Plant Cell Physiol. 39(9): 968-977 (1998)
JSPP © 1998
Distribution and Geometric Isomerism of Neoxanthin in Oxygenic
Phototrophs: 9-Cis, a Sole Molecular Form
Shinichi Takaichi 1 ' 3 and Mamoru Mirauro2
1
2
Biological Laboratory, Nippon Medical School, Kawasaki, 211-0063 Japan
Department of Physics, Biology and Informatics, Faculty of Science, Yamaguchi University, Yamaguchi, 753-8512 Japan
The distribution and geometric form (cis or trans) of
neoxanthin, one of the major carotenoids of oxygenic phototrophs, were systematically examined. The 9-cis form of
neoxanthin, but not the a\\-trans form, was found in chloroplasts of seed plants, ferns, mosses and green algae, all
of which contain chlorophylls a and b. In contrast, neoxanthin was not found in other algal classes, such as Heterokontophyta, Rhodophyta and oxygenic phototrophic prokaryotes. Consequently, with regard to phylogeny, the
appearance of neoxanthin appears to be associated with
the appearance of chlorophyll b. In non-photosynthetic
organs, such as petals and fruits, the presence of neoxanthin was classified into four types; those having only the 9cis form, those with only the all-trans form, those with
both forms, and those without either form. Thus only 9-cis
neoxanthin is found in chloroplasts, and the all-trans neoxanthin is found only in non-photosynthetic organs. Because the absorption spectra of both forms are almost identical, their functions in photosynthesis might be similar.
9'-Cis neoxanthin is not involved in the xanthophyll-cycle,
whereas it is a suitable substrate for abscisic acid synthesis.
Differences in geometric isomerism are discussed in relation
to abscisic acid synthesis.
Key words: Abscisic acid — Carotenoid — Chlorophyll b —
9'-Cis neoxanthin — Neoxanthin — Phylogeny.
Neoxanthin is a common, widely distributed plant
carotenoid first isolated from the green leaves of barley by
H.H. Strain in 1938. Goldsmith and Krinsky (1960) proposed a tentative structure of neoxanthin with three hydroxyl
and one 5,6-epoxy groups, but this structure did not match
with the molecular formula, C4oH5604, by two hydrogen
atoms. Cholnoky et al. (1969) demonstrated that neoxanthin contained an additional allenic group, and proposed
that a major neoxanthin from maple leaves was the 9cis form (9'-cis based on IUPAC-IUB nomenclature, see
Fig. 1), although they illustrated the sW-trans structure in
their paper. Optical isomerism (R or S) of hydroxyl groups
Abbreviation: LHC II, light-harvesting complex of PSII.
Corresponding author: FAX +81 44 722 1231; E-mail takaichi/
[email protected]
3
968
at C-3 and C-3' (Bartlett et al. 1969), an epoxy group
(Goodfellow et al. 1973), and an allenic group (Hlubucek
et al. 1974) have been described, and the chemical structure
was finally confirmed by chemical synthesis (Baumeler and
Eugster 1992). Marki-Fischer and Eugster (1990) identified
two forms of neoxanthin using 'H-NMR analysis; the alltrans form purified from the petals of Trollius europaeus
and yellow rose, and the 9'-cis form from spinach leaves. In
spite of these results, the geometric form (trans or cis) of
neoxanthin in plants is, in general, still believed and illustrated to be the all-/rans form (Young and Britton 1993,
Buchecker and Noack 1995); the only two exceptions are
the reviews of Bjernland (1990) who isolated 9'-cis neoxanthin from spinach leaves and two species of Euglenophytes,
and of Yamamoto and Bassi (1996) who discuss 9'-cis neoxanthin without any explanations or citations.
Neoxanthin is a known component of LHC II in
higher plants, whose three-dimensional structure has been
elucidated (Kiihlbrandt et al. 1994), however the binding
site of neoxanthin has not yet been elucidated. Localization
of neoxanthin in LHC II might be affected by the geometric
form of this pigment, 9'-cis rather all-trans, which consequently results in a difference in the function of the complex.
Neoxanthin is also a known precursor of ABA
(Schwartz et al. 1997b). Because the 9-cis form of ABA is
biologically active (Schwartz et al. 1997b), the 9'-cis neoxanthin is thought to be found in some higher plants only based on the citation of Cholnoky et al. (1969) (Li and Walton
1990, Parry et al. 1990, Marin et al. 1996), however
sufficient evidence for this structure has not been proved.
Because ABA is one of the most important growth regulators in plants and is synthesized from neoxanthin, elucidation of the presence of neoxanthin in plants is worth while.
We therefore systematically examined the distribution
and the geometric form of neoxanthin from higher plants
to oxygenic phototrophic prokaryotes. We found that neoxanthin is present only in photosynthetic organisms containing Chls a and b, and even in those organisms, an
organ specific distribution of the molecular forms was observed. Only the 9'-cis form of neoxanthin exists in chloroplasts but the 9'-cis and/or the all-trans forms exist in the
non-photosynthetic organs, such as petals and fruits.
9'-Cis neoxanthin in oxygenic phototrophs
Materials and Methods
969
sorbance of the Chi a peak was about 1 to 1.5, thus the minimum
molar ratio of detection was about 2 to 3 mmol carotenoids to 1
mol Chi a. In the case of non-photosynthetic organs, such as
flowers and fruits, the main carotenoid peak was also about 1,
thus the maximum resolution of neoxanthin was about 0.3% of
the total carotenoids.
