Carotenoids of Floral Parts and of the Spadix of Arum maculatum

L. R. G. V ALADON and ROSEMARY S. MUMMERY

With 1 figure

Recei ved October 28, 1974

Summary

Contrary to what was expected, the spadix, floral parts and berries of Arum maculatum contained only small amounts of the a-carotene series. The floral parts contained ~-carotene, diepoxy-p-carotene in fairly large quantities and sometimes lutein. The berries at three different stages of maturity contained a whole range of carotenoids, including lycopene but not neurosporene. These results tend to favour lycopene rather than neurosporene as the precursor which is cyclized to eventually give rise to ~-carotene on the one hand and a-carotene on the other. Furthermore, it was shown that as the various parts aged, the control of carotenoid synthesis was removed and epoxy-carotenoids and their derivatives then became apparent.

Key words: Carotenoids, Arum maculatum, Lycopene, Xanthophylls.

Introduction

Based on chemical structure and results obtained from several laboratories (GOODWIN and WILLIAMS, 1965; WILLIAMS, BRITTON and GOODWIN, 1967; WILLIAMS, BRITTON, CHARLTON and GOODWIN, 1967; PORTER and ANDERSON, 1962; YAMAMOTO, NAKAYAMA and CHICHESTER, 1962), VALADON and MUMMERY (1969) proposed the following pathways for carotenoid biosynthesis: phytoene was dehydrogenated to phytofluene, to ~-carotene, and then to neurosporene (Fig. 1). I)-Carotene could be formed by cyclization of neurosporene to jJ-zeacarotene and also by cyclization of lycopene. Whether it is lycopene or neurosporene that is cyclized is still a matter for discussion. McDERMOTT et a1. (1973) were able to show that in different bacteria, different carotenoids accumulated in the presence of certain inhibitors and that in Flavobacterium strain 0147, nicotine inhibition caused the replacement of zeaxanthin by lycopene. These results and those of HOWES and BATRA (1970) suggested that the cyclization reaction was inhibited and that lycopene was the compound that was cyclized. In the fungus Phycomyces blakesleeanus, BRAMLEY (1973) and DAVIES (1973) showed that I)-carotene was almost completely replaced by lycopene in the presence of nicotine but that on removal of nicotine they failed to show a precursor-product relationship between lycopene and j:i-carotene. VALADON

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Carotenoids of Floral Parts and of the Spadix of Arum maculatum 89

and MUMMERY (1974) using the fungus Verticillium agaricinum presented evidence for an in-between situation. There are several steps in the biosynthetic pathway as outlined in Fig. 1 that have still not been proved.

It is well known that as tissues mature, different carotenoids appear while others disappear and it is possible to obtain information on individual steps by studying the sequence of their formation. By identifying different carotenoids from different parts of rose flowers at different stages of maturity, VAL AD ON and MUMMERY (1969) were able to suggest that rubixanthin in these tissues could have been derived from y-carotene.

A closer look at Fig. 1 shows that a- and I)-carotene are on two different pathways: a-carotene cannot be derived from j3-carotene (GOODWIN and WILLIAMS, 1965) and vice versa (WILLIAMS, BRITTON and GOODWIN, 1967). Most of the work to date has been done on the I)-carotene series since very few tissues are known to contain large amounts of a-carotene and of its derivatives. SCHNEPF and CZYGAN (1966) showed that the spadix of Arum maculatum contained large amounts of a-carotene and its derivatives 5,6-monoepoxy-a-carotene, lutein and lutein-5,6-epoxide. This plant material was therefore considered ideal material for studying carotenoid formation of the a-series and especially so since one could obtain various structures on the same organ. The carotenoids of the following structures: the spadix, the pistillodes, the

Fig. 1: Suggested pathways of the synthesis of a- and f3-carotenes and of their derivatives.

Phytoene ~

Phytfluene

C-Carotene ~

N eurosporene

LY'rP,n, "l lycoxanthin

~ lycophyU

f3-Zeacarotene t

y-Carotene

f3-2arotene _ MonoepOXY-f3-

i ~ carotene

Cryptoxanthin i Ze}xanthin Mutatochrome

~th h· Neoxanthin ... --- An~t eraxant m Diepoxy-f3-

Violaxanthin carotene

Au:oxanthin 1

a-Zeacarotene ~

"'-Carotene ~

a-Carotene

L L utem

~t. .d Lutem-S,6-epoxl e

FIJoxanrhin

Monoepoxy-a-carotene

1 Aurochrome Fla vochrome

z. PJlanzenphysiol. Bd. 75. S. 88-94. 1975.

