Recent Advances in Genetic Improvement of Flower Colour

Speaker: Arvind Kumar Verma Ph.D. Scholar, IARI, New Delhi

Chairperson: Dr. S.S. Sindhu (Principal Scientist)

Colour and colour patternsFlower colour: most important trait, dictating consumer attraction

Role of colour:

Attraction of pollinators

Function in photosynthesis

In human health as antioxidants and precursors of vitamin A

Protecting tissue against photooxidative damage

Resistant to biotic and abiotic stress

Symbiotic plant-microbe interaction

Act as intermediary for other compounds

Colour pattern: Differential accumulation of pigment(s)

Pigment Class

Compound Types Compound Examples Typical Colours

Porphyrins Chlorophyll Chlorophyll a and b Green

Flavonoids Anthocyanins Pelargonidin, Cyanidin, Delphinidin, Peonidin

Petunidin, Malvidin

Red, Blue, violet

Anthoxanthins Flavonols Kaempferol, Quercetin, Fisetin, Kaempferide, Morin, Myricetin, Myricitrin, Rutin

Yellow

Flavones Apigenin, Biacalein, Chrysin, Diosmetin, Flavone, Luteolin

Yellow

Isoflavonones Diadzin, Genistein, Enterodiol, Coumestrol, Biochanin

 

Flavonones Eriodictyol, HesperidinNaringin, Naringenin

Colour less co-pigments

Flavans Biflavan, Catechin, Epicatechin,

Colour less co- pigments

Carotenoids Carotenes Lycopene, α-carotene, β-carotene, γ-carotene

Yellow, Orange, Red

Xanthophylls Lutein, Cryptoxanthin, Zeaxanthin, Neoxanthin, Rhodoxanthin, Violaxanthin, Canthaxanthin, Astaxanthin,

 

Major Pigments in Plants

Betalains Betacyanins Reddish to Violet

Betaxanthins miraxanthin and portulaxanthin

Yellow to Orange

Chlorophyllsandcarotenoidsare inchloroplasts

Anthocyaninsare in thevacuole

Site of colour accumulation

Grotewold, 2006

LDOX/ANS= leucoanthocyanidin dioxygenase/anthocyanidin synthase.

Carotenoid biosynthesis pathway

GA3P: glyceraldehyde-3-phosphate, DXR: 1-deoxy-D-xylulose 5-phosphate reductoisomerase.DXS: 1-deoxy-D-xylulose 5-phosphate synthase. MEP: 2-C-methyl-D-erythritol 4-phosphate. IPP: isopentenyl diphosphate. IPI: isopentenyl diphosphate isomerase. GGPP: geranylgeranyl diphosphate. PSY: phytoene synthase(P), CrtB: phytoene synthase (B). PDS: phytoene desaturase (P). ZDS: γ-carotene desaturase (P). ZISO: γ-carotene isomerase (P). CrtISO: carotene isomerase (P). CrtI: phytoene desaturase/isomerase (P). LCY-b: β-cyclase (P). LCY-e: α-cyclase (P). BETA: chromoplast-specific beta-cyclase (P). CYP97B, CYP97C: cytochrome P450 carotene hydroxylases (P). CHY1, CHY2: non-heme carotene hydroxylases (P). CrtO: ketolase (algal/cyanobacterial). CrtW: ketolase (B). ZEP: zeaxanthin epoxidase (P).VDE: violaxanthin de-epoxidase (P). CaCCS: capsanthin/capsorubin synthase (P).

