ACTA BIOLOGICA CRACOVIENSIA

16 TH INTERNATIONAL SYMPOSIUM ON CAROTENOIDS
6.9.
Modification of carotenoid pigments in Iris
germanica L. by overexpression of phytoene
synthase gene (crtB) from Pantoea agglomerans
Zoran Jeknić1 , Sladjana Jevremović2 , Angelina Subotić2 ,
Tony HH Chen1 1Department of Horticulture, Oregon State University, Corvallis,
Oregon, 97331, USA, jeknicz@hort.oregonstate.edu,
chent@hort.oregonstate.edu
2Institute for Biological Research "Siniša Stanković", University of
Belgrade, Bulevar despota Stefana 142, 11060 Belgrade, Serbia,
sladja@ibiss.bg.ac.rs
Iris germanica L. is one of the most horticulturally important,
tall, bearded irises. Flower color in irises is determined by two
distinct biochemical pathways. The carotenoid pathway imparts
yellow, orange and pink while anthocyanin pathway produces
blue, violet and purple flowers. Stoppage in one of the early steps
in either of two biosynthetic pathways is probably responsible for
white flowers. Naturally, there are no truly red iris flowers and
conventional breeding methods have so far failed to generate
them. Genetic engineering provides a means of introducing new
traits into iris by expanding the gene pool beyond what has been
available in iris genome. With a goal of developing red iris flowers,
we transformed the pink iris cultivar 'Fire Bride' with a bacterial
phytoene synthase gene (crtB) from Pantoea agglomerans,
fused to Rubisco small subunit transit peptide from tobacco, and
under the control of several different promoters (pCaMV35S,
E35S-PchsA, AP3 and Llccs). This approach aimed to increase the
flow of metabolites into the carotenoid pathway that would ultimately
lead to the elevated levels of lycopene and a "deeper" red
color. Overexpression of the crtB in iris cv 'Fire Bride' produced a
color change in callus tissue from yellow to orange-red and red.
Carotenoid analysis by HPLC confirmed that the major red pigment
in the transgenic callus was lycopene. Transgenic plants
were regenerated successfully from calli transformed with crtB
driven by AP3 and Llccs promoters and grown to maturity. In both
cases, the most prominent color changes occurred in ovaries and
anthers. Naturally green ovaries turned deep orange and snowwhite
anthers turned pink. Unexpectedly however, the intensity of
pink color in flowers of both types of transgenic plants decreased
compared to wild type. Future studies will focus on determining
tissue specific expression of crtB transgene under AP3 and Llccs
promoters by RT-PCR and correlating expression levels to qualitative
and quantitative changes of carotenoid pigments.
6.10.
Structural and biochemical analyses of the
carotenoid biosynthesis enzymes CRTI and
CRTISO
Halim Jubran, Varda Mann, Joseph Hirschberg
Department of Genetics, Alexander Silberman Institute Life
Sciences, The Hebrew University of Jerusalem, Jerusalem, 91904,
ISRAEL, halim.jubran@mail.huji.ac.il
The plant enzyme carotene isomerase (CRTISO) and the bacterial
phytoene desaturase (CRTI) have structural similarities in size
and amino acid sequence. CRTI catalyzes four desaturation steps
along with cis to trans isomerization of the polyene chain, while
converting phytoene into all-trans lycopene. In contrast, CRTISO
only catalyses the cis to trans isomerization of cis-neurosporene
and cis-lycopene. Our study is aiming at discovering the struc-
SESSION 6
tural features in these enzymes that determine their different
functions. In the absence of crystallographic three-dimensional
structures, we have introduced specific mutations in these
enzymes in order to unravel amino acid residues that are associated
with specific catalytic functions. Chimeric polypeptides of
CRTISO and CRTI were tested in E. coli for desaturation and isomerization
activities. Our results indicate specific domains in
each of the enzymes that are imperative for their activities. A
domain of 44 amino acids near the FAD-binding motif in CRTI
was tested with specific amino acid substitutions. Several amino
acids within this domain were demonstrated crucial for the catalytic
activity of the enzyme. Substitution of these residues with
those that occur in the same position in CRTISO abolished or
reduced enzyme activity and exposed cis-ζ-carotene and transneurosporene
as intermediates in phytoene desaturation by the
mutated CRTI.
6.11.
An EPR study of thylakoid membrane dynamics
in mutants of the carotenoid biosynthesis
pathway of Synechocystis sp. PCC6803
Kinga Kłodawska1 , Przemysław Malec1 , Mihály Kis2 ,
Zoltán Gombos2 , Kazimierz Strzałka1 1Department of Plant Physiology and Biochemistry, Faculty of
Biochemistry, Biophysics and Biotechnology, Jagiellonian
University, 30-387 Krakow
2Institute of Plant Biology, Biological Research Center, Hungarian
Academy of Sciences, 6701, Szeged, Hungary
Thylakoid membranes are the site of light-dependent reactions of
photosynthesis in plants and cyanobacteria.The photosynthetic
activity has been found to be significantly altered in
Synechocystis mutants blocked at different steps of carotenoid
biosynthesis.
The rigidity of thylakoid membranes isolated both from wild
type Synechocystis and from mutants in genes encoding acyl-lipid
desaturases and/or selected enzymes of carotenoid biosynthesis
pathway, was studied by EPR spectroscopy using 5-SASL and 16-
SASL spin probes. Cyanobacteria were cultivated at 25°C and
35°C at different light regimes: photoautotrophically (PAG) and/or
in light-activated heterotrophic conditions (LAHG).
RO mutant is not able to synthesize xanthophylls because of
disruption of genes encoding β-carotene hydroxylase CrtR and βcarotene
ketolase CrtO. ROAD mutant has the same characteristic
with additional disruption of genes encoding acyl-lipid desaturases
A and D responsible for introducing double bonds in Δ12
and Δ6 positions of C 18 fatty acids [1]. ΔcrtH mutant is not able
to synthesize cis to trans carotene isomerase. This mutant cultivated
under LAHG conditions produces cis-carotenes and small
amounts of trans-carotenes but no xanthophylls, whereas under
PAG conditions is not distinguishable from the wild type [2].
ΔcrtH/B mutant is carotenoidless mutant with a disruption in
crtB gene encoding phytoene synthase, in ΔcrtH background [3].
EPR spectra of spin labeled thylakoid membranes were
recorded at temperature range from 5°C to 65°C. Order parameters
and rotational correlation times were calculated from the
spectra.
The values of the measured parameters obtained for membranes
from all analyzed mutants were not significantly different
in comparison to the wild type Synechocystis. These results suggest
that the changes both in carotenoid composition and in the
content of unsaturated glycerolipids have not a substantial effect
on the rigidity of thylakoid membranes in cyanobacteria under
both LAHG and PAG culture conditions.
90 ACTA BIOLOGICA CRACOVIENSIA Series Botanica