1. ABCDE flower model

This presentation deals about the ABC flower model.

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

2. Introduction 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 Indian Agricultural Research Institute, New Delhi

3. 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: M CM1 from the budding yeast,  Saccharomyces cerevisiae , A GAMOUS from the thale cress  Arabidopsis thaliana, D EFICIENS from the snapdragon  Antirrhinum majus S RF 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 Indian Agricultural Research Institute, New Delhi

4. History of Flower Development Model Indian Agricultural Research Institute, New Delhi 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

5. 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 Development Indian Agricultural Research Institute, New Delhi

6. Indian Agricultural Research Institute, New Delhi ABCDE Model of Flower Development (Dornelas & Dornelas 2005)

7. Indian Agricultural Research Institute, New Delhi   c) Sepals Mutants

8. Indian Agricultural Research Institute, New Delhi

9. Mutations in Floral Organ Identify Genes AP1 & AP2 AP3 & PI AG SEP Wild Type Indian Agricultural Research Institute, New Delhi

10. 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

11. 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

12. 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 Indian Agricultural Research Institute, New Delhi

13. 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

14. 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

15. Case studies Indian Agricultural Research Institute, New Delhi

16. Indian Agricultural Research Institute, New Delhi   c) Case Study-1 Objectives: 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. Comparison between Large petaloid stamens induced by silencing both pMADS3 and FBP6 with small petaloid stamens induced by silencing only pMADS3.

17. Introduction Indian Agricultural Research Institute, New Delhi   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

18. 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

19. 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

20. 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.

21. 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

22. 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). VIGS-untreated control flower; Stamens and a carpel of non-VIGS flower; pMADS3-VIGS flower (white and blue mixed color); Petaloid stamens and a carpel of pMADS3-VIGS flower; pMADS3/FBP6-VIGS flower (white); Petaloid stamens and a carpel of pMADS3/FBP6-VIGS flower (white).

23. 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.

24. 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). An opened double flower with a second new flower in whorl 4 An opened second new flower; Fused corolla (left), a carpel (center), and petaloid stamens (right) of the second flower; An ectopic new flower emerging from the axil of whorl 3; 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 2 nd new flower arose instead of a carpel. This process was repeated, generating 3 rd & 4 th new flowers. It exhibited a voluminous and decorative appearance with a high commercial value.

25. 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

26. Inferences Indian Agricultural Research Institute, New Delhi   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

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

28. 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

29. 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.

30. 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)

31. 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

32. 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

33. 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

34. 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.

35. 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

36. 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. Inferences Indian Agricultural Research Institute, New Delhi

37. 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 Conclusion Indian Agricultural Research Institute, New Delhi

38. Indian Agricultural Research Institute, New Delhi   c)