1. ABCDE flower model

IndianAgriculturalResearchInstitute,NewDelhi
ABCDE Model of Flower Development
and its Utility
Abhay Kumar Gaurav
10459
Ph. D. 2nd Year
Seminar: GP 691
Division of Genetics
Indian Agricultural Research Institute, New Delhi

 In 1991, E S. Coen. & E. M. Meyerowitz proposed the ABC Model: To explain
how floral whorls develop in Arabidopsis thaliana and Antirrhinum majus
Flowers of most Eudicot species are composed of 4 floral organ types:
Sepals
Petals
Stamens (Androecium- Male) and
Carpels (Gynoecium- Female)
These 4 components are all arranged in individual whorls around the meristem
Most of the genes of ABCDE model are MADS-box genes
Fig: Arabidopsis showing
4 floral organs
Carpels
Stamens
Petals
Sepals

MADS-box
 The MADS box is a conserved sequence motif found in genes which
comprise the MADS-box gene family
 The MADS box encodes the DNA-binding MADS domain
 The length of the MADS-box are in the range of 168 to 180 base pairs
 Origin:
 MCM1 from the budding yeast, Saccharomyces cerevisiae,
 AGAMOUS from the thale cress Arabidopsis thaliana,
 DEFICIENS from the snapdragon Antirrhinum majus
 SRF from the human Homo sapiens
In plants, MADS-box genes are involved in controlling all major aspects
of development, including male & female gametophyte development,
embryo and seed development, as well as root, flower and fruit
development, floral organ identity and flowering time determination
IndianAgriculturalResearchInstitute,New
Delhi

History of Flower Development Model
Coen, E. S., and Meyerowitz, E. M. The war of the whorls: Genetic interactions
controlling flower development, Nature, 1991, 353: 31 -37.
ABC Model
Colombo, L., Franken, J., Koetje, E. et al., The Petunia MADS box gene FBP11
determines ovule identity, Plant Cell, 1995, 7: 1859-1868
ABCD Model
Theissen, G., Development of floral organ identity: Stories from the MADS
house, Curr. Opin. Plant Biol., 2001, 4: 75-85.
ABCDE Model
Theissen, G., Saedler, H., Floral quartets, Nature, 2001, 409: 469-471
Quartets Model

This model developed on the basis of Arabidopsis thaliana and Snapdragon
mutants. Most of the genes of ABCDE model are MADS-box genes.
Class A genes (APETALA1, APETALA2) controls sepal development & together
with class B genes, regulates the formation of petals. Antirrhinum: LIPLESS 1 and
2
Class B genes (e.g. PISTILLATA, and APETALA3), together with class C genes,
mediates stamen development. Antirrhinum: DEFICIENS (DEF) and GLOBOSA
(GLO)
Class C genes (e.g., AGAMOUS), determines the formation of carpel.
Antirrhinum: PLENA (PLE)
The class D genes (e.g., SEEDSTICK, and SHATTERPROOF) specify the identity of
the ovule. Petunia: FBP7 and FBP11
Class E genes (e.g., SEPALLATA), expressed in the entire floral meristem, & are
necessary. (SEP1, SEP2, SEP3 and SEP4)
Delhi

(Dornelas & Dornelas 2005)

c)
Sepals
Mutants

Delhi

Mutations in Floral Organ Identify Genes
AP1 & AP2 AP3 & PI AG SEPWild Type

A-Class Mutant
APETALA 1 or APETALA2
No Petals (only carpels and stamens)
B-Class Mutant
APETALA 3 or PISTILLATA
No petals and a lot of Pistils

C- Class Mutants
AGAMOUS mutant
No Gametes
(Se-Pe-Pe-Se-Pe-Pe)
E- Class Mutants
SEPALLATTA mutant
(Sepal or leaves)
Trends in Plnt science August 2000, Vol. 5, No. 8
Arabidopsis
D- Class Mutants
SHATTERPROOF mutant