90 L. R. G. VALADON and ROSEMARY S. MUMMERY

staminate and pistillate flowers, and the berries were investigated in an attempt to

clarify certain steps in the biosynthetic pathway as outlined in Fig. 1.

Material and Methods

The following were used in this investigation: spadix (purple), pistillodes (purple), staminate flowers (purple-yellow), pistillate flowers - an early stage (white-yellow) and a later stage (green-yellow), and berries - an early stage (green) and a later stage (orange) of Arum maculatum L. grown in the grounds of the Botany Department, Royal Holloway College.

The various parts were extracted several times with methanol in a Waring Blendor and then usually with methanol-diethyl ether (1 : 1, v/v) until no more colour was extracted. Partition with diethyl ether was performed and all the colour was transferred to the epiphasic layer, which was concentrated under reduced pressure at about 35°C. Procedures for the saponification of the extracts, the removal of some sterols, the phase partition of carotenoids between hexane and aq. 90 Ofo (v/v) methanol, and the separation and identification of carotenoids by column and thin layer chromatography were as described previously (VALADON and MUMMERY, 1967, 1968). Circular chromatography on Silica-Gel paper Whatman SG81 and Aluminium hydroxide Whatman AH81 was as described by VALADON and MUMMERY (1972).

Carotenoids having epoxy groups were characterized by the modified cone. HCI-ether test of ]UNGALWALA and CAM A (1962).

The structural identity of individual carotenoids was established by comparison with authentic samples using various chromatographic methods as already described, visible spectroscopy and in a few cases IR spectroscopy. The cis-trans isomers, chrysanthemaxanthin and flavoxanthin were extremely difficult to separate and the values for these two compounds are grouped together as flavoxanthin.

The concentrations of individual carotenoids were determined by measuring Emax and 1%

comparing it with known E 1 em values at Amax for pure pigments (GOODWIN, 1955).

1"10 For those pigments whose E 1 em values were not known, Amax was assumed to be 2500

(GOODWIN, 1954). All results were calculated on a per gramme dry-weight basis.

Results and Discussion

The results of carotenoids found in the various parts of A. maculatum are given in Table 1. The smallest amount of total carotenoid was observed in the white-yellow staminate flowers (0.05 mg/g) closely followed by the spadix, while the largest amount was found in the orange berries (3.07 mg/g). I)-Carotene was the only carotenoid found in all the various parts studied, 5,6 : 5',6' -diepoxy-fj-carotene was missing in the orange berries only, while a-carotene was found in very small amounts in three cases only. The absence of 5,6-monoepoxy-a-carotene was unexpected since this was found in relatively large amounts in the species investigated by SCHNEPF and CZYGAN (1966). Lutein, one of the typical pigments of green, photosynthetic tissues was not present in the purple tissues of the spadix and of the pistillodes, but was the

Z. Pjlanzenphysiol. Bd. 75. S. 88-94. 1975.

Carotenoids of Floral Parts and of the Spadix of Arum maculatum 91

Table 1: Quantitative distribution of carotenoids in the spadix, floral parts and berries of Arum maculatum.

Values given are percentages of total carotenoids

spadix hairs 0 S? S? berries berries (purple) (purple) staminate pistillate pistillate (green) (orange)

Carotenoid flowers flowers flowers (purple- (white- (yellow) yellow) yellow) (pale berries)

Carotene:

Phytofluene 0.7 2.3 1.5 a-Carotene 1.3 0.6 0.3 fl-Carotene 13.2 24.3 14.8 10.2 17.0 32.6 8.8 ~-Carotene 2.5 2.5 /i-Zeacarotene

, 0.9 0.3 T

{'-Carotene 14.0 9.3 Lycopene 9.1 5704

Total carotenes 13.9 24.3 16.1 10.2 17.0 62.0 80.1

Xanthophyll:

5,6-Monoepoxy-(J -carotene 2.9 3.5

5,6 : 5'6'-Diepoxy-(J-carotene 29.8 38.1 44.1 18.9 6.7 3.1

Cryptoxanthin 0.7 Zeaxanthin 5.9 Antheraxanthin 0.3 Violaxanthin 13.5 20.3 1.9 Neoxanthin 28.0 37.6 14.1 7.7 Mutatochrome 4.0 Aurochrome 66.9 0.5 Lycophyll 1.6 Lutein 39.8 + 41.9 2304 6.6 Lutein-5,6-epoxide 2.0 Flavoxanthin 11.9 004

Total xanthophylls 86.1 75.7 83.9 89.S 83.0 37.7 19.9

Total carotenoids 0.06 0.17 0048 0.05 0.28 0041 3.07 (mg/g)

largest, individual carotenoid (41.9 %) in the more mature pistillate flowers, while it was only just present in young flowers.

