ACTA BIOLOGICA CRACOVIENSIA

16 TH INTERNATIONAL SYMPOSIUM ON CAROTENOIDS
ed in tissue [2]. Carotenoid-binding proteins have been described
extensively in plants and bacteria, however much less is known
about their roles in invertebrates and vertebrates.
A method is being developed for the purification of
carotenoid-binding proteins from the roe of E. chloroticus.
Density-adjusted ultra-centrifugation is used to separate the roe
homogenate into a low-density lipid-rich fraction and a clarified
solution. In addition to large quantities of protein, both fractions
contain significant amounts of the major roe carotenoid echinenone
and are being investigated for the presence of carotenoidbinding
proteins. It is hypothesised that the lipid fraction may
contain lipoproteins that bind non-specifically to large numbers
of carotenoid molecules. The solution fraction is more likely to
contain small soluble carotenoid-binding proteins of the lipocalin
type, in which the interaction between protein and carotenoid is
both specific and stoichiometric.
The techniques of solution phase iso-electric focusing and ion
exchange chromatography have been investigated as purification
procedures and have proved promising in co-purifying carotenoid
and protein. The next phase of the procedure will utilise sizeexclusion
chromatography to increase the purity of carotenoidbinding
proteins. As a final purification step, an immobilised
carotenoid affinity chromatography column [3] will be developed,
to selectively purify carotenoid-binding proteins. It is then hoped
that the isolated proteins will be analysed and identified by
MALDI TOF/TOF mass spectrometry and structures will be elucidated
by crystallographic techniques.
REFERENCES
SYMONDS RC, KELLY MS, SUCKLING CC, YOUNG AJ. 2009. Carotenoids in
the gonad and gut of the edible sea urchin Psammechinus miliaris.
Aquaculture. 288: 120-125.
BRITTON G. 1995. Structure and properties of carotenoids in relation to
function. FASEB J., 9:1551-1558.
RAO MN, GHOSH P LAKSHMAN MR. 1997. Purification and partial characterisation
of a cellular carotenoid-binding protein from Ferret
liver. J. Biol. Chem. 272: 24455-24460.
6.15.
Geranylgeranyl diphosphate synthase isoforms
involved in carotenoid biosynthesis in
Arabidopsis thaliana
M. Águila Ruiz-Sola, Manuel Rodríguez-Concepción
Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-
UAB. Campus UAB Bellaterra, 08193 Barcelona, Spain,
aguila.ruiz@cid.csic.es, manuel.rodriguez@cid.csic.es
Geranylgeranyl diphosphate (GGPP) is the precursor for the
biosynthesis of carotenoids and other important plant isoprenoids
such as gibberellins and the side chain of chlorophylls,
tocopherols, and isoprenoid quinones. GGPP is produced by the
enzyme GGPP synthase (GGDS), encoded by ten genes in
Arabidopsis thaliana. We hypothesize that different GGDS
isozymes might be involved in the production of specific isoprenoids,
including carotenoids, by channeling GGPP to their
corresponding biosynthetic pathways. Carotenoids are synthesized
in plastids and they preferentially accumulate in photosynthetic
tissues of Arabidopsis. Based on these features and on the
role of carotenoids as photoprotectants, GGDS isoforms involved
in their production are expected to be targeted to plastids, highly
expressed in green tissues, and light-induced. Among the seven
Arabidopsis genes encoding true GGDS enzymes with a predicted
plastid-targeting peptide, only At4g36810 (herein referred to
as GGDS1) met the expression criteria. The total loss of GGDS1
function is lethal, but we have identified a mutant (ggds1-1) in
which a 40% decrease in the expression of GGDS1 results in
smaller plants with a significant reduction in carotenoid levels.
These results suggested that GGDS1 is the main (or only) isoform
involved in carotenoid biosynthesis. Alternatively, the expression
patterns of other GGDS isoforms might not be appropriate to rescue
the loss of GGDS1 in the mutants. To test this latter possibility
we have generated constructs for expression of several plastid-targeted
active GGDS isoforms under the control of the
GGDS1 promoter or a constitutive promoter (35S). GFP fusions
will also be expressed for complementation experiments. Lines
complementing the ggds1-1 mutant with GGDS1:GGDS1,
35S:GGDS1 and 35S:GGDS1-GFP constructs are already available,
and immunoprecipitation experiments using those overexpressing
GGDS1-GFP are in progress to identify proteins that
interact with GGDS1 in in vivo complexes. It is expected that we
will find enzymes of the carotenoid pathway among them. Similar
experiments using lines overexpressing other GFP-tagged GGDS
isoforms should provide useful information on whether protein
partners are shared among different GGDS-containing complexes.
Together, these experiments should serve to verify what GGPP
isoforms are specifically involved in the production of
carotenoids, which would optimize future biotechnological strategies
aimed at obtaining new plant varieties rich in these compounds.
Our latest progress along these lines will be presented.
6.16.
α-Carotene and its derivatives have a sole
chirality in phototrophic organisms?
SESSION 6
Shinichi Takaichi1 , Akio Murakami2 , Mari Mochimaru3 ,
Akiko Yokoyama 4
1Department of Biology, Nippon Medical School, Kosugi-cho 2,
Nakahara, Kawasaki 211-0063, Japan, takaichi@nms.ac.jp
2Kobe University of Research Center of Inland Seas, Awaji 656-
2401, Japan
3Department of Natural Science, Komazawa University,
Komazawa, Setagaya 154-8525, Japan
4Graduate School of Life and Environment Sciences, University of
Tsukuba, Tennoudai, Tsukuba 305-8572, Japan
Eukaryotic phototrophic organisms necessarily synthesize not
only chlorophylls but also some carotenoids, which can be divided
into two groups; β-carotene and its derivatives (zeaxanthin,
violaxanthin, neoxanthin, fucoxanthin, peridinin, diadinoxanthin,
etc.), and α-carotene and its derivatives (lutein, loroxanthin,
siphonaxanthin, prasinoxanthin, etc.). Distribution of α-carotene
and its derivatives is reported to be limited in some taxonomic
groups of phototrophic organisms. In addition, only (6'R)-type of
α-carotene and its derivatives have been reported from algae and
land plants, although C-6' in α-carotene, between ε-end group and
conjugated double bonds, is chiral, (6'R)- and (6'S)-types. To confirm
the reliability of chirality, we re-examined distribution of αcarotene
and its derivatives, and analyzed their C-6' chirality
using CD or NMR after purification of the carotenoids.
We found α-carotene and/or its derivatives from Rhodophyceae
(macrophytic group), Cryptophyceae, Euglenophyceae, Chlorarachniophyceae,
Prasinophyceae, Chlorophyceae, Ulvophyceae,
Trevouxiophyceae, Charophyceae, and land plants, while we
could not detected them from Glaucophyceae, Rhodophyceae
(unicellular group), Chryosophyceae, Raphidophyceae, Bacillariophyceae,
Phaeophyceae, Xanthophyceae, Eustigmatophyceae,
Haptophyceae, and Dinophyceae. In addition, loroxanthin and
siphonaxanthin, which are synthesized from lutein, were found
from Euglenophyceae, Chlorarachniophyceae, Prasinophyceae,
Chlorophyceae, and Ulvophyceae. We analyzed chirality of
α-carotene and/or its derivatives from around 40 species
described above, and found they had only (6'R)-type.
92 ACTA BIOLOGICA CRACOVIENSIA Series Botanica