Variations
Figure:
Modified ABC model proposed
by van Tunen et al, 1993 to
explains the flower morphology
of Tulip.
 Class B genes are expressed in
whorl 1 as well as whorls 2 and 3,
thus the organs of whorl 1 have
the same petaloid character as
those of whorl 2.
 The expression pattern of
the class B genes (TGGLO,
TGDEFA, and TGDEFB) from
Tulipa gesneriana.
Te, tepals; Ot, outer tepals;
It, inner tepals; St, stamens;
Ca, carpels.
Evolution of MADS-box genes in Monocots. The Scientific World JOURNAL (2007) 7, 268–279

The function domains are
indicated by colors: A, red,
B, yellow, C, purple, D,
green and E, light-blue.
•Rice homologs of arabidopsis ABC
genes:
•Apetala 1 (A)
• OsMADS14
• OsMADS15
•Apetala 3 (B)
• OsMADS16
•Agamous (C)
• OsMADS3
• OsMADS58
Rice Science, 2013, 20(2): 79−87
http://mob.wmmrc.nl/role-transcription-factors-floral-organ-identity/abcs-rice

Utility
 Development of male sterile line
Mutating B gene. Ex: Antirrhinum (defience)
 Development of double flower in ornamentals
Mutating C gene. Ex; Petunia, Antirrhinum (ple)
 Control of fruit/seed shattering
Mutating D gene (stk & shp)
 Development of unique flower form
Mutating E gene (sepallatta) Ex: Green Rose
Arabidopsis

CASE STUDIES

c)
Case Study-1
Objectives:
1. Study of the effects of the gene silencing of C-class MADS-box genes by using a
VIGS system on flower phenotypes in petunia cultivars.
2. Comparison between Large petaloid stamens induced by silencing both
pMADS3 and FBP6 with small petaloid stamens induced by silencing only
pMADS3.

c)
 Double flowers enhances the commercial value of Petunia hybrida. As
ornamental plants, double flowers with large petaloid stamens and/or
new flowers at inner whorls are desired
 Double flower formation: Mainly due to conversion of stamen and
carpel into petal and new inflorescence
 C-class genes along with B-class genes, specify stamen identity in whorl
3. A/C to ABC model of floral organ identity (Coen and Meyerowitz,
1991)
 Suppressing C-class genes in whorl 3 results in the conversion of stamen
into petal and carpel into new inflorescence
 C-class genes belong to AG-clade of the large MADS-box gene family

Petunia has two genes belonging to the AG-clade:
 euAG- subclade gene PETUNIA MADS-BOX GENE3 (pMADS3) and
 PLENA- subclade gene FLORAL BINDING PROTEIN6 (FBP6)
(Angenent et al., 2009; Tsuchimoto et al., 1993)
 Silencing of either pMADS3 or FBP6 resulted in partial loss of stamen
identity and slightly altered carpel morphology. No double flower
 Flowers with both pMADS3 and FBP6 silenced exhibited near-complete
loss of both stamen and pistil identities . They were completely converted
into large petaloid tissues in whorl 3, new flowers were formed instead of
carpels in whorl 4, and ornamental double flowers were produced

Materials and Methods
Plant materials:
 VIGS treatments of each of the C-class MADS-box genes, pMADS3 and FBP6, and
of pMADS3 & FBP6 conducted in four petunia cultivars, ‘Cutie Blue’, ‘Fantasy
Blue’, ‘Picobella Blue’,and ‘Mambo Purple’
Plasmid construction:
 The tobacco rattle virus (TRV)-based VIGS system (suppression of the anthocyanin
pathway via chalcone synthase silencing as reporter as it produced white flower)
 Vector: pTRV1 and pTRV2 VIGS
 PhCHS was amplified and cloned into the EcoR1 site of pTRV2 vector
 The non-conserved regions of petunia C-class genes, pMADS3 and FBP6, were
amplified using the primers and cloned into the SmaI site of pTRV2 PhCHS vector
individually to generate constructs for silencing pMADS3 and FBP6 separately and
fused to generate a construct for silencing pMADS3 and FBP6 simultaneously

