Genetic and molecular analysis of two new loci controlling flowering in garden pea, Pisum sativum . A. S. M. Mainul Hasan PhD candidate School of Biological Sciences University of Tasmania
General biology of flowering ❖ Plant organ producing flowers is known as inflorescence. ❖ Shoot/Vegetative apical meristem (SAM) transforms into an inflorescence meristem at the reproductive stage. ❖ Timing and synchronization with season is important. ❖ Plants require exposure to specific photoperiods/day length. ❖ Other important environmental factors – ➢ Vernalization (exposure to cold) ➢ Ambient temperature ❖ Endogenous cues- ➢ Circadian clock ➢ Developmental age ➢ Gibberellin ➢ Sugar, Hormones http://slideplayer.com/slide/7009388/
Flowering time mechanism in important crop plants Flowering time mechanism in the model system, Arabidopsis thaliana ❖ Knowledge developed in the past decade. AP1 LFY Shoot apex ❖ Mobile signal called florigen later discovered as a protein, FLOWERING LOCUS T (FT). ➢ Cereals: rice, maize, sorghum, barley, wheat FT ❖ Integrates environmental factors. FD ➢ Legumes: barrel medic, soybean, pea ❖ Expressed in leaves and moves to shoot apex ➢ Nightshades: potato, tomato ❖ Forms a complex with FD to induce floral ❖ The process is somewhat evolutionary FT meristem identity genes. Leaf conserved. FLC CO ❖ Regulated by: Daylength – CONSTANS ( CO ) ? ❖ However, diverged mechanism have been ? Vernalization – FLC evolved in these plants. Vernalization Photoperiod Circadian clock ? ? Amb.temp. Gibberellin Arabidopsis thaliana ? Autonomous ? Age
Pea ( Pisum sativum ) ❖ One of the most agronomically important legume, member of Fabaceae family. ❖ Domesticated in fertile crescent/middle east. ❖ Good source of carbohydrate, proteins and vitamins. ❖ Self-pollinating, annual plant species. ❖ Vernalization responsive, long day plant. Source: http://mapsofworld10.xyz/global-map/ ❖ Model species for flowering time studies in legumes. Source: http://www.slideshare.net/ipga/brian-clancey-presentation Source: http://www.pulseaus.com.au/growing-pulses/bmp/field-pea
Current knowledge on flowering time in pea, Pisum sativum Strategy: Gene discovery by mutant analysis Early - Flowering mutants Late - Flowering mutants late4 Courtesy: Jim Weller Courtesy: Jim Weller A. thaliana : PHYA GIGANTEA FTa1 LUX ELF4 ELF3b Genes that promote flowering Genes that repress flowering
Relevant background information of the project ❖ Two novel loci promoting flowering Long days ≥ 16 hours 39 NFI 40 NFI ( LATE3 and LATE4 ). ❖ LATE3 : 3 recessive mutant alleles ( late3-1 , late3-2 , late3-3 ) ❖ LATE4 : 2 recessive mutant alleles ( late4-1 , late4-2 ) 16 NFI ❖ Extremely late flowering N o d e o f flo w e r in itia tio n (N F I) 5 0 1 5 R o o t d ry w e ig h t (m g ) S te m d ia m e te r (m m ) 5 0 5 L e a f a re a (m m 2 ) 4 0 4 0 4 1 0 3 0 3 0 3 2 0 2 0 2 5 1 0 1 0 1 0 0 0 0 la te 3 -1 la te 3 -2 la te 3 -3 la te 4 -1 la te 4 -2 W T la te 3 -2 la te 4 -1 1 2 3 1 2 W T T la te 3 -1 la te 3 -2 la te 3 -3 la te 4 -1 la te 4 -2 W T - - - - - W 3 3 3 4 4 e e e e e t t t t t a a a a a l l l l l * After growth of 12 days *Leaf area of single leaflet at node 10 *Diameter of stem between node 9 and 10
Goal and strategies Overall goal: Identify the LATE3 and LATE4 genes and understand their function What are the LATE3 and LATE4 genes? 1. Develop genetic map. 2. Identify/evaluate candidate genes. How do they function to regulate flowering? 3. Explore genetic interactions with other flowering loci (especially early mutants). 4. Explore regulatory interactions with other known flowering genes.
1. What are the LATE3 and LATE4 genes? Cyclin dependent kinase 8 ( CDK8 ), Cyclin C ( CYCC1 ) which are components of the eukaryotic mediator complex are positional candidates for Late3 and Late4 respectively cv. Terese x late3-1 late4-1 x cv. Terese ABI3 ABI3 RKP1 RKP1 PAP1 TPR1 TPR1 CYCC1 ATPB1 BTB1 AAP2 MCO1 PAP1 ICE1 CDK8 CYCC1 ATPB1 BTB1 MCO1 ICE1 SPS1 AAP2 CDK8 FRI 2.0 Mb 3cM UNI SPS1 FRI COLa UNI COLa WRI11 0.5 cM 0.25 Mb AAR3 AAR3 WRI11 NIP2 NIP2 PsLGV Mt Chr. 7 PsLGIII Mt Chr. 3
1. What are the LATE3 and LATE4 genes? Eukaryotic mediator complex ❖ Eukaryotic mediator complex is involved in transcriptional regulation and deeply conserved among eukaryotes. It has 4 components: head, tail, middle and CDK8 module. ❖ CDK8 and CYCC1 are components of the CDK/CDK8 module along with MED12 and MED13 . ❖ Head, tail and middle module (core mediator) promote transcription of genes along with general transcription factors by initiating RNA polymerase II activity. ❖ CDK8 module acts in maintaining optimum transcription of genes by blocking re-initiation through binding with core mediator which is Ref: Davis et. al ., 2012 crucial for various biological processes in Ref: Yang et. al. , 2016 eukaryotes.
