1. Moss Systems Biology for Translational Research Ralf Reski

2. Moss Physcomitrella patens: A New Model Plant  Small  Few cell types  Cell lineage  Tightly controlled growth conditions  Predominant generation haploid  Fully sequenced genome  Efficient homologous recombination  targeted gene knockout / replacement unique to higher eukaryotes

3. Modelling Plant Development 1: Light Microscope

4. Modelling Plant Development 2 : Pattern Recognition

5. Modelling Plant Development 3: Simulation branching type of branching F-type branching, left F-type branching, right Y-type of branching Growth! no yes

6. miRNAs Regulate Gene Expression Qickly micro RNAs:  not translated  more efficient than synthesis of regulatory proteins (e. g. repressors)  faster regulatory circuits

7. Biogenesis and Function of micro RNAs AGO RISC CAP AAAAA Dicer Nucleus Cytosol HASTY HEN HYL AGO RISC pri-miRNA pre-miRNA Post-transcriptional regulation of gene expression

8. Animals and Plants: Different Sets of DICER PLANTS ANIMALS

9. Targeted knockout of DCL1a: Drastic Effects WT RT-PCR analysis of miRNAs  DCL1a KO1 KO2WTKO1KO2miRNA156 miRNA160 miRNA166 miRNA390 RT-PCR analysis of target transcripts PpSBP3PpARFPpHDZIPPpHB10PpTAS1 7 6 5 4 3 2 1 0 Rel. Transcript Level WT KO1 KO2 No DCL1a: No miRNAs, enhanced TF levels, serverely affected growth

10. Targeted knockout of DCL1b: Less Drastic Effects  DCL1b Wild Type WT1234  PpDCL1b KO miRNA156 miRNA160 miRNA166 miRNA390 miRNA535 miRNA538 U6 snRNA miRNA Expression miRNAs still present BUT developmental arrest

11.  DCL1b: miRNAs Cannot Cleave Target mRNAs Control PpGNT1 (no miRNA target) WTKO1KO2KO3KO4 PpSBP3 (Squamosa Promoter Binding Protein) PpSBP3 5’ U 3’ GUGCUC CUCUCUUCUGUCA CACGAG GAGAGAAGACAGU miR156 3’ U 5’ PpC3HDZIP (HD leucine zipper) PpC3HDZIP1 5’CU U 3’ GG AUGAAGCCUGGUCCGG CC UACUUCGGACCAGGCU miR165 3’CC C 5’ PpHB10 (HD leucine zipper) PpHB10 5’CU U 3’ GG AUGAAGCCUGGUCCGG CC UACUUCGGACCAGGCU miR165 3’CC C 5’

12. miRNA Target Genes Silenced in  DCL1b Mutant miRNAs cannot cleave target mRNAs in the mutant, nevertheless transcript levels of target genes reduced. Novel feed-back loop: Transcriptional control by miRNAs miRNA target transcripts Controls (no miRNA targets) EF1  PpHDZIP1 PpHB10 PpARF PpGNT1 WT1234  PpDCL1b-Mutants PpSBP3

13. Group 3 PpARF3-1 PpARF3-2 Tandem duplication Arabidopsis: 21 ARF Genes Rice: 24 ARF-Genes Physcomitrella: 15 ARF Genes PpARF3-1 PpARF1-1 PpARF1-2 PpARF1-3 PpARF1-4 PpARF1-5 PpARF1-6 PpARF1-7 PpARF1-8 PpARF1-9 PpARF1-10 PpARF1-11 PpARF1-12 Phypa_61245 Group 1 Group 3 Group 2 Tandem duplication PpARF3-2 miRNA160 regulates ARF Transcription Factors

14. miRNA160: Part of Multilevel Regulatory Network Wild typeARFm Mutant ARF miRNA160 Auxin GH3-2 ARF miRNA160 Auxin GH3-2

15. Precise Manipulation of miRNA160 Network Targeted base-specific mutation of endogenous gene! t ggc atg cag ggg gcc agg ca G M Q G A R t ggc atg caa ggc gcg cga ca Pau I G M Q G A R Abolish miRNA160 binding site without changing amino acids in the transcription factor

16. Dynamic Modelling of 4-Level Regulatory Network Endogenous auxin synthesis Impact of GH3-2 on [Auxin] Auxinapplication Auxin: miR160: Activation of miR160 by auxin Activation of miR160 by ARF GH3-2: Activation of GH3-2 by ARF ARF: Activation of ARF by auxin Inhibition of ARF by miR160  3 =0 in the ARFm mutant

17. Expectations from Dynamic Modelling ARF Mutant 2.0 1.5 1.0 0.5 0 1020304050 [Expression] [Time] GH3-2 GH3-2 miRNA miRNA ARF ARF Steady-State Auxinapplication Wild type

18. Mutant: Elevated ARF mRNA levels ARF transcript levels respond to auxin WTARFm Mutant 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 Control1h4h8h12h24h Auxin treatment Relative Expression

19. Elevated Transcript Levels of Downstream Gene GH3 conjugates (= inactivates) auxin GH3-2 mRNA levels enhanced by auxin Relative Expression 0 0,5 1 1,5 2 2,5 control1h4h8h12h24h Auxin treatment WTARFm Mutant

20. Elevated Levels of Conjugated (= Inactive) Auxin Auxin GH3-2: Auxin conjugating enzyme Free & conjugated Auxin Free Conjugated WTARF Mutant NAA + IAA [ng/g Fresh Weight]

21. Mutant is Hyposensitive to Auxin WT ARFm Mutant [Auxin]0 0.1 µM 0.5 µM 1 µM ARFm mutant confirms prediction. Auxin

22. Extended Model to Include Diurnal Changes

23. Current Modelling Approaches

24. The Players Gotelinde Seumel Basel Kraiwesh Asif Arif Erika Lang Philipp Rödel Marta Tomek Jutta Ludwig-Müller Olaf Rönneberger Stefan Jansen Andreas Schlosser Daniel Lang Marco Vervliet-Scheebaum Wolfgang Frank Jens Timmer