Compositional heterogeneity of protochlorophyllide ester in etiolated leaves of higher plants

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 220, No. 1, January, pp. 286-292, 1983

Compositional Heterogeneity of Protochlorophyllide Ester in Etiolated Leaves of Higher Plants YUZO SHIOI’ Division

of Biology,

Miyazaki

AND TSUTOMU

Medical

College,

Kiyotake,

SASA Miyazaki

889-16, Japan

Received August 2, 1982

The formation and degradation of protochlorophyllide esters, i.e., protochlorophylls, were studied in etiolated leaves of kidney bean in relation to their aging. By the sensitive analysis of the pigments using high-performance liquid chromatography, the presence of four protochlorophylls esterified with phytol, tetrahydrogeranylgeraniol (THGG), dihydrogeranylgeraniol (DHGG), and geranylgeraniol (GG) was detected in kidney bean grown in the dark. Similar components were also observed in the etiolated seedlings of cucumber, sunflower, and corn. The content of each protochlorophyll species changed with the plant species and age of plants. In the case of kidney bean, the content of protochlorophyll phytol reached a maximal level at 9 days, then decreased rapidly during the subsequent development, in spite of the total protochlorophyll content remaining unchanged. In contrast to the degradation of protochlorophyll phytol, the other three protochlorophylls esterified with THGG, DHGG, and GG accumulated. These results may indicate that (i) protochlorophyll phytol is formed from the first esterified protochlorophyll GG through the next three hydrogenation steps as in the case of chlorophyll a phytol formation; (ii) the esterification reaction stops at 9 days and then reaction proceeds in sequence in the reverse direction, leading to the dehydrogenation of the alcohol moiety of protochlorophyll phytol to protochlorophylls THGG, DHGG, and GG.

Etioplasts of the leaves of dark grown higher plants contain chlorophyll precursor pigments, Pchlide’ (Mg-Z-vinyl, 4ethylpheoporphyrin a5) and esterified Pchlide termed Pchl (1). Recently, some reports have appeared (2) on the occurrence of divinyl Pchlide (Mg-2, 4-divinylpheoporphyrin as) in addition to usual Pchl(ide)3 in the etiolated higher plant tissues. On exposure to light, Pchlide having absorption maximum at 650 nm is con’ To whom reprint requests should be addressed. * Abbreviations used: Pchlide, protochlorophyllide; Pchl, protochlorophyll; Chl, chlorophyll; Chlide, chlorophyllide; HPLC, high-performance liquid chromatography; GG, geranylgeraniol; DHGG, dihydrogeranylgeraniol; THGG, tetrahydrogeranylgeraniol. 3 Pchl(ide) refers to the mixture of protochlorophyllide and protochlorophyllide ester. 0003-9861/83/010286-07$03.00/O Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved

verted to Chlide (3, 4) which then undergoes esterification to yield Chl a (5). Several reports (6-8) described the formation of Chl a esterified with different CaO alcohols in the last step of Chl a biosynthetic pathway. Riidiger and co-workers (8-10) demonstrated that the esterification is not a one-step reaction catalyzed by the enzyme chlorophyllase, but Chl a esterified with GG is formed first by the action of enzyme “Chl synthetase” and then is transformed to Chl a esterified with phytol through three hydrogenation steps. Esterification of Pchlide in etiolated leaves of higher plants proceeds during a dark reaction and the esterifying alcohol is generally considered to be phytol, except for GG of Pchl in barley grown in the dark reported by Liljenberg (11). On the other hand, Pchl from inner seed coats of pump286

COMPOSITIONAL

HETEROGENEITY

OF

287

PROTOCHLOROPHYLL

lated leaves of kidney bean. These results were compared with the esterification steps of Chl a and the probable sequence of the biosynthesis and degradation of Pchl species is discussed. MATERIALS

0

20 ProtochlorOPhYlI

40

60

(Dmoles)

