Photosynthesis Research 15:67-73 (1988) O Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands

Regular paper

Violaxanthin de-epoxidase in etiolated leaves E R H A R D P F U N D E L & RETO J. STRASSER Institute of Biology, Department of Bioenergetics, University of Stuttgart, Ulmer Str. 227, 7000 Stuttgart 60, Federal Republic of Germany Received 10 June 1987; accepted in revised form 3 September 1987

Key words: Carotenoid transformation, chloroplast development, etiolated leaf, in vivo spectroscopy, xanthophyll cycle Abstract. In etiolated leaves the occurrence of the enzymatic violaxanthin de-epoxidation to zeaxanthin is shown. The carotenoid transformation is provoked by the infiltration of whole leaves with ascorbate at pH 5 and is susceptible to DTT. Identification of the de-epoxidase activity is achieved by in vivo spectroscopy and pigment analysis (TLC).

Abbreviations: DTT Dithiothreitol, Hepes-N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid, TLC- Thin-layer Chromatography

Introduction

Since the discovery of the xanthophyll or violaxanthin cycle in the leaves of higher plants by Sapozhnikov et al. in 1957 the knowledge about this carotenoid transformation has increased considerably, though its function is still not clear (see Siefermann-Harms 1977 and Yamamoto 1979 for a review). It starts with the enzymatic de-epoxidation of violaxanthin (5,6,5",6"-diepoxizeaxanthin) via antheraxanthin (5,6-monoepoxizeaxanthin) to zeaxanthin induced by strong light; the back reaction is light independent. In isolated chloroplasts, the maximum activity of the de-epoxidase in the dark requires pH 5 and ascorbate (Hager 1966a), whereas the epoxidase needs pH 7.5, NADPH, and molecular oxygen (Hager 1975, Siefermann and Yamamoto 1975a). These properties have led to the idea that the deepoxidation takes place inside the thylakoid and is actuated through photosynthetic proton-pumping, while the epoxidation happens at the outside of the membrane (Hager 1975, Siefermann and Yamamoto 1975b). The appearance of the epoxidase activity has been demonstrated in the course of the flash-induced greening of bean leaves (Siefermann-Harms et al. 1980). The de-epoxidation has been measured spectroscopically in flashed

68 bean leaves, which received a total of a few seconds of light (Strasser 1973). However, it is still not clear when the ability of de-epoxidation is first established in the leaf. In this paper we demonstrate the presence of the de-epoxidase in etiolated leaves, which clarifies the question whether or not the xanthophyll cycle requires functional chlorophyll-containing structures for its activity. Materials and methods

Bean seeds (Phaseolus vulgaris L. var. Commodore) were planted in moist vermiculite-perlite and grown in complete darkness for 15-18 days. Harvesting of the primary leaves and all further handling were performed either in the dark or under a dim green light. Fluorescence spectra were recorded at 77 K using .branched fibre optics connected with a Hamamatsu photomultiplier via a monochromator (H-I 0, ISA-Instruments) (Strasser and Butler 1976a). Excitation light was broadband blue (450 4- 50 nm) of 25 W/m 2 at the surface of the sample. Whole leaves were vacuum-infiltrated in a buffer o f p H 5 (300 mM sorbitol, 50 m M sodium citrate, 10 m M NaC1) or of pH 7.2 (300 mM sorbitol, 50 mM Hepes, 10 mM NaCI) and remained in the corresponding buffer during the experiment. If present, the concentrations of ascorbate and DTT were 80 mM and 0.6 raM, respectively. For absorbance measurements, infiltrated leaves were fixed on an optical diaphragm. During an interval of several hours, the reaction of individual leaves was followed by repeatedly recording the absorbance spectrum with an Aminco DW-2a spectrophotometer on line with a microcomputer. Before calculating the difference spectra, all series of spectra were normalised to the same initial absorbance. For pigment determination, 1 g of leaves was washed in the pH 7.2 buffer and homogenised in a cold mortar together with 1 g of CaCO3. Pigments were extracted with pure acetone and, after removal of acetone by vacuum-evaporation, transferred to diethylether. The pigments were separated using the alkaline thin-layer chromatography developed by Hager and Meyer-Bertenrath, 1966b. Carotene was determined in chloroform and the xanthophylls in ethanol using the extinction coefficients published by Hager and Meyer-Bertenrath, 1967. Lutein and antheraxanthin were obtained together and quantified spectroscopically as described elsewhere (Meck and Strasser 1984). Results

