A Systems Approach to Cellulose Synthesis. Staffan Persson MPI-MP, Potsdam

Transcription

1 A Systems Approach to Cellulose Synthesis Staffan Persson MPI-MP, Potsdam

2 The Cell Wall: A Complex Compartment Inference of gene function from other species difficult Economically important ~ gene products Somerville et al., (2004) Science

3 Main Approach: To Combine Bioinformatic Predictions with Biological Testing CESAX CESAY CESAZ Wild-type irx8 GT8 irx1 CESA8 irx9 GT43 Persson et al. (2005) PNAS; Brown et al. (2005) Plant Cell; Bauer et al. (2006) PNAS; Persson et al. (2007) Plant Cell; Pena et al. (2007) Plant Cell Co-expressed genes for the cellulose synthase genes Functional Annotations cellulose synthase (IRX5) cellulose synthase (IRX1) glycosyl transferase family 8 protein cellulose synthase (IRX3) laccase, putative glycoside hydrolase family 19 (CTL1-like) COBRA homolog COBL4 fasciclin-like arabinogalactan-protein (FLA11) glycosyl transferase family 43 glycogenin glucosyltransferase (glycogenin)-related expressed protein expressed protein germin-like protein (GLP10) no apical meristem (NAM) family laccase, putative fasciclin-like arabinogalactan-protein (FLA12) leucine-rich repeat kinase exostosin family protein unknown expressed protein expressed protein GDSL-motif lipase/hydrolase protein leucine-rich repeat protein kinase rac GTPase activating protein expressed protein expressed protein plastocyanin-like domain-containing protein laccase, putative calmodulin-binding family protein NAD-dependent epimerase homeodomain transcription factor (KNAT7) laccase, putative expressed protein p21-rho-binding domain protein Ras-related GTP-binding family Lysine/histidine transporter kinesin motor protein-related unknown glycoside hydrolase family 28 protein expressed protein

4 Problems with Large-Network Visualizations Division of network necessary! Incomprehensible!

5 Genome Clustering Pipe-line Heuristic/fast Supports weighted edges Very good control of cluster size Mutwil et al. (2009) Plant Physiol.

6 A Typical Co-expressed Gene Cluster from HCCA Mutwil et al. (2009) Plant Physiol.

7 Meta Network Functionally Related Processes Connected! Each node represent a co-expressed gene cluster Edges between nodes represent a strong link between the co-expressed genes clusters Cluster 86: Primary cell walls Mutwil et al. (2009) Plant Physiol.

8 Cluster 86 - Primary wall cellulose synthesis CESA1, 3 and 6 CESA1,3,6 CTL1/POM1 Wild-type + srf s cobra + srf s Bringmann et al. (unpublished) Niittyla et al. (in preparation) Mutwil et al. (2009) Plant Physiol.

9 CTL1 is ubiquitously expressed similar to the primary CESA genes CESA1 CESA2 CESA3 CESA6 CTL1 Sanchez-Rodrigues et al. (in preparation)

10 Sanchez-Rodrigues et al. (in preparation) Cellulose deposition is reduced in ctl1 CESA6 promoter YFP CESA6 genomic Paredez et al., (2006) Science

11 Synergistic effect between ctl1 and prc1-1 (cesa6) mutants Both ctl1 and prc1-1 (a CESA6 null mutant) are cellulose deficient. But, it appears likely that while CESA6 is part of the CESA complex, CTL1 affects cellulose deposition in other ways Thus, double mutants should be additive! ctl1 prc1-1 doubles!! Sanchez-Rodrigues et al. (in preparation)

12 Complete Primary Wall Cellulose Deficiency Leads to Collapsed Pollen ctl1 ctl2 double mutant do not lead to pollen deficiencies Two close CTL homologs In Arabidopsis..perhaps Functional redundancies?? Several mutants affecting Cellulose are dwarfed! Sanchez-Rodrigues et al. (in preparation) Persson et al. (2007) PNAS

13 CTL1 localization 35S CTL1 GFP ctl1 ctl1/ctl1-gfp 20µm 20µm 35S::CTL1-GFP (in ctl1-2) CTL1-gfp plasmolized seedlings (0,5M Suc 45min) Sanchez-Rodrigues et al. (in preparation)

14 CTL1 Trafficking is Actin-Mediated 35S::CTL1-GFP 35S::CTL1-GFP LatB 100nM Sanchez-Rodrigues et al. (in preparation)

15 Up until now.. 1. CTL1 is co-expressed with primary CESA genes 2. Mutations in CTL1 affect CESA movements 3. The function of CTL is not essential for cellulose production 4. CTL1 is localized in vesicles that are trafficked via actin filaments to the cell surface But WHAT does it do??? Expression of enzyme in heterologous system!