FIG. 1. Calibration curve of Pchl obtained after HPLC of isolated pigment. The ordinate presents the areas of Pchl phytol peaks (V cm-*) determined by fluorescence measurements. The abscissa indicates picomol of pigment used. The pigment was eluted with 100% methanol at a flow rate of 1.5 ml/min at 4O’C. Sample volume, 5 ~1. Pchl was detected by fluorescence measurements using excitation and emission wavelengths at 437 and 638 nm, respectively.

kin is esterified with different alcohols (12). Previously, we reported the separation and identification by HPLC of at least 1’7 kinds of Pchls esterified with different alcohols from inner seed coats of three Cucurbitaceae (13). These pigments appeared in relation to maturation of fruits and some of the pigments are considered, as pointed out by Jones (14), to be formed during the degradation process. In the course of our study on the Chl analysis by HPLC, we found the presence of four Pchls esterified with different CzO alcohols in the extract of etiolated leaves of kidney bean (15). At present, however, there is little information on the physiological significance of these pigments and details of the biosynthesis or degradation process involved. The recently developed analytical technique of HPLC provides a useful tool for the resolution of these problems, because of its marked advantages, e.g., high resolving power, sensitivity, and reproducibility. In this paper, data are presented on the quantitative analysis of the Pchl species with special relation to aging of the etio-

AND

METHODS

Plant materials and culture conditicms. Kindey bean seeds (Phaseolus vulgaris L.) were obtained from Asahi Noen Seed Co. Ltd. (Aichi, Japan). Seeds of cucumber (Cucumis sativus L.), sunflower (Helianthus annuus L.), and corn (Zea mays L.) were purchased from Miyazaki Shubyo (Miyazaki, Japan). Seeds of each plant were germinated in wet vermiculite at 26°C in the dark for 1 to 17 days. Extraction and pwijication of pigments. One to three grams of the primary etiolated leaves removed from the seedlings after various culture periods was ground with a small volume of cold absolute acetone.

5-day-Old

4

g-day-old

l4-day-old

17.day-old

P.

1

I

I

0

5

10 Retention

FIG. 2. El&ion

I5 time

I

I

20

25

1

(mln)

profiles of Pchls isolated from kidney bean by HPLC. Pigments were extracted and purified from etiolated bean leaves at the indicated age. Chromatographic conditions of HPLC are the same as in Fig. 1.1, Pchl GG; 2, Pchl DHGG; 3, Pchl THGG; 4, Pchl phytol.

288

SHIOI

AND

TABLE HPLC

CHROMATOGRAPHIC

Kidney Peak

No. 1

2 3 4

PROPERTIES

OF Pchls

SASA

I

ISOLATED

FROM ETIOLATED

(Phaseolus vulgaris)

bean

hia

kfb

12.15 14.09 16.68 19.71

6.32 7.49 9.05 10.87

Pumpkin UC 0.58 0.69 0.83 1.00

LEAVES

(Cucurbita

GG DHGG THGG phytol

BEAN

moschata)d

tR

k’

a

12.24 14.08 16.77 19.77

6.37 7.48 9.10 10.91

0.58 0.69 0.83 1.00

Pchls Pchl Pchl Pchl Pchl

OF KIDNEY

Note. Chromatographic

conditions are the same as in Fig. 1. n Retention time in min. b k’, the capacity factor, is given by k’ = (tR - to)/&,, where tR and b are the retention and unretained solute in given system, respectively, ’ (Y, the ratio of capacitv factor. is calculated bv k’ l/k’ phytol. d Data from Ref. (I3). Extraction was repeated three times in order to ensure maximum extraction. The acetone extractions were combined and concentrated in oucuo at less than 35°C to about 2 ml and finally diluted to 5 ml with absolute acetone for the spectrophotometric analyses of total Pchl(ide). All operations up to extraction were performed under low-intensity green safelight. For the HPLC analysis, pigments were purified according to the method of Omata and Murata (16) as a mixture of Pchls by repeated column chromatography on DEAE-Sepharose CL-6B (0.9 X 5 cm) in order to show the entire elution profiles of Pchl species (cf. Ref. (15)). When crude extract was used, the major Pchlide is cochromatographed and overlapped with the fast-eluting Pchl components. For the analysis of esterifying alcohols, each pigment was obtained pure from ll- to ll-day etiolated leaves of kidney bean by repeated thin-layer chromatography with cellulose plates and solvent mixture of methanol-methylene chloride-water (100:18:20, v/v/v) (17) and semipreparative HPLC under the conditions described in the legend to Fig. 1, except for the injection volume.