Biological materials—More than 170 species were used for
analyses. These species covered 19 divisions and more than 80%
of the classes of oxygenic phototrophs. The remaining division
and classes were covered by references, so that all divisions and approximately 90% of the classes could be surveyed. The major species examined are listed in Tables 2 and 3 and all species examined
Results
are in Table 4, together with references. Unicellular algae were
pure cultures, and marine macroalgae were natural products colConfirmation of the molecular structure of 9'-cis neolected at seaside. Seed plants, ferns, mosses and non-photosynthetxanthin—The molecular weight of purified neoxanthin was
ic organs, such as flowers and fruits, were collected from fields or
600. The 'H-NMR spectrum of neoxanthin was compatible
botanical gardens, or purchased at markets.
with that of 9'-cis neoxanthin (Table 1). The assignments of
Purification of neoxanthin—In order to confirm the chemical
structure of neoxanthin, it was purified from spinach leaves. Pig- all protons, including multiplicity and coupling constants,
were confirmed with 'H-'H COSY and NOE spectra. Espements were extracted with acetone : methanol (7 : 2, v/v) supplemented with 0.1 volume (v/w) of 1 M Tris-HCI (pH 8.0) to pre- cially, the NOE correlations between H-7' and H3-19', H-8'
vent acidification, which causes the rearrangement of 5,6-epoxide
and H-ll', and H - l l ' and H3-20', which are characteristics
of neoxanthin and violaxanthin to furanoid 5,8-epoxide (Eugster
of 9'-cis neoxanthin, could be detected. Thus, neoxanthin
1995), and further extracted with 100% acetone several times. The
in spinach leaves is confirmed to be 9'-cis neoxanthin [9'-cissolvent was evaporated and the pigments were dissolved in n-hex(3S,5R,6R,3'S,5'R,6'S)-5',6'-epoxy-6,7-didehydTO-5,6,5',6'ane. The pigments were loaded on a column of silica gel 60 (Merck, Germany) and washed with rt-hexane and n-hexane: acetone
tetrahydro-/?,/?-carotene-3,5,3'-triol] (Fig. 1).
( 7 : 3 , v/v) successively. Neoxanthin was eluted with n-hexane :
Absorption maxima of 9'-cis neoxanthin in the HPLC
acetone ( 1 : 1 , v/v). This fraction was then separated on a column
eluent were located at 413, 435 and 464 nm, whereas those
of DEAE-Sepharose CL-6B (Pharmacia, Sweden). Neoxanthin
of all-trans neoxanthin were located at 417, 440 and 470
was eluted with n-hexane : acetone (1 : 2, v/v) and finally purified
nm with a red-shift of approximately 5 nm from those of
with TLC with silica gel 60 (Merck), and developed with dichloromethane : ethyl acetate : acetone (5 : 10 : 2, by volume). Molecuthe 9'-cis form. Following separation with HPLC, the retenlar weights were determined by field-desorption mass spection time of 9'-cis neoxanthin was 8.5 min, while that of the
trometry using a double-focusing gas chromatographic mass
all-trans form was 7.9 min. Consequently, the geometric
spectrometer equipped with a field-desorption apparatus (Mforms, 9'-cis and all-trans, of neoxanthin could be distin2500; Hitachi, Japan; Takaichi 1993). 'H-NMR spectra in CDC13
guished
by their absorption spectra and their HPLC retenwere recorded with a VXR-500 spectroscopy (Varian, U.S.A.) ustion times. When the mixture of purified all-trans and 9'-cis
ing tetramethylsilane as an internal standard.
neoxanthin was analyzed, the lower limit of detection of
For isomerization of neoxanthin, iodine dissolved in n-hexthe all-trans form was estimated to be 3% of the total neoane and washed with 0.1 M Tris-HCI (pH 8.0) was added to neoxanthin in n-hexane. White light from a fluorescent lamp (daylight
xanthin content.
type, ca. 20/nnol m~2 s~') was illuminated for 30 min. A solution
Presence of 9'-cis neoxanthin in chloroplasts—The pigcontaining al\-trans neoxanthin was separated with HPLC, and its
ment compositions in green leaves or green organs of seed
retention time and absorption spectrum were analyzed as deplants, mosses and ferns, and whole cells of algae were anascribed below.
Analysis of carotenoids—First, 0.1 volume (v/w) of 1 M lyzed. Because photosynthetic pigments, including Chls
Tris-HCI (pH 8.0) was added to the biological materials to prevent
acidification. The biological materials were homogenized in ca. 5
volumes of acetone : methanol (7 : 2, v/v), followed by centrifugation. This extraction procedure was repeated once more. Approximately 0.1 volume of chloroform and then approximately 1
volume of water were successively added to the extracts. The chloroform fraction was collected, and washed once or twice with
water. After centrifugation, the pigments were separated on an
A\\-trans neoxanthin
HPLC equipped with a ft Bondapak C18 column (Waters,
U.S.A.). When necessary, some modifications in the extraction
procedure were performed.
Neoxanthin and violaxanthin were eluted with methanol:
water ( 9 : 1 , v/v) at a flow rate of 2.0 ml min" 1 , and then other
carotenoids and Chls were eluted with methanol. Chls and carotenoids were identified by their retention times on HPLC and their
absorption spectra in the eluent by a photodiode array detector,
MCPD 3600 (Otsuka Electronics, Japan; Takaichi and Shimada
1992). Under our analyzing conditions, the lower limit for the de9'-Cis neoxanthin
OH
tection of carotenoids by the absorption spectra was about 0.003
on the MCPD. Usually in the sample solutions, the maximum abFig. 1 Structures of all-trans and 9-cis neoxanthin.