All the parts examined with the exception of the berries contained more xanthophylls - in ratios greater than 3: 1 - than carotenes. In the berries, however, the reverse was found to be the case. A close look at the various floral parts

Z. Pjlanzenphysiol. Bd. 75. S. 88-94. 1975.

92 L. R. G. VALADON and ROSEMARY S. MUMMERY

revealed the presence of j3-carotene and 5,6: 5',6'-diepoxy-j3-carotene, sometimes in large amounts. Usually when there was a decrease in J)-carotene content there was an increase in diepoxy-(]-carotene unless another I)-carotene derived compound increased as well. Such an example was the pistillate flowers: J3-carotene was low (10.2 %), diepoxy-J3-carotene (18.9 %) was not as high as in the staminate flowers (44.1 %), yet there was a large amount of aurochrome (66.9 %). Although no monoepoxy-j3-carotene was observed in the flowers, the evidence suggested that j3-carotene gave rise to monoepoxy-j3-carotene (which disappeared fairly quickly), to diepoxy-j3-carotene and both of these in turn to mutatochrome and aurochrome respectively.

Very little information was obtained on the a-carotene pathway as far as the floral parts were concerned. a-Carotene was found only in the staminate flowers together with large amounts of lutein. In the pistillate flowers no a-carotene was present at all but there was a trace of lutein in the early stage increasing appreciably to 41.9 % in the later stage. This information does not help to elucidate that particular pathway. The only other a-carotene derived pigment found at all here was flavoxanthin and then only in the purple spadix.

As the green tissues mature, oxidative processes are known to take place with the result that very few members of the a-carotene series are identified; instead epoxy-carotenoids and their derivatives become the main carotenoids (VALADON and MUMMERY, 1969). The presence of mutatochrome and aurochrome (the furanoid oxides of 5,6-monoepoxy-jJ-carotene and 5,6 : 5',6' diepoxy-j3-carotene respectively) in the early pistillate flowers was rather puzzling since they disappeared in the later stage, being replaced by violaxanthin and neoxanthin. However, the later pistillate flowers were more like berries and were in fact the very early stages of berry formation. In that case the late pistillate flowers were equivalent to the first berries and it was possible to compare the three stages of the berries. The pale berries which were still enclosed in the spadix had little chlorophyll due to the virtual absence of light and proportionately more xanthophylls than carotenes. The next stage showed an increase in total carotenoids with the concomitant accumulation of the more saturated carotenes e. g. jj-zeacarotene, ~ -carotene, lycopene and y-carotene and a decrease in more unsaturated carotenoids such as violaxanthin, lutein and neoxanthin. This was taken a stage further in the more mature orange berries when total xanthophylls were reduced further compared to total carotenes, especially as lycopene was increased by over 600 %. Also 5,6 and 5,6 : 5',6' expoxy carotenes disappeared and 5,6 epoxy lutein appeared for the first time and so did the 5,8 furanoid compounds aurochrome and flavoxanthin. These results are not in disagreement with the scheme of Fig. 1 and further are in agreement with GOODWIN'S (1966) and VALADON and MUMMERY'S (1969) suggestions that as fruits mature very few members of the a-carotene series were present and there was an increase in epoxy and furanoid compounds when the control of carotenoid synthesis was removed.

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Carotenoids of Floral Parts and of the Spadix of Arum macula tum 93

A point worth noting was that most of the carotenoids of Fig. 1 were identified in some or all of the parts of A. maculatum studied. However, neurosporene was not identified although lycopene was found in large quantities (57 Ofo) in the more mature berries. The presence of lycopene in the late stages would suggest that it is an important precursor of other carotenoids and that it only accumulates when it cannot be used any more to form its products - when control of carotenoid synthesis has been removed. On the other hand the absence of neurosporene does not rule it out completely as the precursor that was cyclized. It may well be that turnover was so rapid that it was being used as fast as it was being formed. These results therefore tend to favour lycopene rather than neurosporene but it will not be at all surprising to find that one or other of these compounds or both are important precursors in different tissues.

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94 L. R. G. VALADON and ROSEMARY S. MUMMERY

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Dr. L. R. G. VAL AD ON and Miss R. S. MUMMERY, Department of Botany, Royal Holloway College (University of London), Egham Hill, Egham, Surrey TW 20 OEX, England.

Z. Pjlanzenphysiol. Bd. 75. S. 88-94. 1975.