Determination hols. The content

of Pchls and their esterifying alco-

of total Pchhide) including divinyl derivatives was determined spectrophotometrically using a Shimadzu UV-300 (Kyoto, Japan) and the extinction coefficients of Pchl reported by Koski and Smith (18). Pchlide concentration was calculated by subtraction of total Pchls from total Pchl(ide). Separation, identification and quantitative analysis of Pchl species were carried out with HPLC using Model LC-3A system (Shimadzu) and Du Pont ODS column (250 X 4.6 mm) as described previously (13, 15). Pigments were detected fluorometrically using a Hitachi fluorescence spectrophotometer, Model 650-60 (Tokyo, Japan). Chromatographic conditions are described in the legend to Fig. 1. The calibration curve of Pchl

times

of retained

phytol” is shown in Fig. 1. The response of Pchl phytol to fluorescence detection was linear over the wide concentration range of 2-50 pmol and can be extended to 1 nmol. The quantitative analysis requires a minimum of 2 pmol so that this system is sufficient to detect and quantify the pigments within a small experimental error. Concentration of each Pchl species was calculated using the standard curve for Pchl phytol. The esterifying alcohols were analyzed by gas chromatography after saponification of the purified pigments. Each Pchl fraction was obtained pure after repeated cellulose thin-layer chromatography followed by semipreparative HPLC noted above. The gas chromatographic analysis was performed according to our previous method (15). The injection was carried out using a solvent cut system at 170°C isothermal oven temperature (injection temperature, 190°C). The column used was a silica capillary (12 m x 0.24 m/i.d.) packed with polyethyleneglycol20M on Uniport B and carrier gas nitrogen (60 mllmin). Dnder the conditions used, retention times for the alcohols (in min) were phytol, 13.02; THGG, 19.22; DHGG, 22.83; and GG, 29.03. RESULTS

Separation and IdentiJication Esters

of Pchlide

Figure 2 shows the HPLC elution profiles of the purified Pchls obtained from four representative culture periods of kidney bean. At least four Pchls with retention times 12.15,14.09,16.88, and 19.71 min 4 Pchl GG, Pchl DHGG, tol mean Pchl esterified

Pchl THGG, and Pchl phywith the respective alcohol.

COMPOSITIONAL TABLE

HETEROGENEITY

II

GAS CHROMATOGRAPHIC ANALYSIS OF THE ESTERIFYING ALCOHOLS OF Pchls ISOLATED FROM KIDNEY BEAN Esterifying

COIW3P

Peak No. (Fig. 2) 1 2 3 4

tration (nmol) 123 89 121 137

GG

DHGG

101

78.3

alcohol THGG

(nmol) Phytol

96.6 -

125

Note. Pigments were extracted and highly purified from ll- to 14-day etiolated leaves of kidney bean. Extraction and purification of pigments and gas chromatographic conditions are described in the text. The recovery of alcohols after saponification was approximately 91 to 80%.