Agroinoculation of TRV vectors:
 Virus infection was carried out by
means of the Agrobacterium-
mediated infection of petunias
 Young leaves of 3-week old petunia
plants were inoculated
Virus-induced gene silencing
(VIGS)
Creation of engineered viruses
carrying sequences corresponding to
the host gene to be silenced
Infection leads to synthesis of viral
dsRNA
This activates the anti-viral RNA
silencing pathway
Results in down-regulation of the host
gene transcript.
* The Tobacco Rattle Virus (TRV)
provides the most robust results in
terms of efficiency, ease of application,
and absence of disease symptoms.

Results and Discussion
In ‘Picobella Blue’ and ‘Mambo Purple’: No white flower was noted
(Unknown genetic background, Chen et al., 2004)
In ‘Cutie Blue’ and ‘Fantasy Blue’: Completely white double flowers were
observed, indicating the strong and complete silencing
In flowers inoculated with either pMADS3-VIGS or FBP6-VIGS,
morphologically significant but small conversions in whorls 3 & 4 were
observed
In flowers of pMADS3-VIGS inoculated petunias, anthers converted into
small petaloid tissues but filaments retained their original structure
In flowers of FBP6-VIGS inoculated petunias, the stamens were almost
unaffected
In petunias inoculated with pMADS3/FBP6-VIGS, prominent double flowers
with highly ornamental appearance formed. Complete loss of stamen
identity was observed. Both anthers and filaments were completely
converted into petaloid tissues

Fig. 1. Morphological changes in
flowers of P. hybrida cv ‘Cutie Blue’
inoculated with pTRV2-
PhCHS/pMADS3 (pMADS3-VIGS) and
pTRV2-PhCHS/pMADS3/FBP6
(pMADS3/FBP6-VIGS).
(a) VIGS-untreated control flower;
(b) Stamens and a carpel of non-VIGS
flower;
(c) pMADS3-VIGS flower (white and
blue mixed color);
(d) Petaloid stamens and a carpel of
pMADS3-VIGS flower;
(e) pMADS3/FBP6-VIGS flower
(white);
(f) Petaloid stamens and a carpel of
pMADS3/FBP6-VIGS flower
(white).

Fig. 2. Morphological changes in
flowers of P. hybrida cv
‘Fantasy Blue’, ‘Picobella Blue’,
and ‘Mambo Purple’ inoculated
with pTRV2-
PhCHS/pMADS3/FBP6
(pMADS3/FBP6-VIGS).
(a–c) ‘Fantasy Blue’;
(d–f) ‘Picobella Blue’;
(g–i) ‘Mambo Purple’;
(a, d and g) VIGS-untreated
control flowers;
(b, e and h) pMADS3/FBP6-VIGS
flowers;
(c, f and i) stamens and carpels or
converted new flowers of
pMADS3/FBP6-VIGS flowers.

Fig. 3. New flower formation in whorl 4 and
from axil of whorl 3 in a double flower of P.
hybrida cv ‘Mambo Purple’ inoculated with
(pMADS3/FBP6-VIGS).
(a) An opened double flower with a second
new flower in whorl 4
(b) An opened second new flower;
(c) Fused corolla (left), a carpel (center),
and petaloid stamens (right) of the
second flower;
(d) An ectopic new flower emerging from
the axil of whorl 3;
(e) An unconverted stamen (left) and petal-
like tissues of the ectopic new flower.
Flowers inoculated with pMADS3/FBP6-VIGS in whorl 4, carpels converted into new flower
(Cultivar-dependent)
In 50% of the double flowers of ‘Mambo Purple’, a 2nd new flower arose instead of a carpel. This
process was repeated, generating 3rd & 4th new flowers. It exhibited a voluminous and
decorative appearance with a high commercial value.

