α-tocopherol synthesis by Capsicum Fruit chromoplasts

J Plant Physiol.

\-Of. 150. pp. 509-513 (1997)

-a-Tocopherol Synthesis by Capsicum Fruit Chromoplasts YOLANDA

ARANGo* and

KLAUS-PETER HEISE

Institut fur Biochemie der Pflanze der Universitat Gottingen, Untere Karspiile 2, 0-37073 Gottingen, Germany Received August 20, 1996 . Accepted September 23, 1996

Summary

Chromoplast envelope membranes isolated from mature fruits of the yellow variety of pepper (Capsicum annuum) contain lower carotenoid levels than corresponding ones from red fruits. Their pigment composition is like that of chloroplast envelopes. The present report is concerned with the pathway of a-tocopherol synthesis in this yellow variety of pepper. We report the radioactive incorporation of [methyl)4Cl-S-adenosylmethionine into metabolites of the tocopherol pathway, i.e. 2,3-dimethyl-5-phytylquinol (70-75 %), 'Y- (0-10 %) and a-tocopherol (20-25 %), even without exogenous substrates. The presence of y-tocopherol stimulates a-tocopherol synthesis by a factor of five. Another aspect of this work is concerned with the fractionation of osmotically treated chromoplasts, which resulted in a low (d :;;: 1.06 gl cm3) and a high (d:;;: 1.1 glcm3) density fraction. We show data on the distribution of marker enzymes, the capacities for tocopherol synthesis and the lipid composition of these fractions.

Key words: Capsicum fruits, chromoplasts, a-tocopherol synthesis, compartmentation. Abbreviatiom: SAM :;;: S-adenosylmethionine; TLC :;;: Thin Layer Chromatography. Introduction

In spinach leaf tissues the biosynthesis of a-tocopherol is localized in the chloroplast envelopes (Soll et al., 1980 and 1985). The proposed pathway starts with the prenylation of homogentisic acid by phytyl pyrophosphate, followed by the first methylation of 2-methyl-6-phytylquinol, subsequent cydization of 2,3-dimethyl-5-phytylquinol to y-tocopherol and the second methylation to a-tocopherol (Soll et al., 1985; Marshall et al., 1985). The methylation substrate is S-adenosylmethionine (SAM). Like certain reactions of lipid metabolism (UD P-Gal: diacylglycerol galactosyltransferase and Acyl-CoA thioesterase) the enzyme mechanisms of the above reaction sequence are predominantly (80-90 %) found in the inner envelope membrane (Soll et al., 1985; Block et al., 1983). The occurrence of the terminal steps of a-tocopherol synthesis from 2,3-dimethyl-5-phytylquinol in total chromoplast membrane fractions from Capsicum annuum fruits has been shown by Camara et al. (1982) and the methylation of y-tocopherol (y-tocopherol methyltransferase) has been characterized in more detail by D'Harlingue

* Correspondence. © 1997 by Gustav Fischer Verlag, Jena

and Camara (1985). An association of the enzyme activities with certain chromoplast membrane fractions has not been described, however, except for the fibril assembly (Deruere et al., 1994). Looking for a suitable plant material for studying a-tocopherol metabolism, we have chosen the yellow variety of Capsicum annuum fruits in which during transformation from chloroplast to chromoplast the membrane organization is paralleled by a marked increase in the a-tocopherol content from 86 to 298 Jl,g/g dry weight (Lichtenthaler, 1969), which corresponds to that in the normal fruit species (Camara et al., 1982). In contrast to the red fruit, the increased levels of a-tocopherol are accompanied by reduced amounts of carotenoids, especially of xanthophyll esters (Lichtenthaler, 1969), which facilitates the analytical procedure. In the present study, chromoplasts from the fruits of the yellow Capsicum variety were isolated on discontinuous sucrose gradients (Camara, 1983) and fractionated after hypotonic shock on continuous sucrose gradients (SchUnemann, et al., 1994) in order to isolate envelope membranes. Some interesting phenomena concerning the composition and the a-tocopherol synthesizing capacities of the resulting chromoplast membranes were summarized as well.