Grotewold, 2006

Factors on flower colour perceptionpH of the vacuole:pH of the vacuole is acidic

Small changes of pH have visible effects on flower colour

Some plants genes have been identified influencing pH

Metal ions:Metal complexing has a blueing effect on flower colour

Colour change from red to blue in hydrangea sepals is most likely due to complexing of Dp 3-glucoside with

aluminium ion and 3-caffeoylquinic acid

Co-pigmentation:Co-pigmentation of anthocyanins with flavones, flavonols and other compounds

Primula, where the contribution of flavonols to flower colour is genetically controlled (gene B). Co-

pigmented flowers (BB) give a mauve colour, whereas in the absence of flavonols (bb) maroon flowers are

formed

Co-occurrence of anthocyanins and yellow pigmentsMixtures of Ans and yellow flavonoids were found in the orange-yellow or orange-red flowers of antirrhinum and bronze flower colour of helichrysum

Tanaka et al., 2009

Regulatory region Coding region

Where? When?How Much?

Protein (Enzyme)

Red Pigment

Genes containregulatory region and

coding region

Springob et al., 2003

Genes involved in pigment synthesis1.structural (enzyme) genes2.regulatory genes

Enzyme Enzyme GeneGene Species Species

CHSCHS ChsChs Antirrhinum, Chrysanthemum, Orchid, Rosa, DianthusAntirrhinum, Chrysanthemum, Orchid, Rosa, Dianthus

CHICHI ChiChi Antirrhinum, Petunia, Eustoma, DianthusAntirrhinum, Petunia, Eustoma, Dianthus

F3HF3H F3hF3h Antirrhinum, Calistephus, Chrysanthemum, Dianthus, OrchidAntirrhinum, Calistephus, Chrysanthemum, Dianthus, Orchid

F3’HF3’H F3’hF3’h Antirrhinum, Dianthus, PetuniaAntirrhinum, Dianthus, Petunia

F3’5’HF3’5’H F3’5’hF3’5’h Calistephus, Eustoma, PetuniaCalistephus, Eustoma, Petunia

FLSFLS FlsFls Petunia, RosaPetunia, Rosa

FNSFNS FnsIIFnsII Antirrhinum, GerberaAntirrhinum, Gerbera

DFRDFR DfrDfr Antirrhinum, Calistephus, Gerbera, Orchid, Dianthus, PetuniaAntirrhinum, Calistephus, Gerbera, Orchid, Dianthus, Petunia

ANSANS AnsAns Antirrhinum, Calistephus, PetuniaAntirrhinum, Calistephus, Petunia

GTGT 3Gt3Gt Antirrhinum, GentianaAntirrhinum, Gentiana

GTSGTS Gts Gts PetuniaPetunia

1.structural (enzyme) genes: is a gene that codes for any RNA or protein product other than a regulatory protein.

Vainstein, 2004

Two classes of regulatory genes identifiedTF with MYB domainTF with MYC/bHLH motif

Infuence the type, intensity and pattern of flavonoid accumulation but donot encode flavonoid enzyme

Regulatory genes

PlantPlant GeneGene

Myb MycMyb Myc

Petunia Rosea,Mixta DeliaPetunia Rosea,Mixta Delia

Gerbera Gmyc IGerbera Gmyc I

Perilla Mybp IPerilla Mybp I

Petunia Phmyb3, An2, An4 An1Petunia Phmyb3, An2, An4 An1

(Vainstein, 2004)

GeneGene Enzyme Enzyme DxsDxs

DxrDxr

LpiLpi

GpsGps

FpsFps

GgpsGgps

PsyPsy

ZdsZds

Lcy-bLcy-b

Lcy-cLcy-c

NsyNsy

CcsCcs

Ptox Ptox

Dexoyxylulose 5-phosphate synthaseDexoyxylulose 5-phosphate synthase

Dexoyxylulose 5-phosphate reducoisomeraseDexoyxylulose 5-phosphate reducoisomerase

