Moss Systems Biology for Translational Research Ralf Reski

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

Modelling Plant Development 1: Light Microscope

Modelling Plant Development 2 : Pattern Recognition

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

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

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

Animals and Plants: Different Sets of DICER PLANTS ANIMALS

Targeted knockout of DCL1a: Drastic Effects WT RT-PCR analysis of miRNAs  DCL1a KO1 KO2WTKO1KO2miRNA156 miRNA160 miRNA166 miRNA390 RT-PCR analysis of target transcripts PpSBP3PpARFPpHDZIPPpHB10PpTAS Rel. Transcript Level WT KO1 KO2 No DCL1a: No miRNAs, enhanced TF levels, serverely affected growth

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

 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’

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

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

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

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

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

Expectations from Dynamic Modelling ARF Mutant [Expression] [Time] GH3-2 GH3-2 miRNA miRNA ARF ARF Steady-State Auxinapplication Wild type

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

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

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]

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

Extended Model to Include Diurnal Changes

Current Modelling Approaches

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