510

YOl.ANDA ARANGO

and KuUS-PETER HEISE

Materials and Methods

Plant material Mature Capsicum annuum fruits from the yellow variety were used. They were obtained from the local market. ~aTation ofchromoplasts

The method developed by Camara (1985) for intact chromoplast isolation was used. The chromoplast band taken at the 0.84-1.45 M interphase of the sucrose gradient was diluted with shock buffer (10 mmoUL Tricine-KOH, pH 7.9). This mixture was centrifuged twice at 100,000 &. for 30 min in a TFf 65.13 fixed angle rotor (Kontron). The pellet was resuspended with 5 mL shock buffer. The chromoplast membranes were frozen at -80·C until used.

was taken for lH NMR spectra (300 Mhz, CDCI 3), which contained typical signals as shown by Soli and Schultz (1980): 0 0.85 (12 H, Me chain), 1.22 (19 H, chain), 1.61 (3 H, C = CoMe), 2.02 (2 H, CH 2-allyl to C = C chain), 2.04 (6 H, Me quinone), 3.11 (2 H, CHrallyl to C = C-quinone and chain), 5.14 (1 H, HC = C-chain), and 6.47 (1 H, quinone). Localization of the radioactive substances was verified by radioscanning using a TLC-Scanner (LB 2723 Berthold). The spots were scrapped off the plate and placed in counting vials with 0.6 mL of Scint-Gel plus (Packard). Then, 10 mL of scintillation fluid was added (KJein and Notides, 1969). The radioactivity was determined using a Packard liquid scintillation counter. 2-Methyl-6-phytylquinol and 2,3-dimethyl-5-phytylquinol were synthesized according [0 Soli (1987).

Results ~aTation ofchromoplast SUbfracti01lS

The chromoplast suspension in shock buffer was layered on a continuous sucrose gradient (from 0.35 to 1 M sucrose) before centrifugation at 80,000 &. for 14 h in a TST 28.38 swinging rotor (Kontron). At the completion, two bands were found, which were washed twice with shock buffer, pelleted at 100,000 &. for 45 min and resuspended with shock buffer for the enzyme assays. Markn- mzyme assays

Acyl-CoA-synthetase was measured as marker for outer envelope membranes Goyard and Stumpf, 1981). UDP-Gal: diacylglycerol galactosyltransferase (Douce and Joyard, 1980) and AcyI-CoA thioesterase Goyard and Stumpf, 1980) were indicators for the inner envelope membranes, which according to Soli et al. (1985) are the main sites of a-tocopherol synthesis. Analytical mahods

The assay for a-tocopherol synthesis was based on the methylation of the membrane levels of 2-methyl-6-phytylquinol, subsequent cyclization of 2,3-dimethyl-5-phytylquinol and methylation of the resulting -y-tocopherol in the presence of S-adenosyl-L-lmethyl-14C] methionine (Du Pont. NEN, Germany). The enzymic reaction was carried out for 2 h at 25 ·C in darkness in a medium containing (0.5 mL final volume): 50 mmol/L Tricine-NaOH (pH 7.6), 0.25 moUL sorbitol, 1 mmol/L MgC12' lmethyl.14C]-SAM (59.3 mCi/mmol, 6.8 nmol), 0.095 mmol/L -y-tocopherol and chromoplast membranes with 0.4-0.6mg protein, determined according to the method of Bradford (1976). The reaction was stopped by the addition ofImL chloroform-methanol (1 :2, v/v) and ImL of 0.9% sodium chloride. After shalting with a vortex mixer, the mixrure was cleared for 4 min by centrifugation. The upper phase was removed and the lower phase was washed twice with 1 mL methanol and 1 mL 0.9 % sodium chloride. The interphase was washed once with 1 mL chloroforrnlmethanol (1: 2, v/v). The chloroform layers were withdrawn and concentrated by a stream of nitrogen. The lipid residue was streaked on a Silica gel G-25 chromatoplate and developed with light petroleurnldiethyl ether (10: 1, v/v). In order to facilitate the identmcation of the substances, an aliquot of standard a- and -y-tocopherol was added to the mixrure as carrier. The components were identified by comparing their Rf values with those of reference standards. 2,3-Dimethyl-5-phyty!quinol was identified after multiple rechromatography in different solvent systems according to Soil (1987). Afterwords, the 2,3-dimethyl-5-phyty!quinol spot was scrapped off the plate and removed with 1 mL hexane/isopropanol (3: 2, v/v) and 1 mL 0.9 % sodium chloride. The upper (hexane) phase was concentrated by a stream of nitrogen. The lipid residue

During the isolation of chromoplasts the MgCl2 was excluded from the homogenization medium because it causes an agglutination of heterogeneous membranes (Camara, 1985). Thus, in presence of MgCh, we found relatively large differences in the marker enzyme distribution for envelopes. According to previous publications (Camara et al., 1982; Camara, 1983 and 1985) the chromoplasts were enriched at the 0.84 to 1.45 mollL interface of the discontinuous sucrose gradient. No pellet was found in the gradients after centrifugation. Fractionation of osmotically treated chromoplasts on continuous sucrose gradients (Schiinemann et al., 1994) resulted in a high (d = 1.11g1cm3) and a low (d = 1.06g/cm3) density fraction, which nearly correspond to inner (d = 1.12 glcm 3) and outer (d = 1.08 glcm3) envelope membranes from spinach chloroplasts (Block et al., 1983). Furthermore, the absorption spectrum of a lipid extract from the high density fraction shown in Fig. 1 corresponded to the absorption 442 nm 471 nm

j

10

0,5

J-<

O~---+----+----+----+----*----~

200

250

300

350

400

450

500 nm

Wavelenldl

Fig. 1: Absorption spectrum of a lipid extract of a high density fraction (1.1 glcm3) from Capsicum annuum fruit chromoplast envelopes (yellow variety). The absorption in the ultraviolet region between 250 and 300 nm is mainly due to a-tocopherol.

