The homeo'c gene AGAMOUS (AG) in Arabidopsis. Presenta'on by: Yang Liu and Zhonghang Zhang (Daisy) February 27th, 2018

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1 The homeo'c gene AGAMOUS (AG) in Arabidopsis Presenta'on by: Yang Liu and Zhonghang Zhang (Daisy) February 27th, 2018

2 Arabidopsis Flower George Haughn, UBC

3 Genes Direc'ng Flower Development in Arabidopsis agamous six stamens six petals carpels a new flower repeat this pattern to form more than 70 organs Bowman et al.1989

4 The characterized mutant allele, ag-1, was isolated after EMS mutagenesis. The AG locus has been mapped to chromosome 4. Object: Cloning and characterization of the AG gene, which was facilitated by a T-DNA insertion mutation.

5 T- DNA Based Inser'onal Mutagenesis Transferred DNA of tumorinducing plasmid of Agrobacterium. From bacteria into plants nuclear DNA genome. A tool for studying gene function in plants. Knock down/ Knock out/ Knock on genes expression.

6 Flower Phenotype of Mutants Insertion mutant: Pedicels of inner flowers are elongated; Greater separation of internal flowers Different genetic backgrounds Inser'on mutant ag- 1 Wild- type Insertion mutant: Lack stamens and carpels A flower within a flower.

7 The Arabidopsis ERECTA Gene ERECTA Gene ER encodes a putative transmembrane receptor kinase that regulates organ shape. Arabidopsis Landsberg erecta (Ler) Ler is one of the most popular ecotypes of Arabidopsis and has been widely used for both molecular and genetic studies. It harbors the erecta (er) mutation and shows an altered organ shape. Ler develops a very compact inflorescence with flowers clustering at the top.

8 Flower Phenotype of Mutants Inser+on mutant: Pedicels of inner flowers are elongated; Greater separa'on of internal flowers Different gene'c backgrounds Inser'on mutant in ER background ag- 1 EMS- induced mutant in Ler (er) background Wild- type

9 Inser&on muta&on and ag- 1 muta&on are allelic Complementation test: Precondition: A single nuclear recessive mutation heterozygous for ag- 1 heterozygous for inser&onal muta&on ag-2 F1 ¼ progeny ag mutant flowers ¾ progeny wild- type flowers allelic Why didn t use a classical complementation test? Homozygous mutants are sterile. If they are not the same gene mutation: F1? All progeny have wild-type flowers.

10 The kanamycin resistance and mutant phenotype Cosegregate Genotype Wild type AG-2/AG-2 Heterozygous AG-2/ag-2 Homozygous ag-2/ag-2 Flower Phenotype Normal Normal flower within a flower Kanamycin Resistance No Yes Yes What could we conclude from this cosegreate analysis? Conclusion: T-DNA can be used as a molecular probe to isolate the AG gene for gene cloning in the next step.

11 Plasmid Rescue SalI restric'on site SalI Diges'on T- DNA AG gene DNA from homozygous ag-2 mutant plants Self Liga'on T- DNA Transforma'on into E. coil and selected on Kanamycin plates Kanamycin rescued clones pcit505

12 Restric'on fragment length polymorphism (RFLP) analysis P ag-1(ler)/ag(ler) heterozygous AG(Nd-O)/AG(Nd-O) Wild-type F1 ag-1(ler)/ag(nd-o) AG (Ler)/AG(Nd-O) F2 ⅛ ag phenotype Self-fertilized ⅞ Wild-type DNA extracted from each mutant; Prepare DNA blots and hybridized with probes.

13 Restric'on fragment length polymorphism (RFLP) analysis EcoR1 digested DNA from each individual BglII digested DNA from each individual

14 Restric'on fragment length polymorphism (RFLP) analysis In 118 chromosomes scored, no crossover were observed between a pcit505 and the ag-1 mutation. The map distance for the linkage of the pcit505 and AG gene is less than 2 CentiMorgan. 2/120 2% T-DNA inserted in or near the AG gene; The plant sequence contained by pcit505 was derived from the AG genomic region.

15 Construc'on of Two Libraries?? The rest sequence information of the ag gene? Construction of DNA library and cdna library of wild-type Arabidopsis Why? pcit505 was used as a probe for screening

16 DNA Library Nuclear DNA fragments (from Arabidopsis Columbia and Landsberg erecta ecotypes) + Vector pcit30 = A cosmid DNA Library of wild-type Arabidopsis sequences Meyerowitz et al. 1992

17 cdna Library λ gt10 insertion vector can carry 8kb into a EcoR1 site in CI gene. EcoR1 site λ λ λ CI gene EcoR1 linker - - cdna fragments - - EcoR1 linker (immature floral buds cdna from wild type Arabidopsis) EcoR1 EcoR1 λ λ λ a cdna fragment λ cdna library of wild- type Arabidopsis flowers

18 Library Screening (pcit505) Clones are isolated and sequenced

19 Transgene Complementa'on + pcit540 ag-2 mutant ag-2 plants transformed with pcit540

20 How many methods used to test that gene cloned was AG gene? 1. RFLP analysis. 2. Transgene complementation.

21 Nucleotide sequence probe from the region flanking the T-DNA insertion site (pcit505) cdna clones from WT 3 10^6 Sequencing 27 of the longest cdnas several overlapping cdna clones corresponding genomic region

22 Nucleo'de sequence of AG cdnas How was the coding region of AG gene deduced? - Deducing from the stop codon and find the longest ORF position of intron sequences

23 Structure of AG gene > 9 exons ranging in size from 42 to 235 bases 8 introns ranging in size from 84 to 2,985 bases the 35kb insertion is in 2,985-base intron

24 Are these sequence represent AG gene? Sequencing ag-1 mutant (Ler) wild type gene (Ler) a single nucleotide change: AG AA at 138- base intron(same gene in ag- 2)

25 The incomplete cdna clones None of the cdna clones are of full length! The deduced ORF con'nues the 5' end of the longest cdna No transla'onal ini'a'on codon has been found The full- length AG protein could be longer than 285 amino- acid Why were the researchers so concerned about obtaining a longer cdna?

