Download Lecture 15 Genetic Manipulation of Flower pigmentation

Lecture 15
Genetic Manipulation of Flower pigmentation
Anthocyanins
As an alternative to traditional breeding techniques, uniquely colored flowers can
be developed by manipulating the genes for enzymes in the anthocyanin biosynthesis
pathway. Anthocyanins, which are a class of flavonoids, are the most common type of
flower pigment. They are synthesized from the amino acid phenylalanine by a series of
enzyme-catalyzed reactions. The color of the flower is determined by the chemical side
chain substitutions of different chemical structures, with the cynanidin derivatives
producing more red and the delphinidin derivatives producing more (Fig.1).
While the petunia enzyme dihydroflavonol 4-reductase can convert colorless
dihydroquercetin to red cyanidin -3-glucoside and colorless dihydromyricetin to blue
delphinidin -3-glucoside, it cannot use colorless dihydrokaempferol as a substrate.
(Fig 1).However, when petunias were transformed with a dihydroflavonol 4-reductase
gene from maize, the flowers of the transgenic plants was brick red –orange. This unique
color, which had never been seen before in petunias, was due to the production of
pelargonidin-3-glucoside by the transgenic plants.
Four plants(roses,caranations,tulips,and chrysanthemums) account for approximately
70%of the cut-flower industry worldwide.Moreover,since genetic transformation
protocols have been worked out for these four plants, they should become the focus of
efforts to develop plants with unique colors. For example, transgenic chrysanthemums
with both sense and antisense constructs of the chrysanthemum chalcone synthase cDNA
have been produced. Chalcone synthase catalyzes the first step in anthocynanin
biosyntheis .(Fig 1).It is expected that both the sense and the antisense cDNA will
suppress chalcone synthase gene expression in transgenic plants and produce white
flowers instead of the normal pink. “Sense suppression”, which is also called
“cosuppression”, occurs when an additional copy of an endogenous gene prevents the
accumulation of the mRNA from the endogenous gene. On the other hand, the antisense
chalcone synthase RNA should block translation of endogenous chalcone synthase
mRNA.
Fig.1 Biosynthesis of anthocyanins.
CHS- chalcone synthase; CHI-chalcone isomerase ; F3H-flavonone 3-hydroxylase
F3’H-flavonoid 3’-hydroxyase; F3’5’H-flavonoid 3’5’-hydroxylase;
DFR-dihydroflavonol 4 –reductase; 3GT, UDP-glucose: flavonoid 3-O-glucosyl
transferase
The sense and antisense constructs were placed under the control of the
cauliflower mosaic virus 35S promoter on a binary Ti plasmid vector and then introduced
into plant cells. Three of the 133 sense transformants and three of the 83 antisense
transformants produced white flowers, which indicated that endogenous chalcone
synthase gene expression and, as a consequence, anthocyanin synthesis had been
suppressed. The white-flowering plants were propagated vegetatively through cuttings,
and approximately 90 to 98% of the plants continued to produce white flowers when
planted in the field. This work is an important step toward the development of
commercial flower varieties with novel colors.
Manipulation of carotenoids astaxanthin
The carotenoids astaxanthin ,which provides the characteristic pink color to
salmon,trout,and shrimp,is synthesized by marine bacteria and micro algae and then
passed on to fish through the food chain. More important, astaxanthin protects salmon
and trout eggs from damage by UV radiation and improves the survival and growth rate
of juveniles. Most likely, these properties of astaxanthin are related to its function as a
powerful antioxidant. However, when fish are grown in aquaculture they are separated
from the natural food chain and astaxanthin must be added to their feed in order to impart
the typical pink color to their flesh. Currently, astaxanthin is chemically synthesized and
accounts for approximately 15% of the total cost of salmon farming.
To produce astaxanthin biologically, one group of researchers first cloned a
cDNA encoding the enzyme β-carotene Ketolase (β-C-4 oxygenase) from the unicellular
green alga Haematococcus pluvialis. When this cDNA was expressed in an organism that
contains β-carotene and the gene for β-carotene hydrolase, astaxanthin was synthesized.
By appropriate genetic manipulation, astaxanthin has been synthesized in tobacco
flowers. The cDNA for the algal β-carotene ketolase was fused to DNA encoding a
chloroplast transit peptide and used to transform tobacco plants. To limit expression of
astaxanthin to flowers and fruits, the cDNA for the algal β-carotene ketolase was fused to
the promoter of the tomato pds gene, which encodes phytoene desaturase.To increase the
expression of the cDNA for the algal β-carotene ketolase, the DNA fragment carrying
the pds promoter was reduced in size and then fused to a β-glucuronidase gene.
The construct, which had a 305-bp deletion from the5’ terminus of the pds promoter,
showed a decrease in β-glucuronidase activity in leaves, sepals, and petals and a very
large increase in activity in flower ovaries (nectaries).To obtain maximal gene
expression ,305-bp deleted pds promoter was placed upstream of the cDNA for the algal
β-carotene ketolase. The net result of all these genetic manipulations was that, once the
introduced T-DNA had been inserted into the plant genomic DNA, the algal β-carotene
ketolase together with a transit peptide was inserted through the chromoplast
membrane,with the transit peptide being removed in the process .Once inside the
chromoplast , the algal β-carotene ketolase worked in concert with the endogenous
β-carotene hydroxylase to convert the β-carotene to astaxanthin,which accumulated in the
flower nectaries.The advantage to producing asataxanthin in plants can store large
amounts of carotenoids inside cells in lipid vesicles within the plastids. Thus, plants can
accumulate 10- to 50-fold greater concentrations of carotenoids than microorganisms,
whose membranes are damaged by high concentrations of carotenoids. These
manipulations have the potiental to dramatically lower the cost of the astaxanthin
that is used in salmon farming.