9-Cis neoxanthin in oxygenic phototrophs
970
Table 1
'H-NMR spectrum of 9'-cis neoxanthin in CDC13 from spinach leaves
roton(s)
H3-I6
H3-I7
H3-I8
H3-I9
H3-2O
H 2 -2a
P
H-3ax
HO-3
H 2 -4o
P
H-8
H-10
H-ll
H-12
H-14
H-15
H3-I6'
Hj-17'
H3-I8'
H3-I9'
H3-2O'
H2-2' a
P
H-3'
HO-3'
H2-4' a
P
H-7'
H-8'
H-10'
H-ll'
H-12'
H-14'
H-15'
8 Values in ppm, multiplicity and coupling constant in Hz
9'-cis neoxanthin
9'-cis neoxanthin"
.068 s
1.334 s
.350 s
1.800 s
.957 s*
.93
.36
4.32 m
1.29 d (1.5)
2.263 ddd (2.1, 4.1, 12.9)
1.406
6.032 s
6.116 d (10.5)
6.544 dd (11.5, 15.0)
6.339 d (15.0)
6.242 d (9.5)
6.62 m
1.008 s
1.165 s
1.255 s
1.933 s
1.963 s*
1.63 dd
1.235 d (3.0)
3.92 m
~1.26
2.408 ddd (1.8, 5.0, 14.3)
1.66 dd
5.935 d (15.5)
6.836 d (15.5)
6.075 d (11.5)
6.759 dd (11.8, 14.8)
6.292 d (15.0)
6.242 d (9.5)
6.62 m
1.07
1.34
1.35
1.81
1.96
1.95
1.34
4.32
2.27
1.41
6.04
6.12
6.61
6.35
6.25
6.63
1.01
.17
.21
.93
.96
.63
1.26
-3.91
2.40
1.65
5.94
6.84
6.08
6.76
6.29
6.26
6.63
0
Marki-Fischer and Eugster (1990), Englert (1995).
* Corresponding assignments may be reversed.
and carotenoids, are known to be synthesized in chloroplasts and localized, in general, in photosynthetic organs, it
is probable that the analyzed pigments came preferentially
from chloroplasts.
Only 9'-cis neoxanthin, and not the all-trans form, was
found in chloroplasts of seed plants, ferns, mosses and
green algae (Table 2), all of which also contained Chls a
and b. Euglenophyta and Chlorarachniophyta, which are
postulated to have originated by the secondary symbiosis
of Chls a/b containing algae, also contained only the 9'-cis
form. The contents of 9'-cis neoxanthin in the above mentioned organisms were 10 to 20% of the total carotenoids.
All of the seed plants, ferns and mosses contained alltrans violaxanthin in addition to lutein, Chi b, Chi a and /?carotene, which were eluted in this order on reverse phase
HPLC. Antheraxanthin, zeaxanthin and ct-carotene were
found as minor components in most of these species.
Relative pigment contents were similar among these species. Some Chlorophyta, such as Dunaliella, Scenedesmus,
Ulva, Spirogyra and Nitella, had a pigment composition
9'-Cis neoxanthin in oxygenic phototrophs
971
Table 2 Presence of 9'-cis neoxanthin in chloroplast
Division
Class
Subclass
Infraclass
Chlorarachniophyta
Euglenophyta
Euglenophyceae
Chlorophyta
Prasinophyceae
Chlorarachniophyceae
Chlorophyceae
Ulvophyceae
Charophyceae
Bryophyta
Jungermanniidae
Marchantiidae
Bryidae
Order
Genus and species
Chlorarachniales
Eutreptiales
Euglenales
Chlorarachnion sp. NIES
Mamiellales
Pseudoscourfieldiales
Chlorodendrales
Pyramimonadales
Dunaliellales
Chlamydomonadales
Chlorococcales
Ulvales
Siphonocladales
Caulerpales
Zygnematales
Charales
Metzgeriales
Jungermanniales
Marchantiales
Dicranales
Grimmiales
Isobryales
Hypnobryales
Polytrichales
Psilotales
Lycopodiales
Selaginellales
Isoetales
Microphyllophyta
Psilotopsida
Aglossopsidae
Glossopsidae
Sphenophyta
Sphenopsida
Equisetales
Pterophyta
Marattiopsida
Ophioglossopsida
Leptosporangiopsida
Marattiales
Ophioglossales
Filicales
Salviniales
Cycadophyta
Ginkgophyta
Coniferophyta
Gnetophyta
Cycadales
Ginkgoales
Eutreptiella gymnastica"
Euglena gracilis strain Z
Mantoniella squamata
Pseudoscourfieldia marina CS208
Tetraselmis chuii CS26
Pyramimonas parkeae NIES254
Dunaliella salina
Chlamydomonas reinhardii
Chlamydomonas parkeae NIES441
Scenedesmus obliquus
Ulva pertusa
Ulva arasakii
Chaetomorpha crassa
Bryopsis maxima
Spirogyra sp.
Nitella sp.
Pellia endivi/olia
Plagiochila sp.
Conocephalum conicum
Marchantia sp.
Dicranum japonicum
Rhacomitrium lanugerosum
Climacium japonicum
Pleuroziopsis ruthenica
Hypnwn tristo-viride
Polytrichastrum formosum
Psilotum sp.
Lycopodium sp.
Selaginella uncinata
Isoetes japonica
Equiselum arvense
Equisetum hiemale
Angiopteris lygodiifolia
Botrychium ternatum
Neottopteris antiqua
Dryopteris erythrosora
Salvinia natans
Cycas revoluta
Taxodiales
Podocarpales
Ginkgo biloba
Pinus sp.