were separated from all culture stages of samples by HPLC, although the content of each component varied with age. In situ fluorescence spectroscopic analysis of the HPLC elute of these four peaks showed identical excitation maximum at 435 nm for Soretband, indicating that four pigments are typical monovinyl Pchl. Furthermore, the HPLC properties, retention times, capacity factors (k’), and ratios of capacity factor (a) of these pigments from peaks 1 to 4 were coincident to the Pchl GG, Pchl DHGG, Pchl THGG, and Pchl phytol, previously observed in inner seed coats of three Cucurbitaceae (13) (Table I). These results were further supported by the gas chromatographic analyses of esterifying alcohols obtained after saponification of a mixture of purified pigments of each peak. The gas chromatograms of alcohol fraction from a mixture showed the presence of only four alcohols, and they were identified in terms of their retention times with reference to the standards as GG, DHGG, THGG, and phytol. As shown in the experiments of pure fractions (Table II), peaks 1 to 4 were confirmed further as Pchl GG (peak l), Pchl DHGG (peak 2), Pchl THGG (peak 3), and Pchl phytol (peak 4). Minor peaks at approximately 3.0 and 8.6 min in their retention times could be detected only in aged etiolated leaves (Fig. 2). However, on the basis of in situ excitation maximum

289

OF PROTOCHLOROPHYLL

of these peaks, these pigments were not due to Pchl species as described below. Similar HPLC elution profiles of pigments were obtained from cucumber, sunflower, and corn etiolated leaves as shown in Fig. 3. Four Pchls with retention times of 12.2, 14.0, 16.7, and 19.7 min were detected in cucumber, sunflower, and corn as in the reference sample of kidney bean (14 days old). The chromatographic properties of these Pchls were in good agreement with those of the standard, kidney bean Pchls (cf. Table I), indicating the presence of the same molecular species in these plants. Thus, peaks 1 to 4 were identified by their retention times as Pchl GG (peak l), Pchl DHGG (peak 2), Pchl THGG (peak 3), and Pchl phytol (peak 4). The

A. Kldnev

bean il4-day-old)

~

AAl2

3 i1, L-

B. Cucumber (kdav-old1

C. Sunflower

('l-day-old)

0. Corn (5.day-old)

I

I

I

1

I

I

0

5

10

I5

20

25

Retention

time Wn1n1

FIG. 3. Elution profiles of Pchls isolated from etiolated leaves of cucumber, sunflower, and corn by HPLC. Conditions of extraction and purification of pigments are described in the text. HPLC chromatographic conditions are the same as in Fig. 1. 1, Pchl GG; 2, Pchl DHGG; 3, Pchl THGG; 4, Pchl phytol.

SHIOI

.

AND

SASA

ProtochlormhYIIide 1 e

Protochlorophyll

0

4

8

12

16

20

0

4

8

Age (Days)

12

16

20

Age (Days)

FIG. 4. Pchl(ide) content of kidney bean grown in the dark. Pigments were extracted and purified at the indicated age of seedlings and analyzed quantitatively by spectrophotometry and HPLC. Data are average of three experiments. (A) Contents of Pchlide and total Pchls; (B) individual content of four Pchls.

content of each Pchl was changed with culture periods and was of course dependent on the plant species. In contrast to kidney bean, sunflower, and corn, very low content of Pchl phytol was noted in cucumber (Fig. 3B). The minor components with retention times 3.0,6.9, and 8.0 min in cucumber, sunflower, and corn were due to contaminated porphyrin substances on the basis of both in situ excitation maximum of the peaks and spectrophotometric determination of isolated pigments. Isolated components showed absorption maxima at 425, 446, 597, and 650 nm. These pigments were also found in the acetone extract of nongerminated seeds of each plant. Accumulation

of Different

a predominance of Pchlide at later stages. These results are in agreement with previous observations (19, 20). With respect to Pchls, the content of Pchl phytol increased rapidly and reached a maximal level after 9 days and then began to decrease (Fig. 4B), whereas the con1

Pchls

The content of both Pchlide, including divinyl species and total Pchl accumulation in kidney bean, on a fresh leaf weight basis increased rapidly after germination and reached stationary levels after 7 days for Pchlide and after 11 days for total Pchl (Fig. 4A), but the content of Pchlide was much higher than that of Pchl, leading to

0'

0

I 5

I IO

15

20

Age (Davs)

FIG. 5. The ratios of sum of Pchls esterified with GG, DHGG, and THGG to total Pchls during the development stage of kidney beans grown in the dark. The values plotted were calculated from the data of Fig. 3B.