The surface areas of petaloid
stamens in pMADS3/FBP6-VIGS
plants were more than 10 times
as large as those in pMADS3-
VIGS plants
Upper limb-like region of the
large petaloid stamens in
pMADS3/FBP6-VIGS plants
accounted for > 90% of the total
area, so it was mostly due to the
development of this region
The average sizes of epidermal
cells in plants inoculated with
pMADS3/FBP6-VIGS were only
1.5 times as large as those in
plants inoculated with pMADS3-
VIGS

c)
 Double flowers can be induced by virus-induced gene silencing
(VIGS) of two C-class MADS-box genes, pMADS3 and FBP6
 Large petaloid stamens induced by pMADS3/FBP6-VIGS were
compared with small petaloid stamens induced by pMADS3-VIGS
 New flower formation in the inner whorl of flowers silenced in both
pMADS3 and FBP6 gene is cultivar-dependent
 They are valuable for future breeding of petunia cultivars bearing
decorative double flowers with large petaloid stamens and inner
new secondary flowers

c)
Case Study-2
Objectives:
1. To study the functions of GmMADS28 gene which plays pivotal roles in the
regulation of floral organ number and petal identity in soybean.
2. Induction of male sterility caused by the ectopic expression of GmMADS28
gene and its use in plant breeding

 Soybean (Glycine max [L.] Merr.) is an imp crop that provides protein and oil
 Understanding of the processes of its reproductive development and the
identification of the genes responsible for this is imp for soybean breeding
 For this 28 flower-enriched transcription factors in soybean is identified
through a microarray analysis
 Out of 28, a soybean homolog of Arabidopsis AGL9/SEP3 (GmMADS28 )was
selected for further analysis
 GmMADS28 plays pivotal roles in the regulation of floral organ number and
petal identity
 The sterility caused by the ectopic expression of GmMADS28 offers a
promising approach to genetically produce new sterile material that could
potentially be applied in crop hybrid breeding

Results
GmMADS28 is a member of Class E MADS-box genes
 GmMADS28 is 1,026 bp in length and contains an ORF of 732 bp.
 Comparison of the GmMADS28 cDNA and soybean genomic DNA sequences
suggested that it contains 8 exons & 7 introns
 GmMADS28 and other plant SEP genes share the same numbers of exons and
introns; each exon size is highly conserved, whereas the intron size is
divergent
 To address relationship of GmMADS28 to other plant SEP proteins, a
neighbor-joining phylogenetic tree was constructed based on the alignment of
amino acid sequences.
 GmMADS28 was grouped into the plant SEP3 subfamily. In particular,
GmMADS28 was closer to Lotus japonicas LjSEP3 and Arabidopsis SEP3.
 GmMADS28 is an E gene; based on the tree and highest sequence identity
between GmMADS28 and SEP3.

Figure: Characterization of GmMADS28. (a) Real-time qPCR analysis of GmMADS28 in soybean leaves, roots,
flowers and pods. (b) Exon-intron structures of GmMADS28 and Lotus japonicas LjSEP3, Arabidopsis SEP3
and maize ZAG2. The black and white boxes represent exons and introns, respectively. (c) Phylogenetic
analysis of GmMADS28 and other plant SEP proteins. (d) Subcellular localization of GmMADS28-GFP fusion
protein. (GmMADS28 is localized in the nucleus)

Transcripts of GmMADS28 accumulate
predominantly in reproductive organs
 The expression analysis showed
that the GmMADS28 mRNA was
mainly detected in the
reproductive organs viz. flower,
seed, & pod, but not in the leaf or
root
 GmMADS28 was detected in all
four organs and shows highest
expression in petals
Fig: GmMADS28 expression in different
tissues of soybean by semi-quantitative RT-
PCR analysis. Actin gene was used as the
reference gene.
Figure: In situ hybridization of GmMADS28 in soybean
developing flowers. (a-e) The longitudinal sections of
flowers at differential developmental stages hybridized
with an antisense (b-f) or sense (a) probe. (e) The cross
section of flowers at the mature stage. fm, floral
meristem; sp, sepal primordium; pp, petal primordium;
stp, stamen primordium; cp, carpel primordium; st,
stamen; pe, petal; ca, carpel; an, anther; o, ovule.
In situ localization of GmMADS28
transcript