Podocarpus macrophylla
Ephedrales
Gnetales
Ephedra sinica
Gnetum gnemon
(continued)
9-Cis neoxanthin in oxygenic phototrophs
972
Table 2
(continued)
Division
Anthophyta
Class
Subclass
Infraclass
Dicotyledonopsida
Archichlamiidae
Magnoliatae
Hamameliatae
Caryophylliatae
Dilleniiatae
Rosiatae
Sympetalidae
Monocotyledonopsida
Alismatidae
Arecidae
Commelinidae
Zingiberidae
Liliidae
Order
Genus and species
Piperales
Ranunculales
Urticales
Fagales
Caryophyllales
Polygonales
Violales
Capparales
Rosales
Apiales
Solanales
Asterales
Houttuynia cordata
Ranunculus sp.
Ficus carica
Quercus acutissima
Spinacia oleracea
Polygonum filiforme
Viola sp.
Brassica rapa
Rosa sp.
Petroselinum sativum
Lycopersicon esculentum
Chrysanthemum coronarium
Hydrocharitales
Alismatales
Arecales
Arales
Commelinales
Cyperales
Bromeliales
Zingiberales
Liliales
Orchidales
Elodea sp.
Alisma sp.
Trachycarpus sp.
Colocasia antiquorum
Tradescantia sp.
Zea mays
Ananas comosus
Zingiber mioga
Narcissus pseudo-narcissus
Cymbidium hybridum
Bjornland (1990).
similar to that described above. There are several species
with unique carotenoid compositions; namely, Chlorarachnion and Pyraminomas contain loroxanthin ester (Sasa et
al. 1992), Chlamydomonas parkeae and Bryopsis contain
siphonaxanthin ester, and Chlamydomonas reinhardii and
Chaetomorpha contain loroxanthin. Euglenophyta was
exceptional in that it lacked violaxanthin and lutein, but
contained diadinoxanthin, which was confirmed by its absorption spectrum and the molecular weight, and diatoxanthin (Bjernland 1982).
Absence of neoxanthin in other algal classes—In contrast, neoxanthin in either a cis- or a trans-form was not
found in other algal classes (Table 3) with an accuracy of
detection within 0.3% of the total carotenoids. Those were
Haptophyta, Heterokontophyta, Dinophyta and Cryptophyta containing Chls a and c, Rhodophyta containing
Chi a, and the oxygenic phototrophic prokaryote, Prochlorophyta and Cyanophyta. Glaucophyta (Chapman 1966)
reportedly does not contain neoxanthin.
Violaxanthin, a precursor of neoxanthin synthesis
(Fig. 2), is found in Raphidophyceae, Phaeophyceae and
Eustigmatophyceae in Heterokontphyta and is also reported in Chrysophyceae in Heterokontphyta (Withers et
al. 1981). Pigment compositions in the above mentioned
algae were very different from those in organisms containing 9'-cis neoxanthin.
Neoxanthin in non-photosynthetic organs—The geometric isomerism and distributions of neoxanthin were
also examined in non-photosynthetic organs that contain
carotenoids. All samples examined are indicated in Table
4. We found four types of distribution of neoxanthin. One
type contained only the 9'-cis form, such as carnation and
dandelion petals, corn seed, and an etiolate of wheat,
whereas the second type contained only the all-trans form,
such as rose and narcissus petals, pumpkin and tomato
fruit, and carrot root. The third type contained both forms
of neoxanthin, such as pansy and garbera petals. Neoxanthin was not found in the fourth type, such as sunflower
petals and pineapple fruit. The all-trans form was found in
approximately half of the samples. The content of neoxanthin were a few percent in these samples examined.
The carotenoid composition in non-photosynthetic
9'-Cis neoxanthin in oxygenic phototrophs
973
Table 3 Absence of neoxanthins in algal classes
Division
Class
Order
Genus and species
Cyanophyta
Cyanophyceae
Chroococcales
Oscillatoriales
Synechococcus sp. PCC7942
Gloeobacter violanus ATCC29082
Oscillatoria agardhii NIES204
Prochlorophyta
Prochlorophyceae
Prochlorales
Prochloron didemni
Glaucophyta
Glaucophyceae
Glaucocystales
Cyanophora paradoxa "
Glaucocystis nostochinearum"
Rhodophyta
Protoflorideophycidae
Porphyridiales
Cyanidium caldarium
Porphyridium purpureum SAG 1380-la
Asterocytis ramosa"
Nemalion multifidum
Gelidium amansii
Grateloupia turuturu
Pachymeniopsis elliptica
Ahnfeltia paradoxa
Delisea fimbriata
Champia expansa
Chondria crassicaulis
Laurencia undulata
Florideophycidae
Goniotrichales
Nemaliales
Gelidiales
Cryptonemiales
Ahnfeltiales
Bonemaisoniales
Rhodymeniales
Ceramiales
Cryptophyta
Cryptophyceae
Cryptomonadales
Chroomonas nordstedtii
Dinophyta
Dinophyceae
Gymnodiniales
Gonyaulacales
Symbiodinium sp. CS163
Alexandorium tamarense
Heterokontophyta
Chrysophyceae
Chromulinales
Synurales
Raphidomonadales
Chromulina ochromonoides CCAP909/1 *
Synura peterseniib
Chattonella antiqua NIES1 c
Heterosigma akashio
Thalassiosira eccentricad
Phaeodactylum tricornutum
Ectocarpus sp.
Petalonia fascia
Myelophycus simplex
Eisenia bicyclis
Undaria pinnatifida
Dictyota dichotoma
Sargassum fulvellum
Pleurochloris meiringensis SAG860-3'
Vischeria stelata SAG887-2
Vischeria punctata SAG887-1
Nannochloropsis oculata CCAP849/1
Raphidophyceae
Bacillariophyceae
Phaeophyceae
Xanthophyceae
Eustigmatophyceae
Haptophyta
Haptophyceae
" Chapman (1966).