The low temperature fluorescence spectrum of our dark grown bean leaves

69 657 I

674 I

632

'

6

~'~~,,o,~5 7 2

0

Wovetength (rim)

Fig. 1. Low temperature (77 K) fluorescence spectrum ofetiolated bean leaves. Samples of the dark grown leaves were frozen in liquid nitrogen immediately after harvesting. Fluorescence spectra were measured using branched fibre optics. Excitation light was 450 _ 50 nm of

25 W/m 2.

is characterised by a distinct maximum at 657 nm, accompanied by 5 smaller rises (Fig. 1), as is already known for etiolated leaves (Sironval et al. 1968). Repeatedly recorded absorption spectra of an etiolated leaf taken after infiltration with ascorbate at pH 5 are shown in Fig. 2. Difference spectra calculated from these reveal a large absorbance increase at 505 nm and have defined isosbestic points. The absorbance changes provoked by the infiltration with ascorbate in a pH 5 buffer are prevented by the co-infiltration with

1 5 0 Mla. t10 ~ln. 8 0 ~IN,

'

8 1 5 0 MIN, MINUS ~0 MIN. 110 ~[N. MINUS q0 MIN, -

-

80 MIN,

MINUS ~D m N ,

q 5a39LI 1

~ength {nm)

Fig. 2. Absorption spectra of an etiolated leaf vacuum-infiltrated with ascorbate at pH 5. Spectra were recorded at the indicated times after vacuum-infiltration with a pH 5 buffer containing 80 mM ascorbate, 300 mM sorbitol, 50 mM sodium citrate and 10 mM NaC1. The resulting difference spectra were enlarged 6-fold.

70

®

/,l~

PH5.O~+ASCORBATE: -OTl

.io

-ASCORBATE;

-DT1

© PH 5.0; +ASCOrBATE; ÷DTI

I

t~O0

I

I

1.SO 500 Wovetength (nml

t

550

Fig. 3. Absorbance changes of etiolated bean leaves vacuum-infiltrated with different buffer solutions. Difference spectra were calculated from the absorbance spectra at 4 hours minus l hour after vacuum-infiltration and plotted at a 6-fold increase of sensitivity. Infiltration was with a pH 5 buffer (curves A-C) containing 300mM sorbitol, 5 0 m M sodium citrate and 10mM NaC1 or a pH 7.2 buffer (curve D) containing 3 0 0 m M sorbitol, 5 0 m M Hepes and 10 m M NaCI. If present, the concentrations of ascorbate and D T T were 80 m M and 0.6 mM, respectively.

DTT (Fig. 3C), and do not occur if ascorbate is given in a pH 7.2 buffer or if the leaf is infiltrated at pH 5.0 without ascorbate within an interval of about 4 hours (Figs. 3B and C). Prolonged incubation of an etiolated leaf without ascorbate at pH 5.0, however, leads to an absorbance change with the same spectral features as already shown in Fig. 2. This delayed 505 nm change starts in the range of 4 to 5 hours after infiltration and amounts to 50% of the change observed after ascorbate infiltration at low pH (Fig. 4). E

~ .20 c~

.~.1S

]~[LTP,A I5 . 0I; I+ASCOABATE ~ P H

~.10

~u o .05 ~ "e 8

i~ I

I

I

I

PH5,0s -ASCORBATE I I I I

1 2 3 t. s 6 7 Time after infiltration (hours)

8

Fig. 4. Kinetics of the 505 nm absorbance increase of etiolated bean leaves vacuum-infiltrated with a pH 5 buffer. Whole leaves were infiltrated with a pH 5 buffer (300 m M sorbitol, 5 0 m M sodium citrate, 10 m M NaC1) containing 80 m M ascorbate or no ascorbate, respectively. The plotted values are derived from the difference spectra.

