stereoisomer at C1 position of pFCC-1 (W. Mühlecker and B. Krautler, personal communication).

If allowed to react jointly with PaO, RCC reductases from various sources invariably produced only one of the two pFCCs. Metabolic channeling with stereoselective access of the reductase to RCC complexed with the oxygenase could be responsible for this phenomenon. Thus, two types of RCC reductase differing in the access to the substrate, RCC, seem to occur in the plant kingdom. As judged by the pFCCs found in the 18 plant species tested so far, both forms occur in dicots as well as in monocots. Uniformity of types of pFCC has been observed at the leve1 of plant families (Table IV), but much broader screening will be

30

20

RCC-I (mM-l) o 200 400 600 2

Table IV. Types oiprimary FCCs produced b y enzyme preparations from various plant species

Gerontoplast membranes loaded with stroma protein (including RCC reductase) were isolated from senescent leaves and analyzed for FCCs produced in the assay of PaO activity, as described by Horten- steiner et al. (1995). pFCCs were identified by retention time on reverse-phase HPLC using Bn-FCC-2 and Ca-FCC-2 as references.

pFCC-1 (Bn-FCC-2 type) pFCC-2 (Ca-FCC-2 type)

RCC (PM) Figure 3. Saturation kinetics of RCC reductase. Amounts of FCC assessed after incubation of the purified enzyme in the presence of different concentrations of RCC represent the integrated FUs of all three FCCs produced. The Lineweaver-Burk plot (inset) indicates a K, value of 605 pM.

Brassicaceae B. napus Brassica oleracea Arabidopsis thaliana

Phaseolus vulgaris P. sativum

Fabaceae

3

Poaceae H. vulgare F. pratensis

Apiaceae Apium graveolens Petroselinum crispum

Asteraceae Lactuca sativa

Chenopodiaceae Beta vulgaris Spinacia oleracea

Abutilon striatum

C. annuum Lycopersicum esculentum Nicotiana rustica

Allium porrum Tulipa sp.

Malvaceae

Solanaceae

Liliaceae



682 Rodoni et al. Plant Physiol. Vol. 11 5, 1997

necessary to evaluate the taxonomic and evolutionary sig- nificance of the two types of RCC reductase.

Received April 11, 1997; accepted July 1, 1997. Copyright Clearance Center: 0032-0889/97/ 115/0677/ 06.

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