Characterization of the transcription factor ANAC058 and its role in suberin regulation Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Katharina Markus aus Köln Bonn, Februar 2018 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn. 1. Gutachter: Dr. Rochus Franke 2. Gutachter: Prof. Dr. Volker Knoop Tag der Promotion: 14. 06. 2018 Erscheinungsjahr: 2018 I Contents List of abbreviations…………………………………………………………………………………... VI 1 Introduction…………………………………………………………………………………………… 1 1.1 Border tissues as apoplastic diffusion and uptake barriers in plants………................ 1 1.1.1 Cuticle…………………………………………………………………………………. 1 1.1.2 Casparian strip and suberin lamella in roots……………………………………… 2 1.1.2.1 Casparian strip……………………………………………………………………….. 3 1.1.2.2 Suberin lamella……………………………………………………………………….. 4 1.1.3 Effect of Casparian ctrip and suberin lamella on water and solute uptake into plants……………………………………………………………………. 7 1.1.4 Different stresses which induce suberin deposition……………………………… 8 1.2 Transcription Factors……………………………………………………………................. 9 1.2.1 NAC transcription factor gene family………………………………………………. 10 1.3 Aims of this work…………………………………………………………………………….. 12 2 Materials and Methods……………………………………………………………………………… 14 2.1 Materials……………………………………………………………………………………… 14 2.1.1 Chemicals…………………………………………………………………………….. 14 2.1.2 Software………………………………………………………………………………. 14 2.1.3 In silico analysis, phylogenetic analysis and databases…………………………. 15 2.1.4 Statistical analysis of data…………………………………………………………... 17 2.2 Plants…………………………………………………………………………………………. 18 2.2.1 Genotypes…………………………………………………………………………….. 18 2.2.2 Cultivation…………………………………………………………………………….. 18 2.2.3 Stress treatments…………………………………………………………………….. 19 2.2.4 Measurement of root hydraulic conductivity (Lpr)………………………………… 20 2.3 Molecular biology methods………………………………………………………………… 22 2.3.1 Extraction of genomic DNA from plant material…………………………………... 22 2.3.2 Extraction of RNA from plant material……………………………………………... 22 2.3.3 Determination of nucleic acid concentration………………………………………. 22 2.3.4 Polymerase Chain Reaction (PCR)………………………………………………… 23 2.3.4.1 Reverse Transcription (RT)-PCR…………………………………………………... 24 2.3.4.2 Organ-specific expression analysis………………………………………………… 27 2.3.4.3 Identification of T-DNA insertion and enhancer trap lines……………………….. 27 2.3.5 Gel electrophoresis…………………………………………………………………... 28 2.4 Microbiological methods……………………………………………………………………. 28 2.4.1 Transformation of chemically competent Escherichia coli cells………………… 28 2.4.2 Transformation of chemically competent Agrobacterium tumefaciens cells….. 28 2.4.3 Extraction, restriction and sequencing of plasmid DNA………………………….. 29 2.4.4 Long-term storage of transformed bacteria……………………………………….. 29 II 2.5 Generation of transgenic A. thaliana……………………………………………………… 29 ® 2.5.1 Cloning an entry clone with the Gateway system……………………………….. 30 ® 2.5.2 Cloning an expression clone with the Gateway system………………………… 31 2.5.3 Generation of the artificial microRNA interference construct……………………. 32 2.5.4 Transformation of A. thaliana……………………………………………………….. 33 2.5.5 Selection of transformed A. thaliana plants for positive transformants………… 34 2.6 Chemical analysis…………………………………………………………………………… 35 2.6.1 Suberin sample preparation………………………………………………………… 35 2.6.2 Transesterification with BF3:methanol…………………………………………….. 36 2.6.3 Wax sample preparation…………………………………………………………….. 36 2.6.4 GC-MS and GC-FID analysis………………………………………………………. 36 2.7 Histology…………………………………………………………………………………….. 38 2.7.1 GUS assay…………………………………………………………………………… 38 2.7.2 GFP fluorescence observation……………………………………………………... 39 2.7.3 Fluorol Yellow 088 staining and observation……………………………………… 39 3 Results…………………………………………………………………………………………………. 41 3.1 Protein and expression analysis…………………………………………………………… 41 3.1.1 In silico co-expression analysis…………………………………………………….. 41 3.1.2 Organ-specific expression of ANAC058…………………………………………… 42 3.1.3 Activity of the putative ANAC058 promoter……………………………………….. 42 3.1.3.1 Induction of putative ANAC058 promoter activity by wounding…………………. 46 3.1.3.2 Induction of putative ANAC058 promoter activity by ABA application…………. 47 3.1.4 NAC binding sites in suberin gene promoters…………………………………….. 47 3.1.5 In silico ANAC058 protein structure and domain analyses……………………… 49 3.2 Analysis of ANAC058 knock-out and knock-down mutants…………………………….. 50 3.2.1 Selection and generation of knock-out and knock-down lines………………….. 50 3.2.2 Analytical investigation of suberin in anac058 mutants………………………….. 54 3.2.3 Histological investigation of suberin in anac058 mutants……………………….. 58 3.2.4 Expression analysis of suberin genes in anac058 mutants……………………... 