26 Finding the complete cdna Many agempts has been done: Nothing! The longest cdnas are of 1,043 bp The polyadenylated messenger RNA is close to 1.1kb This longest cdna is actually near full length why?

27 Hypothesis Since nearly all of the isolated cdnas end within 5 region of a few nucleo'des, sugges'ng that the RNA may have a secondary structure that prevented extending the complete cdna.

28 Riechmann et al.1999

29 Possible AG initiation codons 5 -RACE: can overcome RNA secondary structure problem to extend a few more nucleotides no AUG codon nor interrupted ORF Not AG alleles Non-AUG codon Riechmann et al.1999

30 Non-AUG codon initiation translation AUG codon serves as transla'on start site in vast majority of eukaryo'c genes Non- AUG ini'a'on codon are rare in eukaryo'c cellular CUG, ACG, GUG (several animal and viral mrna) Riechmann et al.1999

31 Two possibility: CUG and ACG *Sequence of 5' region of AG cdna Triplets that differ from ATG in only one nucleotide are boxed Efficiency test: codons fusion with a reporter gene ORF lacking AUG Riechmann et al.1999

32 In vitro test of CUG and ACG AG: ATG codon at 5 end of AG cdna AGATG2: ATG codon at CTG position AGATG2: ATG codon at ACG position Riechmann et al.1999

33 In vitro test of CUG and ACG Engineer ATG at AG cdna CTG position ACG position ACG codon appears to be an efficient initiation codon Riechmann et al.1999

34 In vitro test of CUG and ACG Engineer ATG at AG cdna CTG position ACG position DNA binding test: only the product synthesized from ACG position was active in DNA binding Riechmann et al.1999

35 AG genomic constructs with mutated codons in vivo test Riechmann et al.1999

36 Complementa'on experiment Riechmann et al.1999

37 AGACGm failed to rescue the ag-3 mutant Two possibilities: ACG is the translation initiation codon or Nucleotide change affected RNA stability and/or processing Riechmann et al.1999

38 Complementa'on experiment Riechmann et al.1999

39 Complementa'on experiment CTG CTT: WT ACG ACC: ag-3 phenotype ATGACGm: WT ATGCTGmACGm:WT ACG should be the translation initiation codon! Riechmann et al.1999

40 Immunological detection of AG protein AG translation in vivo is initiated exclusively by the ACG codon. (Found it!) Riechmann et al.1999

41 AG gene expression pattern Different poly(a)+ RNAs hybridize with an AG gene cdna immature floral buds floral stems vegetative tissue

42 AG gene expression pattern strongest signal with immature floral buds 1%signal intensity of that in flower from inflorescence stems and whole plants was detected after long exposure could because a mixture of small number of early developing flowers

43 Organ specific expression? in situ hybridiza'on What is the in situ hybridiza'on? Precise localization of a specific segment of nucleic acid within a histologic section detected by a labeled reporter molecule. principle: complementary base pairing

44 Organ specific expression? in situ hybridiza'on of AG sec'on of WT floral apices + 35 S- labelled an'sense RNA of AG cdna

45 Results from in situ hybridization hybridiza'on was localized at stamens and carpels In consistent with the RNA blot result (flower) AG gene is preferentially expressed in stamens and carpels

46 Comparison of Amino- acid sequence AG is 80% similar with the shared region of these proteins. AG protein is a transcription factor.

47 MADS-BOX

48 Plant MADS-box A conserved mo'f encodes DNA binding domain of eukaryo'c transcrip'on factors Vital role in controlling floral organ development

49 56 amino acids 9 identical among all family members Rounsley et al. 1995

50 AGAMOUS Protein Structure of protein of AGAMOUS gene in Arabidopsis AG encodes a C-class transcription factor of the MADS-box family that plays crucial roles in Arabidopsis thaliana development by regulating the stamens and carpels, the repression of A-class genes, and floral meristem determinacy. The MADS domain: DNA binding domain of eukaryotic transcription factors The I (interverning)and K domains (coiled-coil structure): involved in protein protein interactions

51 MADS- box gene and ABC Model The homeotic genes in ABC model: A genes: APETALA 2 (AP2), AP1; C gene: AGAMOUS (AG); B genes: APETALA 3 (AP3) and PISTILLATA (PI). With the exception of AP2, all other genes are MADS-box genes family and encode MADS domain proteins that function as transcription factors.

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53 Flower shape modifica'on by suppressing AG gene The flower shape of chrysanthemum (Chrysanthemum morifolium) was modified by suppressing the chrysanthemum-agamous (CAG) gene, which might be a C gene, with an antisense transgene. Aida et al. 2008

54 Summary Identification of AG T-DNA insertion mutant Cloning of AG gene by plasmid rescue Identification of AG gene nucleotide sequence AG is a flower-specific homeotic gene AG protein resembles transcription factors

55 Ques&on?