1. What are the LATE3 and LATE4 genes? Multiple independent functionally significant mutations in candidate genes confirms identity PsCDK8 late3-1 : Mutation at 5’UTR causing potential alternative start codon late3-2 : Mutation at donor splice site causing skipping of entire exon 7 late3-3 : Mutation at acceptor slice site causing retention of 7 bp of intron 4 PsCYCC1 late4-1 : Mutation at 5 th position of intron 5 causing generation of 5 different splice variants late4-2 : Mutation at exon 4 causing introduction of premature stop codon and generation of 4 different splice variants
2. How do LATE3 and LATE4 function to regulate flowering? PsCDK8 and PsCYCC1 act in the same regulatory pathway PsCDK8 and PsCYCC1 proteins physically interact LD >= 16h N o d e o f flo w er in itia tio n (N F I) 2. Strong positive interaction control LD >= 16h 5 0 4 0 10. PsCDK8 bait + PsCYCC1 prey 3 0 2 0 11. PsCYCC1 bait + PsCDK8 prey 1 0 0 la te 3 -1 la te 4 -2 la te 3 -1 la te 4 -2 W T * Yeast two hybrid assay, mated diploid yeast cells grown on selective medium for 4 days at 30°C 1 5 L e a f a re a (m m 2 ) 1 0 5 0 la te 3 -1 la te 4 -2 la te 3 -1 la te 4 -2 W T * Yeast two hybrid assay, mated diploid yeast cells *Leaf area of single leaflet at node 10 grown on selective medium for 7 days at 30°C
2. How do LATE3 and LATE4 function to regulate flowering? PsCDK8 and PsCYCC1 promote expression of key pea flowering genes F T a 1 in le a f V E G 1 in a p e x W ild ty p e P IM in ap ex 1 5 3 0 late 3-1 2 5 0 % T FIIa tra n script % T FIIa tra n script % T FIIa tra n script late 4-1 2 0 0 1 0 2 0 1 5 0 1 0 0 5 1 0 5 0 0 0 0 0 2 0 4 0 6 0 8 0 1 0 0 0 2 0 4 0 6 0 8 0 1 0 0 0 2 0 4 0 6 0 8 0 1 0 0 D a y s a fte r s o w in g D a y s a fte r s o w in g D a y s a fte r s o w in g V E G 2 in a p e x F T b 2 in le a f F T c in a p e x 1 0 0 8 5 0 % T FIIa tra n script % T FIIa tra n script % T FIIa tra n script 8 0 4 0 6 6 0 3 0 4 4 0 2 0 2 2 0 1 0 0 0 0 0 2 0 4 0 6 0 8 0 1 0 0 0 2 0 4 0 6 0 8 0 1 0 0 0 2 0 4 0 6 0 8 0 1 0 0 D a y s a fte r s o w in g D a y s a fte r s o w in g D a y s a fte r s o w in g
2. How do LATE3 and LATE4 function to regulate flowering? PsCDK8 and PsCYCC1 suppress expression of important pea flowering repressor gene LD >= 16h N o d e o f flo w e r in itia tio n (N F I) 5 0 PsCDK8,PsCYCC1 4 0 LF is epistatic 3 0 to PsCDK8 and 2 0 PsCYCC1 1 0 0 LF ( PsTF1c ) lf la te 3 -2 la te 4 -1 lf la te 3 -2 lf la te 4 -1 W T L F in a p ex 2 0 0 % T FIIa tra n script W ild ty p e late 3-1 1 5 0 late 4-1 1 0 0 5 0 0 0 2 0 4 0 6 0 8 0 1 0 0 D a y s a fte r s o w in g
Proposed flowering model of pea in relevance to PsCDK8 and PsCYCC1 PsCDK8 and PsCYCC1 are crucial regulators of pea flowering mechanism which acts through strong mutual interaction to mediate expression of various key pea flowering genes. LF VEG1 PIM Shoot apex ? ? FTc PsCDK8 VEG2 PsCYCC1 FTa1 FTa2 FTc FTb1 FTb2 FTs PsCDK8 Leaf ? PsCYCC1 No CO? Circadian clock Photoperiod Pisum sativum
Acknowledgements ❖ Dr. Jim Weller ❖ Dr. Valerie Hecht ❖ Jacqueline Vander Schoor ❖ Dr. Frances Sussmilch Future Fellowship FT120100048 (JW) School of Biological Sciences ❖ Michelle Lang Discovery Project DP160100793 (JW) ❖ Tracey Winterbottom ❖ Pea flowering group members
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