59 3.2.5 Physiological effect of decreased suberin amounts in anac058 mutants……… 60 3.2.5.1 Susceptibility of anac058 mutants to salt and osmotic stress………………….. 60 3.2.5.2 Root hydraulic conductivity of anac058 mutants…………………………………. 65 3.3 Overexpression of ANAC058………………………………………………………………. 66 3.3.1 Tissue-specific overexpression of ANAC058 in PromRALPH::ANAC058 plants… 66 3.3.1.1 Expression of ANAC058 and suberin genes in roots of PromRALPH::ANAC058 plants………………………………………………………... 66 3.3.1.2 Histological investigation of suberin in PromRALPH::ANAC058 plants…………… 67 3.3.2 Induced overexpression of ANAC058 in TRANSPLANTA (TPT) lines………… 68 3.3.2.1 Expression of ANAC058 in induced TPT.D and TPT.G plants………………….. 69 3.3.2.2 Chemical analysis of root suberin in induced TPT.G plants…………………….. 69 3.3.2.3 Histological investigation of suberin in induced TPT.G plants………………….. 70 III 3.3.2.4 Analysis of gene expression in roots of induced TPT.G plants…………………. 72 3.3.2.5 Analysis of ANAC058 expression and wax composition in leaves of induced TPT.D and TPT.G plants………………………………………………................. 74 4 Discussion…………………………………………………………………………………………….. 76 4.1 Identification of ANAC058 as a suberin involved transcription factor candidate……... 76 4.2 Localization of ANAC058 expression and activity of the putative ANAC058 promoter………………………………………………………………………..................... 77 4.2.1 ANAC058 is expressed during plant development in roots but not in leaves…. 77 4.2.1.1 The putative promoter of ANAC058 is active in the root endodermis………….. 78 4.2.1.2 Activity of the putative ANAC058 promoter is found in the root cap and the root base………………………………………………………………………………. 78 4.2.2 The putative promoter of ANAC058 is induced by wounding and ABA……….. 81 4.3 ANAC058 function inferred by bioinformatical analyses………………………………... 83 4.3.1 NAC binding sequences were found in suberin gene promoters………………. 83 4.3.2 Prediction of ANAC058’s secondary structure and functionally important protein regions……………………………………………………………………….. 83 4.4 anac058 mutants…………………………………………………………………………..... 85 4.4.1 Suberin amounts are not affected in whole root systems of anac058 mutants.. 85 4.4.2 Suberin amounts are decreased in apical root sections of anac058 mutants…. 86 4.4.3 Suberin deposition is delayed in anac058 mutants………………………………. 87 4.4.4 Expression of suberin-associated genes is decreased in anac058 mutants….. 88 4.4.5 Physiology of anac058 mutants…………………………………………………….. 89 4.4.5.1 Susceptibility of anac058 mutants to salt stress is increased…………………… 89 4.4.5.2 Susceptibility of anac058 mutants and wild type to osmotic stress is the same. 93 4.4.5.3 Root hydraulic conductivity of anac058 mutants…………………………………. 94 4.5 Overexpression of ANAC058………………………………………………………………. 95 4.5.1 Endodermal-specific overexpression of ANAC058 delays suberin deposition... 95 4.5.2 Conditional overexpression of ANAC058 in roots results in suberin effects…... 96 4.5.2.1 Conditional overexpression of ANAC058 results in ectopic suberin deposition. 96 4.5.2.2 Conditional overexpression of ANAC058 results in increased expression of 98 suberin-associated genes………………………………………………………....... 4.5.2.3 Conditional overexpression of ANAC058 affects expression of CS and 98 lignin-associated genes, not cutin-associated genes………………………........ 4.5.2.4 Conditional overexpression of ANAC058 affects expression of an aquaporin 99 gene slightly…………………………………………………………………………... 4.5.2.5 Conditional overexpression of ANAC058 affects expression of various TFs…. 99 4.5.3 Conditional overexpression of ANAC058 in leaves has no effect on leaf wax... 100 5 Summary………………………………………………………………………………………………. 103 6 Zusammenfassung………………………………………………………………………………….. 105 IV 7 Lists and References……………………………………………………………………………….. 108 Bibliography………………………………………………………………………………………….. 108 List of Figures………………………………………………………………………………………… 119 List of Tables………………………………………………………………………………………… 122 8 Supplemental………………………………………………………………………………………… 123 8.1 Solutions and media………………………………………………………………………… 123 8.2 Primers used in this work…………………………………………………………………… 125 8.2.1 Primer generation……………………………………………………………………. 125 8.2.2 Calculation of primer melting temperature………………………………………… 125 8.2.3 List of used primers………………………………………………………………….. 126 8.3 In silico co-expression analysis……………………………………………………………. 129 8.4 In silico organ and tissue-specific expression……………………………………………. 130 8.5 ANAC058's putative promoter activity…………………………………………………….. 131 8.6 NAC binding sites (NACBS) in promoters of suberin-associated genes……………… 131 8.7 Phylogeny of the NAC family………………………………………………………………. 133 8.8 Conserved motifs specific for the TRD of phylogenetic