* Withers et al. (1981).
c
Kohata and Watanabe (1988).
d
Pennington et al. (1988).
' Biichel and Wilhelm (1993).
Centrales
Raphidineae
Ectocarpales
Scytosiphonales
Dictyosiphoriales
Laminariales
Dictyotales
Fucales
Mischococcales
Eustigmatales
Isochrysidales
Coccosphaerales
Prymnesiales
Pavlovales
Emiliania hyxleyi EMH01
Calyptrosphaera spheroidea CS01
Prymnesium sp. PRY02
Pavlova lutheri CS23
9'-Cis neoxanthin in oxygenic phototrophs
974
Table 4 Geometrical isomerism of neoxanthin in non-photosynthetic organs
DIVISION
Class
Subclass
Infraclass
CONIFEROPHYTA
ANTHOPHYTA
Dicotyledonopsida
Archichlamiidae
Magnoliatae
Order
Genus and species
Organ
or
tissue
Neoxanthin
type"
Taxales
Taxus sp.
Fruit
none
Ranunculales
Nandina domestica
Ranunculus sp.
Trollius europaeus
Dianthus caryophyllus
Mirabilis jalapa
Opuntia sp.
Portulaca oleracea
Actinidia sp.
Carica sp.
Cucurbita sp.
Cucurbita sp.
Viola sp.
Brassica sp.
Diospyros kaki
Kerria japonica
Rosa sp.
Rosa sp.
Sedum lineare
Oenothera odorata
Cornus florida
Citrus unshiu
Citrus paradisi
Impatiens balsamina
Oxalis corniculata
Daucus carota
Capsicum annuum
Lycopersicon escukntum
Lantana camara
Osmanthus asiaticus
Osmanthus fragrans
Chrysanthemum frutescens
Chrysanthemum morifolium
Cosmos bipinnatus
Helianthus annuus
Gerbera hybrida
Taraxacum sp.
Sonchus sp.
Fruit
Petal
Petal
Petal
Petal
Petal
Petal
Fruit
Fruit
Petal
Fruit
Petal
Petal
Fruit
Petal
Petal
Petal
Petal
Petal
Fruit
Fruit
Fruit
Petal
Petal
Root
Fruit
Fruit
Petal
Petal
Petal
Petal
Petal
Petal
Petal
Petal
Petal
Petal
Triticum aestivum
Zea mays
Ananas comosus
Canna generalis
Musa paradisiaca
Iris pseudoacorus
Narcissus pseudo-narcissus
Etholate
Seed
Fruit
Petal
Fruit skin
Petal
Petal
Caryophylliatae
Caryophyllales
Dilleniiatae
Theales
Violales
Rosiatae
Capparales
Ebenales
Rosales
Fabales
Cornales
Sapindales
Sympetalidae
Geraniales
Apiales
Solanales
Lamiales
Scrophulariales
Asterales
Monocotyledonopsida
Commelinidae
Cyperales
Zingiberidae
Bromeliales
Zingiberales
Liliidae
Liliales
cis, 9'-cis form; trans, al\-trans form; none, no neoxanthin.
* Marki-Fischer and Eugster (1990).
skin
skin
skin
skin
cis
trans
trans b
cis
cis
cis
cis
cis & trans
trans
cis & trans
trans
cis & trans
cis
none
cis & trans
trans
trans *
cis & trans
trans
none
none
cis
cis
trans
trans
cis & trans
trans
cis
cis
none
cis
cis & trans
cis
none
cis & trans
cis
none
cis
cis
none
cis & trans
cis
cis & trans
trans
9'-Cis neoxanthin in oxygenic phototrophs
organs could be classified into three types. One type contained pigments similar to those in chloroplasts, including
small amounts of Chls a and b, and these contained only 9'cis neoxanthin, such as carnation and impatiens petals. In
the second type, carotenoid fatty acid esters were major
components, as determined by their HPLC elution profiles,
such as rose and garbera petals, and pineapple fruit. In the
third type, one major carotenoid accounted for more than
50% of the total carotenoids, such as violaxanthin in narcissus petals, lycopene in tomato fruit, and /?-carotene in
carrot root.
Discussion
The distribution of neoxanthin in phototrophic organisms was limited to seed plants, ferns, mosses and green
algae, all of which contain Chls a and b (Table 2), whereas
neoxanthin was not found in other algal classes; Chls a/c
algae (Haptophyta, Heterokontophyta, Dinophyta and
Cryptophyta), Chi a-phycobilin algae (Rhodophyta, Glaucophyta and Cyanophyta), and Prochlorophyta (Table 3).
With regard to the phylogeny of photosynthetic organisms,
the appearance of Chi b is somehow linked to the synthesis
of 9'-cis neoxanthin, except for Prochlorophyta. It is not
clear, however, whether this linkage is inevitable or just
coincidence. Dinophyta have symbiotic algae, such as
Cryptophyceae, Bacillariophyceae and Prasinophyceae, so
in this algae the presence and the geometric type of neoxanthin might depend on the symbiotic algae.
The presence of neoxanthin in Chi a/b algae and the
absence in other algal classes has been reported, whereas in
some Chi a/c algae, the presence of neoxanthin has also
been reported (Goodwin 1980, Bjornland and LiaaenJensen 1989, Rowan 1989), even though the geometric
forms of these algae were not mentioned. The content of
neoxanthin in the Chi a/c algae is reportedly always low (at
most, a few percent of the total). The classification of algae
has been rapidly changing in recent years, thus it is appropriate to re-examine the presence and the geometric form
of neoxanthin using new techniques, as well as to identify
the species. In this sense, our analyses on Chi a/c algae produced better results. The distribution of neoxanthin is consistent with the current phylogeny of algae.