Ectopic expression of GmMADS28 in Tobacco
Fig: Promotes early flowering. (a) Selected
transgenic plants at flowering stage. (b)
no. of leaves (c) plant height of wild-type
& 35S:GmMADS28 plants when flowering
Fig: Phenotypes at reproductive stage. (a, e) WT flower with
five petals and stamens. (b, c, f) 35S: GmMADS28 flowers with
6 petals and stamens. (d) 35S: antisense GmMADS28 flower
with 4 petals. (g) WT with 5 sepals. (h) 5S:GmMADS28 with 6
sepals. (i) 35S:GmMADS28 converts sepal to petal. (j)
35S:GmMADS28 converts stamen to petal. Arrows: stamens

35S:GmMADS28 plants are sterile due to physical alterations in floral structure
Fig: 35S:GmMADS28 develops the shortened and curly filaments.
(a, b) WT stamens can touch the stigma.
(c, d) 35S:GmMADS28 stamens are shortened and curly and cannot touch the stigma.
(e) Comparison of filament length of WT and 35S:GmMADS28. The blue column:
length of curly filaments while the red column: straightened filaments.
(f, g) The epidermal cells of WT and 35S:GmMADS28 filaments were analyzed by SEM

Fig: 35S:GmMADS28 fails to release the pollens.
(a) WT anthers with pollens. (b) 35S:GmMADS28 anthers
without pollens covering. (c) Comparison of anther
development b/w WT and 35S:GmMADS28. The anther
dehiscence was observed at stage 4 of WT anthers .Ep,
epidermic cell; M, middle layer cells; En, endothecium
cells; P, pollens; C, parenchyma cells.
• Tobacco homologs of the flowering time genes
SOC1 and LEAFY, A gene AGL8/FUL, and B gene
DEF accumulated more transgenic compared to
WT leaves.
• GmMADS28 might directly regulate the
expression of these genes, and control flowering
time and petal identity.

Figure: Expression of GmMADS28 in soybean mutant NJS-
10Hfs. (a) The flower without petals of the mutant. The
arrow indicates the conversion of the stamens into petals.
(b) Real-time qPCR analysis of GmMADS28 expression in
petals of the mutant NJS-10Hfs and those of WT. (c) Real-
time qPCR analysis of GmMADS28 expression in stamens of
the mutant NJS-10Hfs and those of WT.
 The 35S:GmMADS28 transgenic tobacco plants
exhibited the conversion of stamens to petals, which
is also the phenotype of soybean mutant NJS-10Hfs
 The expression of GmMADS28 in the stamens and
petals in NJS-10Hfs was higher than in wild-type NJS-
10Hff
 Up-regulation of GmMADS28 may play a critical
role in the conversion of stamens to petals in this
soybean mutant
GmMADS28 is involved in the conversion of
stamens to petals in a soybean mutant

 Through microarray analysis, a flower-enriched gene GmMADS28 encoding a
MADS-box transcription factor was cloned from soybean
 GmMADS28 belongs to E-type gene and play a wide role in reproductive
development
 Constitutive expression of GmMADS28 in tobacco caused a number of
reproductive development changes, like early flowering, conversion of
stamens and sepals to petals, increased numbers of sepal, petal and stamens
etc
 Ectopic expression of GmMADS28 caused sterility due to the shortened and
curly stalks and the failure of pollen release from the anthers.
 It is thus a potential target gene for engineering the male sterile plants.

 The ABCDE model has been successful in explaining how a small number of
regulatory genes, acting alone and in combination, specify the identity of the
floral organs
 By silencing the genes of ABCDE model one can developed flowers with
altered flower morphology like double flower, green rose, male sterile plant
etc
 Male sterile plants can be of greater use in hybrid seed production
 If SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2) are mutated, the
seed pods fail to shatter, or burst. They can be inserted into rapeseed or
other Cole crops to prevent pods from shattering

c)

1. ABCDE flower model

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