COMPOSITIONAL

HETEROGENEITY

OF

291

PROTOCHLOROPHYLL

GGCPP) ----D>v~nyi

FIG. means

Pchlide

r

PChilde

6. A scheme for the sequence GG or GG pyrophosphate.

Z&I

Pchl

GG zx

of the formation

tents of the other three Pchl species were low up to 11 days, and then increased as Pchl phytol content decreased. As shown in Fig. 5, the ratios of the sum of three Pchls to total Pchls calculated from the data of Fig. 3B remained almost constant up to 11 days, and then increased linearly with subsequent aging. These results suggest that after 11 days, Pchl phytol is progressively converted to three different Pchl species esterified with THGG, DHGG, and GG. A similar accumulation of Pchl species was observed in the aged etiolated leaves of cucumber and corn. DISCUSSION

Our results indicate that Pchl in etiolated leaves of higher plants is not homogenous with respect to their esterifying alcohol (Figs. 2 and 3, Table II). Four Pchls esterified with CZoditerpene alcohols could be detected in plants grown in the dark by the sensitive HPLC analysis, but the content of each component varied with the growth period and was strongly dependent on the plant species (Fig. 3). In preliminary results, we also identified two Pchl species esterified with GG and DHGG in the acetone extract of the Chl-less mutant of Chlorella regularis (YG-23) grown in the dark (21). The similarity in distribution of these Pchl suggests that similar esterification process is equally operative in higher plants and algae. The findings of Pchls esterified with GG, DHGG, and THGG in the early stage of development seem to suggest involvement of a Pchl phytol formation. Low levels of these pigments up to 9 days in kidney bean seem to be indicative of a rapid turnover occurring toward synthesis of Pchl phytol. If this is true, the presence of the intermediates in the esterification step is indicative of homology to the Chlide esterification in Chl a syn-

Pchl

DHGG

z

Pchl

and degradation

THGG

E

Pchl

of Pchlide

DhYtol

esters.

GG(PP)

thetic pathway (8-10). Thus, Pchl esteritied with GG is formed first from Pchlide and GG by the action of an enzyme followed by hydrogenation of the alcohol moiety to phytol. The esterification step between Pchlide and GG in kidney bean was probably inactivated after reaching the maximal level of Pchl phytol at 9 days, since the total Pchl level remains unchanged in the subsequent developing stage of 11 to 17 days. Subsequently, Pchl phytol was progressively converted to the Pchls esterified with THGG, DHGG, and GG (Figs. 4B and 5). These results suggest that analogously to the degradation of monovinyl Pchlide to divinyl Pchlide, as discussed by Jones (14), phytol is dehydrogenated by the reverse reaction of the enzyme concerned in the synthesis of Pchl phytol to yield its precursors, Pchls esterified with THGG, DHGG, and ultimately GG. Low levels of Pchl phytol during development in cucumber seedlings seem to indicate the inhibition of the hydrogenation step between Pchl THGG and Pchl phytol (Fig. 3B). This indicates that esterification and hydrogenation are subsequent steps catalyzed by different enzyme as previously reported in Chlide esterification (10). A scheme for the sequence of Pchls formation and degradation is presented in Fig. 6. In conclusion, our findings show that four Pchls esterified with GG, DHGG, THGG, and phytol are widely distributed in the etiolated leaves of higher plants. Consequently, it strongly suggests that Pchl esterified with phytol may not be formed directly from the esterification of Pchlide and phytol but by three hydrogenation steps of the alcohol moiety after esterification with Pchlide and GG. The reverse reaction, dehydrogenation of phyto1 moiety, was observed in aged leaves, indicating the possible route of degradation of the esterifying alcohols.

292

SHIOI

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AND

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C. (1974)

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K. (1976)

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