The geometric form of neoxanthin in chloroplasts was
unique among carotenoids. Only the 9'-cis form without
the all-trans form was found in chloroplasts. Other carotenoids, such as violaxanthin, lutein and /?-carotene, take
only all-trans forms (data not shown; Yamamoto and Bassi
1996). With regard to carotenogenesis, the synthesis of neoxanthin from violaxanthin always accompanies the cis
isomerization, because intermediates, i.e., all-trans neoxanthin and 9-cis violaxanthin, could not be found in chloroplasts (Fig. 2). It is not known that 9-cis neoxanthin is metabolized to other carotenoids, thus 9'-cis neoxanthin is the
975
/? -Carotene
X
/3 -Carotene hydroxylase
Zeaxanthin
Violaxanthin de-epoxidase j j l
Zeaxanthin epoxidase
A\\-trans violaxanthin
Neoxanthin synthase
v'
^^
A\\-lrans neoxanthin
Co-isomerase
Cu-isomerase
9-Cis violaxanthin
^a
^
Neoxanthin synthase
9'-Cis neoxanthin
L
Cleavage enzyme (chloroplast)
Cis-xanthoxin
1
ABA2 (cytoplasm)
Civ-ABA-aldehyde
I
ABA3 (cytoplasm)
C/.v-ABA
Fig. 2 Biosynthetic pathway of carotenoids and ABA. I .carotenoid synthesis (chloroplast thylakoid); ft, xanthophyll-cycle
(violaxanthin de-epoxidase, chloroplast lumen); •, ABA synthesis
(Schwartz et al. 1997a, b).
end product of carotenoid synthesis in Chi a/b plants and
algae.
In non-photosynthetic organs, the presence of neoxanthin can be divided into four types; only the 9'-cis form,
only the all-trans form, both forms, or neither (Table 4).
Neoxanthin content was less than a few percent of the total
carotenoid content. It is difficult to find a correlation between the phylogeny and distribution and the geometric
form of neoxanthin in non-photosynthetic organs. Diversity in carotenoid compositions and the content in the nonphotosynthetic organs might be independent of the classification of plants.
The functions of neoxanthin remain unclear. Because
the absorption spectra of both the cis and the trans forms
are almost identical, their spectroscopic and physicochemical characteristics might be similar, which might be responsible for light-harvesting and photoprotection. Neoxanthin
is mainly distributed to PSII in chloroplasts, especially in
LHCII. The Chi b-less mutant of Chlamydomonas reinhardii, which lacks LHC II, does not contain neoxanthin
(Knoetzel et al. 1988), suggesting that neoxanthin is preferentially associated with LHC II. A three-dimensional structure of LHC II has been proposed and two lutein and
12 Chls molecules are found in the LHC II monomer
(Kiihlbrandt et al. 1994). Two lutein molecules are located
in the central part of the LHC II, and stabilize the helix
structure that binds Chls a and b. Although neoxanthin
and violaxanthin are present in sub-stoichiometric amounts
(Bassi et al. 1993), the binding sites of 9'-cis neoxanthin and
all-trans violaxanthin are unknown. There are two possibilities for the binding site(s) of 9'-cis neoxanthin; one is a
976
9'-Cis neoxanthin in oxygenic phototrophs
different site from that occupied by carotenoids in the alltrans form, since 9'-cis neoxanthin and other al\-trans carotenoids have different stereochemical structures (Fig. 1).
The other possibility is the same site, shown for the reaction-center-B865 complex of the aerobic photosynthetic
bacterium, Erythrobacter longus, containing zeaxanthin
(all-trans form) and bacteriorubixanthinal (9'-cis form)
(Noguchi et al. 1992). The overall structure of the complex,
however, is very different from that of LHC II, thus, the
binding site might not be the same.
Neoxanthin associates with several types of LHC II in
addition to the Chi a/b-lutein type; those are, Chi a/bsiphonaxanthin type from Bryopsis maxima (Nakayama
and Okada 1990), Chi a/6-diatoxanthin type from Euglena
gracilis (Cunningham and Schiff 1986), and Chi a/bprasinoxanthin type from Mantoniella squamata (Wilhelm
and Lenartz-Weiler 1987). In these complexes, the major
carotenoid species is variable, however neoxanthin is
always found in the complexes, suggesting that neoxanthin
has a specific, but cryptic, function in LHC II.
Xanthophyll-cycle functions to dissipate light energy
in chloroplasts of higher plants, where violaxanthin de-epoxidase converts violaxanthin to zeaxanthin under high
light conditions (Fig. 2). In vitro, this enzyme also converts
all-trans neoxanthin to all-trans deepoxyneoxanthin, however the 9'-cis form can not be a substrate (Yamamoto and
Higashi 1978). Because all-trans neoxanthin was not found
in chloroplasts, formation of all-trans deepoxyneoxanthin
under high light conditions was also not to be the case.
Even if all-trans deepoxyneoxanthin is formed from an intermediate, it does not seem to be effective for dissipation
of light energy, because its energy level is similar to that of
antheraxanthin (data not shown). The 9-cis form is not involved in the xanthophyll-cycle, thus its accumulation
might partly correlate with other physiological functions.
In contrast, a cleavage enzyme of 9'-cis neoxanthin has
been found in ABA synthesis (Schwartz et al. 1997b) and
its substrate is only the 9'-cis form not the all-trans one,
which results in the production of cis-ABA through cis-xanthoxin (Fig. 2). Only cis-ABA, not trans-ABA, is biologically active (Schwartz et al. 1997b). Therefore, 9'-cis neoxanthin is a suitable substrate for ABA biosynthesis.
