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ABCDE Model of Flower Development and its Utility

Abhay Kumar Gaurav10459

Ph. D. 2nd Year

Seminar: GP 691

Division of GeneticsIndian Agricultural Research Institute, New Delhi

Introduction In 1991, E S. Coen. & E. M. Meyerowitz proposed the ABC Model: To explain how floral whorls develop in Arabidopsis thaliana and Antirrhinum majusFlowers of most Eudicot species are composed of 4 floral organ types:

Sepals Petals Stamens (Androecium- Male) andCarpels (Gynoecium- Female)

These 4 components are all arranged in individual whorls around the meristemMost of the genes of ABCDE model are MADS-box genes

Fig: Arabidopsis showing 4 floral organs

Carpels

Stamens

Petals

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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

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History of Flower Development ModelIn

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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)

ABCDE Model of Flower DevelopmentIn

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ABCDE Model of Flower Development

(Dornelas & Dornelas 2005)

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Sepals

Mutants

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Mutations in Floral Organ Identify Genes

AP1 & AP2

AP3 & PI AG SEPWild TypeIndi

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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 MutantsSEPALLATTA mutant

(Sepal or leaves)

Trends in Plnt science August 2000, Vol. 5, No. 8

Arabidopsis

D- Class MutantsSHATTERPROOF mutant

VariationsFigure: 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 ofthe 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

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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

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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.

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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 plantsUpper 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 regionThe 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

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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

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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

Introduction

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.

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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

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