ACTA BIOLOGICA CRACOVIENSIA

BIOSYNTHESIS, GENETICS, AND METABOLISM OF CAROTENOIDS
REFERENCES
TASAKAY, GOMBOS Z, NISHIYAMA Y, MOHANTY P, OHBA T, OHKI K, MURATA N.
1996. EMBO J 15: 6416-6425.
MASAMOTO K, WADA H, KANEKO T, TAKAICHI S. 2001. Plant Cell Physiol
42: 1398-1402.
SOZER Ö, KOMENDA J, UGHY B, DOMONKOS I, LACZKÓ-DOBOS H, MALEC P,
GOMBOS Z, KIS M. 2010. Plant Cell Physiol 51: 823-835.
6.12.
Activation by light of carotenoid biosynthesis
in the fungus Neurospora crassa
Eva M. Luque, Luis M. Corrochano
Departamento de Genética, Universidad de Sevilla, Apartado
1095, 41080 Sevilla, Spain, eluque2@us.es, corrochano@us.es
The ascomycete fungus Neurospora crassa accumulates the
carotenoid neurosporaxathin in vegetative mycelia after exposure
to blue light, presumably to protect the cell from excessive light.
Blue light is perceived by the White Collar Complex (WCC), a photoreceptor
and transcription factor complex that binds the promoters
of light-regulated genes to activate transcription. The WCC
is composed of the blue-light photoreceptor WC-1 and its partner
WC-2. Additional photoreceptors have been characterized in
Neurospora crassa. These include two red-light photoreceptors
(the phytochromes PHY-1 and PHY-2), a blue-light photoreceptor
(the cryptochrome CRY-1), and the rhodopsin NOP-1. The WCC
is the main photoreceptor in Neurospora as wc mutants are
defective in all the responses to light. The other photoreceptors
should play secondary roles in Neurospora photoreception as
deletion of the corresponding genes does not impair fungal vision.
Mycelia of the wild-type strain of N. crassa accumulates
143 μg/g dry mass of carotenoids after one day of light exposure,
compared to 3 μg/g dry mass of carotenoids in mycelia kept in the
dark. The accumulation of carotenoids after one day of light in
the photoreceptor mutants was similar to that in the wild-type
strain, with the exception of wc-1 or wc-2 mutants that failed to
accumulate any carotenoids, as expected. A clear reduction in the
amount of carotenoids accumulated after light exposure was
observed in a strain with a mutation in the ve-1 gene, a homolog
of a repressor of photomorphogenesis in the fungus Aspergillus
nidulans. Our results confirmed the secondary role for the
Neurospora photoreceptors in the activation by light of
carotenoid accumulation.
The activation of carotenoid accumulation by light is a complex
response. Carotenoid accumulation is observed with light
intensities higher than 0.1 J/m 2 but the response saturates and
increases again after exposing mycelia to light of 100 J/m 2 . The
presence of two components in photocarotenogenesis suggested
the action of additional photoreceptors optimized to operate at
different light intensities. We have assayed the presence of the two
components in the photocarotenogenesis of the photoreceptor
mutants of N. crassa. Our results show that the secondary photoreceptors
play a key role in the reception of low-intensity light
in combination with the WCC.
Vol. 53, suppl. 1, 2011
17–22 July 2011, Krakow, Poland
6.13.
Purification of biologically active violaxanthin
de-epoxidase from Escherichia coli
Monika Olchawa-Pajor1 , Dariusz Latowski1 ,
Paulina Kuczyńska1,2 , Monika Bojko1 , Kazimierz Strzałka1 1Department of Plant Physiology and Biochemistry, Jagiellonian
University, Gronostajowa 7, 30-387 Kraków, Poland,
olchawa.pajor@gmail.com, dariuszlatowski@gmail.com,
kuczynska.paul@gmail.com, M.Bojko@uj.edu.pl,
kazimierzstrzalka@gmail.com
2Department of Plant Physiology, Institute of Biology, Pedagogical
University, Podchorążych 2, 30-084 Kraków, Poland,
kuczynska.paul@gmail.com
Violaxanthin de-epoxidase (VDE) is one of two enzymes of the
xanthophyll cycle, an important and widely distributed photoprotective
mechanism in plants. VDE catalyzes de-epoxidation of violaxanthin
(Vx) to zeaxanthin (Zx) via the intermediate antheraxanthin
(Ax).
Traditionally, VDE is isolated mainly from plants thylakoids.
However, plants have low contents of this enzyme and therefore a
lot of plant materials is needed for VDE isolation.
Moreover, the existing isolation procedures are not satisfactory
since the amount of the obtained enzyme and the level of its
purity are low.
In the presented studies isolation and purification of VDE
from transgenic E coli strain C43 have been developed. E. coli
was transformed by the gene of VDE from Arabidopsis thaliana
tagged with 6xHis. After induction, VDE gene expression resulted
in high concentration of the enzyme as evidenced by SDS-PAGE
and Western blot analysis.
Purification of VDE has been performed using Ni-NTA Affinity
Resin. Analysis by SDS-PAGE indicated presence of VDE both in
washed and eluted fractions. In the eluted fraction concentration
of VDE was lower, but purity of sample was the highest.
Purified enzyme has been used to catalyze conversion of Vx to
Zx in the in vitro system. Biological activity VDE was tested by
HPLC-method. The de-epoxidation of Vx and Ax, catalyzed by
obtained enzyme was observed both for enzyme with 6xHis tag
and after its removal by thrombin digestion.
6.14.
A search for carotenoid-binding proteins in the
New Zealand sea urchin Evechinus chloroticus
(Kina)
Jodi Pilbrow, Daniel Garama, Alan Carne
Department of Biochemistry, University of Otago, 710 Cumberland
Street, Dunedin, New Zealand 9016, jodi.pilbrow@otago.ac.nz,
daniel.garama@otago.ac.nz, alan.carne@otago.ac.nz
The sea urchin Evechinus chloroticus, locally known as Kina, is
endemic to costal New Zealand. Kina are harvested commercially
as their edible roe (gonad) is considered a delicacy on both local
and international markets. The revenue attained by roe on the
market is in proportion to the desirability of pigmentation, which
appears to be due to varying quantities of carotenoids chemically
modified in and transported from the viscera via protein interactions
[1].
Carotenoid-binding proteins are likely to have an important
role in the pigmentation of sea urchin roe. In addition to acting as
metabolic enzymes, carotenoid-binding proteins may have a variety
of important roles, including; cell surface receptors, transmembrane
transporters and stabilisation of carotenoids deposit-
91