16 CTL1 is predicted to be a chitinase but no chitinase activity detected Amino acids associated with activity mutated in CTL1! SS CTL1 6 His CTL1 prediction Expression of CTL1 in pichia Induction time No observed activity using ~ 20 different substrates! Zhang et al. (2004) Plant Mol Biol Perhaps some form of structural protein??

17 Suppression of the ctl1 may put the gene product in functional context!? Suppressor screen!! EMS, fast neutron etc WT Suppressor of ctl1-2 (M3) ctl1 Self, And screen for elongated mutants Outcome: Identify mutation and confirm! ctl1-2 Mutated ctl1 Sanchez-Rodrigues et al. (in preparation)

18 Similarities between primary and secondary CESA functions? CTLs Cellulose Synthases COBRA-like Kinases MYB TFs And others.. Arabidopsis secondary cell wall network Arabidopsis primary cell wall network

19 Network comparison algorithm was developed for more thorough comparative analysis of co-expression networks. Ruprecht/Mutwil et al. (in preparation) The algorithm returs MN-value which quantifies the network similarity. Higher score = higher similarity.

20 Gene modules involved in cellulose biosynthesis have specialized to certain cell types and are present multiple times in co-expression network. Comparison of Arabidopsis primary cell wall network CESA1 to Arabidopsis genome scale network revealed cellulose biosynthesis like networks in other tissues. Seed node, expression profile At2g37090 glycosyltransferase (secondary cell wall network) At2g32380 (expressed protein) pollen At5g15890 (expressed protein) roots Score 2.98 (p=<0.001) 2.36 (p=<0.001) 1.46 (p=<0.01) PCW Pollen Pollen Roots SCW Ruprecht/Mutwil et al. (in preparation)

21 Gene modules involved in cellulose biosynthesis have specialized to certain cell types and are present multiple times in co-expression network. Comparison of Arabidopsis primary cell wall network CESA1 to Arabidopsis genome scale network revealed cellulose biosynthesis like networks in other tissues. COBL-10 Seed node, expression profile At2g37090 glycosyltransferase (secondary cell wall network) At2g32380 (expressed protein) pollen At5g15890 (expressed protein) roots Score 2.98 (p=<0.001) 2.36 (p=<0.001) 1.46 (p=<0.01) PCW Pollen Pollen Roots SCW Ruprecht/Mutwil et al. (in preparation)

22 Building More Networks Other Plant Species Mutwil et al. (in preparation)

23 Arabidopsis Barley

24 Expanding the network Comparative approaches! Medicago Arabidopsis Barley Rice Commonalities?! Across Within Poplar

25 Comparative analyses across species Secondary Cell Walls Ruprecht/Mutwil et al. (in preparation) Red: > 75% Orange: >50 75% Pink: >25 50%

26 Cellulose biosynthesis is conserved within and between species. Consensus coexpression network For primary and secondary cellulose biosynthesis in the 5 plant species. <25% <50% <75% >75% Ruprecht/Mutwil et al. (in preparation)

27 However, network composition and architecture differ somewhat between primary/secondary and monocot/dicot cellulose co-expression networks. Ruprecht/Mutwil et al. (in preparation) Monocot specific = present in 2/2 monocots (rice, barley) Dicot specific = present in 3/3 dicots (arabidopsis, medicago, poplar) Present in both = present in 3/5 plant species

28 Brachypodium Model Organism for 2 nd Generation Biofuel Crops Currently performing >400 microarrays for Brachypodium Genome Sequence Microarray platform Goal: To infer gene functions by combining transcriptional coordination with sequence similarity

29 Summary 1. We have generated an interactive co-expression network for five plant model organisms. 2. We have used these to identified novel components possibly involved cell wall synthesis, here examplified using CTL1. 3. We have developed an algorithm for comparison of modular structures of genes within and across networks.

30 CTL1 story UC Berkeley Chris Somerville BOKU (Vienna) Marie-Theres Hauser INRA Kian Hematy MPI-MP Microscopy Facility Eugenia Maximova Green Team Acknowledgements GenNet Bjorn Usadel Federico Grigio Oliver Ebenhoh Moritz Schuette Zoran Nikolovski UNC at Charlotte Ann Loraine AG Persson Daniela Geisler Lutz Neumetzler Clara Sanchez-Rodrigues Marek Mutwil Arun Sampathkumar Mattias Ehrlich Anja Froehlich Anett Doerring Colin Ruprecht Carola Paepke Norma Funke Martin Bringmann Ting Wang Max-Planck-Gesellschaft GABI-FUTURE (Plant-KBBE)