a·Tocopherol in Capsicum

511

Table 1: Comparison of relative (%) marker enzyme and lipid composition* between membrane fractions of similar density from Capsicum annuum fruit chromoplasts (yellow variety) (A) and spinach chloroplasts** (B).

A Chromoplasts Enzyme

light (d: 1.06g1cm3)

heavy (d: 1.1 glcm3)

AcyI-CoA synthetase AcyI-CoA thioesterase UDP-GaI: diacylglycerol galactosyltransferase 2,3-dimethyl-5-phytylquinol synthesis SAM: y-tocopherol methyl transferase

55 50±5

Lipids Monogalactolipid Digalactolipid Phosphatidylcholine

B Chloroplasts (fibrils)***

outer envelope (d: 1.08g1cm3)

inner envelope (d: 1.12g1cm3)

45 50±5

81 13

19 88

50±10

50±10

12

88

55±1O

45±10

24

76

50±10

50±10

8

92

15 16 36

13 17 33

17 29 32

50 30 6

(62) (32) (5)

* Percent of total polar lipids. ** According to Block et aI. (1983). *** Isolated from red Capsicum annuum fruit chromoplasts according to Deruere et aI. (1994).

spectrum found in the spinach chloroplast envelope (Lich- exogenous supply with y-tocopherol (Fig. 3: A-C). By intenthaler et al., 1981). The absorption maxima between 400 creasing the [methyl-14C]-SAM concentration during incubaand 500 nm are typical of carotenoids while a-tocopherol ab- tion of chromoplast envelope membranes, in the presence sorbs in the ultraviolet range (292 nm). In these studies, the and absence of y-tocopherol, we were enabled to calculate the presence of prenylquinones was not investigated. In contrast K.n and V max values for the methylation of 2-methyl-6-phyto outer and inner chlo~plast envelopes, both membrane tylquinol and y-tocopherol under formation of 2,3-dimethylfractions from chromoplasts did not differ significantly with 5-phytylquinol and a-tocopherol, respectively. The K.n values respect to their marker enzyme and a-tocopherol synthesis as for SAM were 30 Jlmoi/L each and the Vmax values were 140 well as to their lipid composition (Table 1). The latter ap- and 200 pmol. h- I . mg protein-I, respectively (Fig. 3 C). peared to agree with that of outer chloroplast envelopes. After variation of the y-tocopherol concentration at constant Since we observed that the membrane fractions were quite SAM levels, a K.n for y-tocofherol of 138 JlmollL and Vmax of homogenous, we performed the following investigations on 435 pmol. h -I. mg protein-were calculated (Fig. 4). ex-tocopherol synthesis with combined membrane fractions of osmotically treated chromoplasts that we designated as envelope membranes. Discussion a.-Tocopherol synthesis of chromoplast envelopes was documented by radio TLC of their lipid extracts (Fig. 2) as well Experiments on the envelope isolation from chromoplasts as by incorporation kinetics of [methyl}4C]-SAM into inter- of Capsicum annuum fruits (yellow variety) resulted in two mediates of a-tocopherol biosynthesis. The parameters used membrane fractions with differences in density and pigment were time (Fig. 3A), membrane proteins (Fig. 3B), [methyl- patterns but without the expected differences in their marker 14]_SAM (Fig. 3 C) and y-tocopherol (Fig. 4) concentrations. enzyme and lipid composition (Table 1). Thus, both low (d = In COntrast to earlier findings from Camara et al. (1982) with 1.06 glcm3) and high (d = 1.11 glcm3) density chromoplast chromoplast membranes of Capsicum annuum fruits of the envelope membranes showed lipid compositions comparable original species, we found in the yellow varie~ a significant to those of outer envelopes from chloroplasts. Lipid patterns in~rporation (about 75 %) of [metbyl-14C]-SAM into such as in inner envelopes, however, have recently been found 2,3-dimethyl-5-phytylquinol and to a lower degree (up to in fibril structures isolated from chromoplasts of red Capsi25 %) into a-tocopherol, even in the absence of exogenous cum annuum fruits (Deruere et al., 1994). Fibril assemblies precursors like 2-methyl-6-phytylquinol and 2,3-dimetbyl-5- are a place of carotenoid overaccumulation (i.e. xanthophyll phytylquinol. Furthermore, since the supply with these inter- esters) (Lichtenthaler, 1969), responsible for the red colour, mediates did not appear to stimulate tocopherol synthesis, but appear to playa subordinate role in chromoplasts of the their complicated solubilization, which depends on the con- yellow variety (Laborde and Spurr, 1973). The conformance centration of these lipophilic substrates (Soll, 1987), could be of both chromoplast envelope membranes with respect to avoided. The intermediary formation of y-tocopherol, if their marker and tocopherol synthesizing enzymes (Table 1) measurable, was relatively low «10%). The high turnover of may be due to membrane differentiation during transformathis product was documented by the evident stimulation of tion from chloroplast to chromoplast (Lichtenthaler, 1969). cx-tocopherol production (by a factor 5) as consequence of an These changes in membrane structure and function accom-