71 Table 1. Carotenoid content of etiolated bean leaves vacuum-infiltrated with ascorbate at

pH 5. Time after infiltration

10 min. pH 5; ASC

Pigment content (nmole/g fresh weight) Carotene 7 Lutein 33 Neoxanthin 6 Violaxanthin 36 Antheraxanthin 13 Zeaxanthin 3

5 hours pH 5; ASC

5 hours pH 5; ASC; DTT

9 34 5 3 16 38

11 39 5 28 16 8

18-day-old leaves of the same culture were infiltrated with a pH 5 buffer containing 80mM ascorbate, 300ram sorbitol, 50ram sodium citrate and 10mM NaC1. If present, the concentration of DTT was 0.6 mM. The pigments were extracted after different periods of time as indicated in the scheme and separated using the alkaline TLC-system developed by Hager and Meyer-Bertenrath (1966b). Each column of data results from one TLC-analysis. Repeated pigment assays of equally treated leaves show varying pigment contents but a highly reproducible stoichiometry of the pigments of the xanthophyll cycle.

The carotenoid determination achieved by TLC shows a more than tenfold increase of zeaxanthin in leaves extracted 5 hours after ascorbate infiltration at pH 5.0 compared to samples taken after ten minutes. This increase goes along with a nearly stoichiometrical decrease of violaxanthin, while the other pigments do not change in level significantly (Table 1). In addition, leaves which are co-infiltrated with DTT do not show this drastic alteration in the pigment content (Table 1).

Discussion

This paper revealsthe activity of the violaxanthin de-epoxidase in etiolated leaves, that means in a tissue lacking any active photosynthetic apparatus. The low temperature fluorescence spectra of our plant material (Fig. 1) coincide with publised data (Sironval 1968) and prove that our samples are completely etiolated. By vacuum-infiltration of whole etiolated leaves with a pH 5 buffer containing ascorbate we could observe the typical absorbance changes (Fig. 2) which first have been described in vivo by Strasser and Butler, who induced them by continuous illumination of flashed bean leaves (Strasser 1973, Strasser and Butler 1976b). This phenomenon could unequivocally be related to the action of the violaxanthin de-epoxidase by Siefermann-Harms et al. 1980. The equivalence of the absorbance changes in etiolated and flashed leaves is stressed by the correspondence of the isosbestic points as well as the extrema of the difference spectra (Fig. 2) to

72 values found in the literature (Strasser and Butler 1976b, Siefermann-Harms et al. 1980). The fact that these absorbance changes require both ascorbate and pH 5, on the one hand, and that they are inhibited by DTT on the other hand, has been described for chloroplasts (Yamamoto et al. 1972a, Yamamoto and Kamite 1972b) and further demonstrates the presence of the de-epoxidase. The difference in pigment composition before and after the full-scale spectral shift, as revealed by TLC, may obviously be interpreted as the transformation of violaxanthin to zeaxanthin, while the intermediate (antheraxanthin) remains relatively constant (Table 1). The zeaxanthin formation is inhibited by DTT (Table 1, column 3), which is in accordance with the DTT effect obtained by in vivo spectroscopy (Fig. 3C). The sigmoidal shape and the long time requirement of the ascorbateinduced 505 nm change (Fig. 4) seem to be due to the diffusion barriers imposed by the cell wall and cell membrane. It remains to be clarified whether the further delay and lower extent of the absorbance change in the case of infiltration of an ascorbate-free pH 5 buffer (Fig. 4) reflects the accessibility of a limited amount of endogenous ascorbate for the deepoxidase. We conclude that there is no doubt about the occurrence of the violaxanthin de-epoxidase well before the formation of chlorophyll-containing thylakoids and the onset of photosynthetic activity. This extends the scope in which the function of the xanthophyll cycle may be searched for and suggests the idea of its participation in membrane formation and chloroplast development.