LytB proteinLytB protein

Geranyl diphosphate synthaseGeranyl diphosphate synthase

Fernsyl diphosphate synthaseFernsyl diphosphate synthase

Geranylgeranyl diphosphate synthaseGeranylgeranyl diphosphate synthase

Phytoene synthasePhytoene synthase

ββ-Carotene dessaturase-Carotene dessaturase

Lycopene bita-cyclaseLycopene bita-cyclase

Lycopene – Lycopene – ββcyclasecyclase

Neoxanthin synthaseNeoxanthin synthase

Capsanthin capsorubin synthaseCapsanthin capsorubin synthase

Plastid terminal oxysidasePlastid terminal oxysidase

Genes involved in carotene pigment synthesis

(Vainstein, 2004)

Genetic Improvement: involves changing the plant’s genetic makeup

Making deliberate crosses between two parents

Mutation

Polyploidy

Introducing genes of desired traits into recipient plant by

methods other than sexual crosses

Genetic Improvement of Flower Colour

Conventional breeding

Hybridization:=x

Traditional doner

Desired gene

Commercial variety New variety

Many genes are transferred

Dominance Co dominance

CASE STUDYStudies on inter specific hybridization for transferring yellow colour in Dianthus plumarius (2n=6x=90).

Gatt  et al. (2005) Observation and result x =

Analysis of the flower pigments :Yellow flower colour of D. knappii resulted from flavone and flavonol glycosides.

Yellow carnations were chalcones.

Thus, the F1 hybrids with D. knappii were yellow because they contained the same pigments as D. knappii

but the hybrids with the carnations were pink due to their ability to convert chalcones through

dihydroflavones and then to anthocyanins.

Dianthus plumarius Yellow carnation

x =

D. knappiiDianthus plumarius

Many different genes are involved incontrolling the synthesis of the pigments. In a multi-step process.A B C D E G

H I J L

If a single enzyme is not present and earlystep in the synthetic pathway will not happen.

A x B C D E G

H I J L

Mutation:

a c

ed

b Two types of flower in a single branch

Datta et al., 2001

CASE STUDY

A quick method for establishment of solid mutant

Phenotypic expression in flower after mutationPhenotypic expression in flower after mutation

Ajay

Prasad et at., 2008Pusa AnmolA success story at IARI…………………

M1

M2

In vitro isolation, purification, rapid bulking and field establishment of a radio-mutant

Polyploidy

Auto polyploidy Allo polyploidy

Natural origin or colchiploidy

Mitotic doubling of chromosome number- colchiploidyFailure of meiotic reduction i.e. unreduced gamete- natural

CASE STUDYThe effect of induced polyploidy on the flavonols of Petunia ‘Mitchell' Griesbach and Kamo, 1996

increasing the relative concentration of the major metabolite quercetin-3-sophoroside and decreasing the relativeconcentration of the minor metabolite quercetin-3,7-diglucoside.

Analytical HPLC data for the flavonols of haploid, diploid and tetraploid cytotypes of Petunia

Polyploidy was inducedthrough in vitro colchicine treatment

Q3 = quercetin-3-glucoside, Q32 = quercetin-3-sophoroside, Q7 = quercetin-7-glucoside, Q3,7 = quercetin-3,7-diglucoside, 32,7 = quercetin-3-sophoroside-7-glucoside, Qc32,7 - quercetin-3-caffeoylsophoroside-7-glucoside.

Colour modification Over expression of structural genes Use of sense or antisense enzyme construct Inhibit production of key biosynthetic enzyme Add an enzyme of a particular biosynthetic step

Why transgenic crops are important ?

Limitations of conventional breeding for attaining the desirable traits

Development of organisms that express a “novel” trait: normally not

found in the species

Genetic engineering: Manipulation of plant genome through recombinant DNA technology to alter plant characteristics

Chandler and Brugliera, 2011

Gene transfer methods

Indirect Direct

Most widely usedMore economicalMore efficientTransformation success is 80-85%

Agrobacterium mediated gene transfer

Particle bombardment or

micro projectile

Direct DNA delivery by

Microinjection or PEG

mediated uptake

Ultrasonication

Electroporation

Electroporotic uptake

Chandler and Brugliera, 2011

Gene transformation

Colour modification through antisense RNA technology

Inhibition of gene expression by antisense RNA

Flower color modification of Petunia hybrida commercialvarieties by metabolic engineering

Flower colour changed from purple to almost white by the down-regulation of the CHS gene

Surfinia Purple Mini

CASE STUDY

Tsuda et al., 2004

Surfinia Pure White

Flowers of transgenic Surfinia Purple Mini plant harboring antisense DFR gene

Expression of DFR gene change the expression of the flavonol synthase and flavone synthase gene

Contd…..