512

YOLANDA ARANGO

and

KLAUS-PETER HEISE

B

1

I.

~1

300

~{

200

S'S .8 a

f!!a

A

400

$oI.So

1

A

100

o

A

30

60

90

120 150 180

B

II. o

2

4

me protein

Fag. 2: Radio-TLC of tocopherol metabolites obtained after incubation of Capsicum chromoplast envelope membranes of the yellow variety with labeled [methyl- 14CJ-SAM (I. without y-tocopherol and II. with y-tocopherol addition). After chromatography on silica gel G-plates with petroleumether/diethylether (10: 1; v/v), a-tocopherol (band A) and 2,3-dimethyl-5-phytylquinol (band B) were identified as predominandy radioactive products by corresponding standards.

panied by a varying turnover and availability of pigment precursors like terpenoids might further explain the significant differences in tocopherol synthesizing capacities between chloroplast (spinach leaves) and chromoplast (Capsicum annuum) envelope membranes. Thus, chromoplast envelopes showed lower rates in 2,3-dimethyl-5-phytylquinol formation (up to 140 pmol. h-I . mg protein-I) but significantly higher rates of y-tocopherol methyltransferase (200 pmol . h -I . mg protein-I) (Figs. 3 and 4) (Camara, 1985) than chloroplast envelopes with 750 and 14 pmol. h- I . mg protein-I, respectively (Soll et aI., 1985). Furthermore, in chloroplast envelope membranes these enzyme activities are preferentially associated with the inner envelope (>75 %) (SolI et aI., 1985), while in chromoplast envelopes corresponding compartmentation studies are lacking (Table 1). It was shown for the first time that Capsicum chromoplasts are able to form 14C-Iabelled 2,3-dimethyl-5-phytylquinol, and y- and a-

o

20

40

60

pM SAM

Fig.3: 14C-Incorporation from [methyl-14C]-SAM by chromoplast envelope membranes from Capsicum annuum fruits (yellow variety) into 2,3-dimethyl-5-phytylquinol (0) and a-tocopherol (_, e) in dependence on time (A), protein (B) and SAM concentration (C). The additional increase in a-tocopherol synthesis (e) has been achieved by exogenous supply with y-tocopherol. Incubation mixtures and lipid extraction were as in Materials and Methods.

tocopherol from [methyl-,4C]-SAM (Figs. 2 and 3). The accumulation of label in 2,3-dimethyl-5-phytylquinol (~70%) simultaneously supports earlier findings with intact chloroplasts (5011 et aI., 1980) and purified envelopes (5011 et aI., 1985), showing the cyclization of 2,3-dimethyl-5-phytylquinol to be the slowest step in a-tocopherol synthesis. Whether this step is controlled by inappropriate cyclization conditions in vitro {i.e. by limiting NADPH-Ievels or by missing anaerobic conditions, as supposed for carotene cyclase by Beyer et aI. (1994)) or indeed by increasing levels of 2,3-dimethyl-5-phytylquinol (Fig. 3 C) needs much further

a-Tocopherol in Capsicum 400

.~

300

~

ff

t

200

100

0,2 y

0,4

0,6

-Tocopherol (mM)

Fig.4: a-Tocopherol synthesis from [methyJ.l4C]-SAM by chromoplast envelope membranes from Capsicum annuum fruits (yellow variety) dependent on the y-tocopherol concentration. Incubation conditions and lipid extraction were as in Materials and Methods.

research. As compared with the enzyme properties of the purified y-tocopherol methyltrartsferase from the red Capsicum annuum chromoplasts (D'Harlingue artd Camara, 1985), the substrate requirements of a-tocopherol synthesis calculated for corresponding membranes from the yellow variety for SAM (K", = 30 ~mol/L) and y-tocopherol (K", = 138 ~mol/L) were higher by exactly a factor of ten. This observation explains the reduced enzyme accessibility for these substrates within the membrarte. Acknowledgements

This work was supported by grants of the «Nieders. Vorab der VW-Stifrung».

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