Acknowledgements The authors thank Mrs Ellen Buff for critically reading the manuscript. This work was supported by the Alfried Krupp von Bohlen und Halbach-Stiftung and by the Deutsche Forschungsgemeinschaft (STR 235/2).

References Hager A (1966a) Die Zusammenh~ingezwischenlichtinduziertenXanthophyll-Umwandlungen und Hill-Reaktion. Ber Dtsch Bot Ges 79:94-107 Hager A and Meyer-BertenrathT (1966b) Die Isolierungund quantitative Bestimmungder Carotinoide und Chlorophyllevon B1/ittern,Algen und isoliertenChloroplasten mit Hilfe dtinnschichtchromatographischerMethoden. Planta 69:198-217 HagerA and Meyer-BertenrathT (1967) Die Identifizierungder an Dfinnschichtengetrennten

73 Carotinoide grfiner Bl/itter und Algen. Planta 76:149 168 Hager A (1975) Die reversiblen, lichtabh/ingigen Xanthophyllumwandlungenim Chloroplasten. Ber Dtsch Bot Ges 88:27-44 Meck E and Strasser RJ (1984) Zeaxanthin in a fraction with an excess of lutein. In: Sybesma C (ed.) Advances in Photosynthesis Research. Vol. 1, pp 717-720. The Hague: Martinus Nijhoff/Dr W. Junk Publishers Sapozhnikov DI, Krasovskaya TA and Mayeskaya AN (1957) Changes observed in the relation between the main carotenoids in the plastids of green leaves exposed to light. Dokl Akad Nauk 113:465-467 Siefermann D and Yamamoto HY (1975a) NADPH and oxygen-dependent epoxidation of zeaxanthin in isolated chloroplasts. Biochem Biophys Res Commun 62:456461 Siefermann D and Yamamoto HY (1975b) Properties of NADPH and oxygen-dependent zeaxanthin epoxidation in isolated chloroplasts. Arch Biochem Biophys 171:70-77 Siefermann-Harms D (1977) The xanthophyll cycle in higher plants. A review. In: Tevini M and Lichtenthaler HK (eds) Lipids and Lipid Polymers in Higher Plants, pp 218-230. Berlin: Springer-Verlag Siefermann-Harms D, Michel J-M and Collard F (1980) Carotenoid transformations underlying the blue absorbance change in flashed leaves during the induction of oxygen evolution. Biochim Biophys Acta 315-323 Sironval C, Brouers M, Michel J-M and Kuiper Y (1968) The reduction of protochlorophyllide into chlorophyllide. Photosynthetica 2:268-287 Strasser RJ (1973) Induction phenomena in green plants when the photosynthetic apparatus starts to work. Arch Int Physiol Biochim 81:935-955 Strasser RJ and Butler WL (1976a) Energy transfer in the photochemical apparatus of flashed bean leaves. Biochim Biopys Acta 449:412-419 Strasser RJ and Butler WL (1976b) Correlation of absorbance changes and thylakoid fusion with the induction of oxygen evolution in bean leaves greened by brief flashes. Plant Physiol 58:371 376 Yamamoto HY, Kamite L and Wang Y-Y (1972a) An ascorbate-induced absorbance change in chloroplasts from violaxanthin de-epoxidation. Plant Physiol 49:224-228 Yamamoto HY and Kamite L (1972b) The effects of dithiothreitol on violaxanthin deepoxidation and absorbance changes in the 500-nm region. Biochim Biophys Acta 267: 538-543 Yamamoto HY (1979) Biochemistry of the violaxanthin cycle in higher plants. A review. Pure Appl Chem 51:639-648

Violaxanthin de-epoxidase in etiolated leaves.

In etiolated leaves the occurrence of the enzymatic violaxanthin de-epoxidation to zeaxanthin is shown. The carotenoid transformation is provoked by t...
326KB Sizes 0 Downloads 0 Views