C

Generating White colour

Down regulation of an anthocyanin biosynthesis structural gene (Using an antisense gentian CHS gene)

Petunia (Van der Krol et al., 1988), Gerbera (Elomaa et al., 1993), Chrysanthemum (Courtney-Gutterson et al., 1994), Rose (Gutterson, 1995), Carnation (Gutterson, 1995), Lisianthus (Deroles et al., 1998;Kato et al., 2001) Torenia (Aida et al., 2000a;)More recently, gentian (Nishihara et al. 2003)

Generating red to orange flowersIntroduction maize Dfr gene Petunia (Meyer et al.,1987)

Isolated the blue gene in 1991 and patented in 1992.

Petunia gene  didn’t work in roses.

Transplanting genes is easy to say but hard to do in the lab, so they used their techniques on carnations- a much easier species to manipulate than roses.

In 1996, Florigene developed mauve-coloured carnation, FLORIGENE Moondust and it was the world's first genetically modified flower on sale

In 1997, developed second genetically-modified carnation, FLORIGENE Moonshadow with a richer and true purple colour.

Successfully developed a range of transgenic violet carnations by introduction of a F3′5′H gene together with a petunia DFR gene into a DFR-deficient white carnation

Blue carnation

Fukui et al., 2003

BLUE-VIOLET CARNATION Introduction of a F3’ 5’H gene together with a petunia DFR gene into DFR

deficient white carnation

 FLORIGENE MOONSERIES

 FLORIGENE Moonshadow

 FLORIGENE Moonvista

 FLORIGENE Moonlite

 FLORIGENE Moonshade

 FLORIGENE Moondust

 FLORIGENE Moonaqua

 FLORIGENE Moonique

 FLORIGENE Moonpearl

 FLORIGENE Moonvelvet

 FLORIGENE Moonberry

Blue rose

Why a natural rose could not have the true blue colour?

"Flavonoid 3', 5'- hydroxylase" is one of the key enzymes involved in the flavonoid

biosynthesis for blue colour development deficient in rose

Did’t have Dp

pH of cell sap is 4.0-4.5

Cell sap is govern by 7 genes and each gene contributes 0.5 pH

Blue Gene Technology

In April of 2005, Suntory Ltd. and Florigen Ltd. announced the production of a blue

rose by introducing three transformation constructs simultaneously into roses:

www.suntory.com and www.florigene.com.au

Blue Gene Technology

www.suntory.com and www.florigene.com.au

The transgenic rose variety ‘‘Applause’’ was commercially released in Japan in 2009 (Tanaka et al., 2009)

Suntory selected APPLAUSE as the name for its blue rose as a symbol of congratulations for those whose dreams have come true, as well as of encouragement for those pursuing a dream, whatever it may be.

The price for a single blue rose is about $22 to $33. Source: The Japan Today

Flower colour modifications by regulating flavonoid biosynthesis

Conclusion:Classical breeding methods have been extensively used to develop cultivars with

flowers varying in both the colour and its intensity

Spectral difference in flower colour is mainly determined by the ratio of different classes of pigments and other factors such as vacuolar pH, co-pigmentation and metal ion complexation

Knowledge of flower colouration at the biochemical and molecular level has made it possible to developed novel colour

Genetic engineering overcome almost all the limitations of traditional breeding approaches

The expression of genes transferred across genera is not always predictable and so requires considerable trial to arrive at stable phenotype of commercial interest