All oxygenic phototrophs contain cis-ABA (Hirsch et
al. 1989). On the other hand, two ABA biosynthetic pathways are already known; the carotenoid pathway, i.e.,
cleavage of 9-cis neoxanthin in chloroplasts (Fig.2) and
the sesquiterpene pathway in mold (Zeevaart and Creelman
1988). It remains unclear, however, whether the sesquiterpene pathway also produces cis-ABA in plants and
algae that contain neoxanthin, and whether algae that do
not contain neoxanthin produce cis-ABA by the sesquiterpene pathway or the carotenoid pathway. Discrepancies between the presence of 9'-cis neoxanthin and the biosynthetic pathway(s) of cis-ABA will be the next subject to
be analyzed.
The authors thank M. Ito and Y. Yamano, Kobe University
of Pharmacy, for 'H-NMR measurements, and Alia, I. Enami, M.
Erata, T. Ikawa, T. Katoh, H. Kawai, K. Masamoto, Y. Minami,
H. Miyashita, N. Murata, K. Nakayama, A. Nishigaki, S. Nishitani, T. Ogata, M. Okada, G. Sandmann, the late T. Sasa, N.
Sato, K. Shimada, F. Shimozono, Y. Shiraiwa, T. Tada, M. Takaichi, M. Takamiya, Y. Tamura, M.M. Watanabe, T. Watanabe,
and Y. Yamamoto for cultivation, collection or identification of
biological materials. This work was supported in part by a Grantin-Aid (no. 10640639) to S.T. from Ministry of Education,
Science, Sports and Culture of Japan.
References
Bartlett, L., Klyne, W., Mose, W.P., Scopes, P.M., Galasko, G.,
Mallams, A.K., Weedon, B.C.L., Szabolcs, J. and T6th, G. (1969) Optical rotatory dispersion of carotenoids. J. Chem. Soc. (C) 2527-2544.
Bassi, R., Pineau, B., Dainese, P. and Marquardt, J. (1993) Carotenoidbinding proteins of photosystem II. Eur. J. Biochem. 212: 297-303.
Baumeler, A. and Eugster, C.H. (1992) Synthesis of (6J?,all-£)-neoxanthin
and related allenic carotenoids. Helv. Chim. Ada 75: 773-790.
Bjernland, T. (1982) Chlorophylls and carotenoids of the marine alga
Eutreptiella gymnastica. Phytochemistry 21: 1715-1719.
Bjarnland, T. (1990) Carotenoid structures and lower plant phylogeny. In
Carotenoids: Chemistry and Biology. Edited by Krinsky, N.I.,
Mathews-Roth, M.M. and Taylor, R.F. pp. 21-37. Plenum Press, New
York.
Bjernland, T. and Liaaen-Jensen, S. (1989) Distribution patterns of carotenoids in relation to chromophyte phylogeny and systematics. In The
Chromophyte Algae: Problems and Perspectives. Edited by Green, J.C.,
Leadbeater, B.S.C. and Diver, W.L. pp. 37-60. Clarendon Press, Oxford.
Buchecker, R. and Noack, K. (1995) Circular dichroism. In Carotenoids,
Vol. IB, Spectroscopy. Edited by Britton, G., Liaaen-Jensen, S. and
Pfander, H. pp. 63-116. Birkhauser, Basel.
Biichel, C. and Wilhelm, C. (1993) Isolation and characterization of a photosystem I-associated antenna (LHC I) and a photosystem I-core complex from the chlorophyll c-containing alga Pleurochloris meiringensis
(Xanthophyceae). J. Photochem. Photobiol. B: Biol. 20: 87-93.
Chapman, D.J. (1966) The pigments of the symbiotic algae (cyanomes) of
Cyanophora paradoxa and Glaucocystis nostochinearum and two
Rhodophyceae, Porphyridium aerugineum and Asterocytis ramosa.
Arch. Mikrobiol. 55: 17-25.
Cholnoky, L., Gyorgyfy, K., R6nai, A., Szabolcs, J., T6th, G., Galasko,
G., Mallams, A.K., Waight, E.S. and Weedon, B.C.L. (1969) Carotenoids and related compounds. XXI. Structure of neoxanthin (foliaxanthin). J. Chem. Soc. (C) 1256-1263.
Cunningham, F.X. and Schiff, J.A. (1986) Chlorophyll-protein complexes
from Euglena gracilis and mutants deficient in chlorophyll b. Plant.
Physiol. 80: 223-230.
Englert, G. (1995) NMR Spectroscopy. In Carotenoids, Vol. IB, Spectroscopy. Edited by Britton, G., Liaaen-Jensen, S. and Pfander, H. pp. 147260. Birkhauser, Basel.
Eugster, C.H. (1995) Chemical derivatization: microscale tests for the presence of common functional groups in carotenoid. In Carotenoids, Vol.
1A, Isolation and Analysis. Edited by Britton, G., Liaaen-Jensen, S. and
Pfander, H. pp. 71-80. Birkhauser, Basel.
Goldsmith, T.H. and Krinsky, N.I. (1960) The epoxide nature of the carotenoid, neoxanthin. Nature 188: 491-493.
Goodfellow, D., Moss, G.P., Szabolcs, J., T6th, G. and Weedon, B.C.L.
(1973) Configuration of carotenoid epoxides. Tetrahedr. Lett. 3925-3928.
Goodwin, T.W. (1980) The Biochemistry of the Carotenoids, Vol. 1,
Plants, 2nd Ed. Chapman and Hall, London.
Hirsch, R., Hartung, W. and Gimmler, H. (1989) Abscisic acid content of
algae under stress. Bot. Acta 102: 326-334.
9'-Cis neoxanthin in oxygenic phototrophs
Hlubucek, J.R., Hora, J., Russell, S.W., Toube, T.P. and Weedon,
B.C.L. (1974) Carotenoids and related compounds. XXIX. Stereochemistry and synthesis of the allenic end group. Absolute configuration
of zeaxanthin. J. Chem. Soc, Perkin Trans. 848-852.
Knoetzel, J., Braumann, T. and Grimme, L.H. (1988) Pigment-protein
complexes of green algae: improved methodological steps for the quantification of pigments in pigment-protein complexes derived from the
green algae Chlorelta and Chlamydomonas. J. Photochem. Photobiol.
B.Biol. 1:475-491.
Kohata, K. and Watanabe, M. (1988) Diel changes in the composition of
photosynthetic pigments and cellular carbon and nitrogen in Chattonella
antiqua (Raphidophyceae). / . Phycol. 24: 58-66.
Kiihlbrandt, W., Wang, D.N. and Fujiyoshi, Y. (1994) Atomic model of
plant light-harvesting complex by electron crystallography. Nature 367:
614-621.
Li, Y. and Walton, D.C. (1990) Violaxanthin is an abscisic acids precursor
in water-stressed dark-grown bean leaves. Plant Physiol. 92: 551-559.
Marin, E., Nussaume, L., Quesada, A., Gonneau, M., Sotta, B.,
Hugueney, P., Frey, A. and Marion-Poll, A. (1996) Molecular identification of zeaxanthin epoxidase of Nicotianna plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus
of Arabidopsis thaliana. EMBO J. 15: 2331-2342.
Marki-Fischer, E. and Eugster, C.H. (1990) Neoflor and 6-epineofior from
flowers of TroIIius europaeus; highfleld 'H-NMR spectra of (all-£)-neoxanthin and (9'Z)-neoxanthin. Helv. Chim. Ada 73: 1637-1643.
Nakayama, K. and Okada, M. (1990) Purification and characterization of
light-harvesting chlorophyll a/6-protein complexes of photosystem II
from the green alga, Bryopsis maxima. Plant Cell Physiol. 31: 253-260.
Noguchi, T., Hayashi, H., Shimada, K., Takaichi, S. and Tasumi, M.
(1992) In vivo states and functions of carotenoids in an aerobic photosynthetic bacterium, Erythrobacier longus. Photosynth. Res. 31: 21-30.
Parry, A.D., Babiano, M. J. and Horgan, R. (1990) The role of cis-carotenoids in abscisic acid biosynthesis. Planta 182: 118-128.
Pennington, F., Guillard, R.R.L. and Liaaen-Jensen, S. (1988) Carotenoid
distribution patterns in Bacillariophyceae (diatoms). Biochem. System.
Ecol. 16: 589-592.
Rowan, K.S. (1989) Photosynthetic Pigments of Algae. Cambridge Univer-
977
sity Press, Cambridge.
Sasa, T., Takaichi, S., Hatakeyama, N. and Watanabe, M.M. (1992) A
novel carotenoid ester, loroxanthin dodecenoate, from Pyramimonas
parkeae (Prasinophyceae) and a chlorarachniophycean alga. Plant Cell
Physiol. 33: 921-925.
Schwartz, S.H., Leon-Kloosterziel, K.M., Koornneef, M. and Zeevaart,
J.A.D. (1997a) Biochemical characterization of the aba! and aba3
mutants in Arabidopsis thaliana. Plant Physiol. 114: 161-166.
Schwartz, S.H., Tan, B.C., Gage, D.A., Zeevaart, J.A.D. and McCarty,
D.R. (1997b) Specific oxidative cleavage of carotenoids by VP14 of maize. Science 276: 1872-1874.
Takaichi, S. (1993) Usefulness of field desorption mass spectrometry in
determining molecular masses of carotenoids, natural carotenoid derivatives and their chemical derivatives. Org. Mass Spectrom. 28: 785-788.
Takaichi, S. and Shimada, K. (1992) Characterization of carotenoids in
photosynthetic bacteria. Methods Enzymol. 213: 374-385.
Wilhelm, C. and Lenarz-Weiler, I. (1987) Energy transfer and pigment
composition in three chlorophyll Z?-containing light-harvesting complexes isolated from Mantoniella squamata (Prasinophyceae), Chlorella
fusca (Chlorophyceae) and Sinapis alba. Photosynth. Res. 13: 101-111.
Withers, N.W., Fiksdahl, A., Tuttle, R.C. and Liaaen-Jensen, S. (1981)
Carotenoids of the Chrysophyceae. Comp. Biochem. Physiol. 68B: 345349.
Yamamoto, H.Y. and Bassi, R. (1996) Carotenoids: location and function.
In Oxygenic Photosynthesis: The Light Reactions. Edited by Ort, D.R.
and Yocum, C.F. pp. 539-563. Kluwer Academic Publishers, Dordrecht.
Yamamoto, H.Y. and Higashi, R.M. (1978) Violaxanthin de-epoxidase:
lipid composition and substrate specificity. Arch. Biochem. Biophys.
190: 514-522.
Young, A. J. and Britton, G. (1993) Appendix. In Carotenoids in Photosynthesis. Edited by Young, A.J. and Britton, G. pp. 458-488. Chapmann
& Hall, London.
Zeevaart, J.A.D. and Creelman, R.A. (1988) Metabolism and physiology
of abscisic acid. Annu. Rev. Plant Physiol. Plant Mol. Biol. 39: 439473.
(Received April 27, 1998; Accepted July 7, 1998)