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Molecular and Biochemical Studies of the Craterostigma Plantagineum Cell Wall During Dehydration and Rehydration

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Molecular and Biochemical Studies of the Craterostigma Plantagineum Cell Wall During Dehydration and Rehydration Molecular and biochemical studies of the Craterostigma plantagineum cell wall during dehydration and rehydration Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Niklas Udo Jung aus Adenau, Deutschland Bonn, 2019 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn. 1. Gutachter: Frau Prof. Dr. Dorothea Bartels Institut für Molekulare Physiologie und Biotechnologie der Pflanzen Kirschallee 1 53115 Bonn, Germany 2. Gutachter: Herr Prof. Dr. John Paul Knox Centre for Plant Sciences, Faculty of Biological Sciences University of Leeds Leeds LS2 9JT, UK Tag der mündlichen Prüfung: 11.02.2020 Erscheinungsjahr: 2020 ABBREVIATIONS I. ABBREVIATIONS At: Arabidopsis thaliana CDTA: 1,2-cyclohexanediaminetetraacetic acid CL: cardiolipin Cp: Craterostigma plantagineum D: desiccated GA: galacturonic acid GLP: germin-like protein GRP: glycine-rich protein HG: homogalacturonan Lb: Lindernia brevidens Ls: Lindernia subracemosa mAb: monoclonal antibody PA: phosphatidic acid PD: partially dehydrated PE: phosphatidyl-ethanolamine PC: phosphatidylcholine PIP2: phosphatidylinositol-(4,5)-bisphosphate PME: pectinmethylesterase PMEI: pectinmethylesterase inhibitor R 1: rehydrated 24 h R 2: rehydrated 48 h RWC: relative water content RG-I: rhamnogalacturonan-I RG-II: rhamnogalacturonan-II U: untreated WAK: wall-associated protein kinase FIGURES AND TABLES II. FIGURES AND TABLES List of figures and tables page Figure 1 Summary figure 2 Figure 2 Phylogenetic tree of the Linderniaceae family 4 Figure 3 Composition of pectin 7 Figure 4 Actions of pectinmethylesterases 11 Figure 5 Morphological characterisation of C. plantagineum leaf 28 structures using scanning electron microscopy Figure 6 Amplification of CpGRP1 fragments from pET28a_CpGRP1His 30 plasmid Figure 7 C. plantagineum CpGRP1 amino acid sequence and protein 31 domains Figure 8 Overexpression of CpGRP1 full-length protein and polypeptides 32 Figure 9 Electrostatic surface model of the recombinant CpGRP1 protein 32 Figure 10 Analysis of CpGRP1-pectin interaction by Blue-native page gel- 33 shift assays Figure 11 Effect of different calcium and magnesium concentrations on the 33 interaction of CpGRP1 and pectin Figure 12 Quantification of the protein-pectin interaction using ELISA 34 assays Figure 13 Evaluation of CpGRP1 interaction with C. plantagineum, L. 37 brevidens and L. subracemosa pectin fractions during dehydration and rehydration by dot-blot analyses Figure 14 Evaluation of CpGRP1-pectin interaction in comparison to the 38 apoplastic proteins CpWAK1 and CpGLP1 Figure 15 Quantification of CpGRP1-pectin interaction after pectin de- 39 methylesterification Figure 16 Evaluation of CpGRP1 migration behaviour in SDS-page 40 Figure 17 Evaluation of the effect of cysteine mutations on the CpGRP1 41 migration behaviour in SDS-page Figure 18 Protein-lipid overlay assays with CpGRP1 42 FIGURES AND TABLES Figure 19 Liposome-binding assays with CpGRP1 43 Figure 20 Transcriptome data of different C. plantagineum contigs which 45 were identified as pectinmethylesterases or pectinmethyl- esterase inhibitors Figure 21 Alignment of AtPME31 and Cp_V2_contig_11593 amino acid 46 sequences Figure 22 3D-modeling of Cp_V2_contig_11593 with Phyre2 47 Figure 23 RT-PCR analysis of Cp_V2_contig_11593 transcript abundance 48 Figure 24 Amplification of Cp_V2_contig _11593 coding sequence from C. 49 plantagineum cDNA Figure 25 Alignment of pJET1.2_CpPMEClone9 sequencing result with 49 the CpPME reference sequence Figure 26 Amplification of Cp_V2_ contig_11593 coding sequence from 50 pJET1.2_CpPMEClone9 Figure 27 Amplification of the full insert from pDONR201_Cp_ 50 V2_contig_11593Clone9 Figure 28 CpGRP1-CpWAK1-pectin complex 62 Figure S1 Standard curve to calculate protein concentrations 64 Figure S2 LM25 binding to different dilutions of the KOH fraction 64 Figure S3 Standard curve to calculate the galacturonic acid content 65 Figure S4 Protein blast for Cp_V2_contig_11593 65 Figure S5 Sequencing result of pJET1.2_CpPMEClone9_F and 66 pJET1.2_CpPMEClone9_R Table 1 Monoclonal antibodies used in this study for pectin and 23 hemicellulose characterisation Table 2 Analysis of changes in the cell wall composition of C. planta- 29 gineum leaves in a desiccation/rehydration cycle Table 3 Quantification of galacturonic acid content 35 Table 4 Analysis of changes in cell wall methylesterification of L. 36 brevidens and L. subracemosa leaves in a desiccation /rehydration cycle Table S1 List of primers used in this study 63 TABLE OF CONTENTS III. TABLE OF CONTENTS 1. SUMMARY .......................................................................................................... 1 2. INTRODUCTION ................................................................................................. 3 2.1 The importance of water for plant survival and desiccation tolerance ............... 3 2.2. The Linderniaceae family ................................................................................. 4 2.3 Resurrection plants and adaptations to survive desiccation .............................. 5 2.4 Plant cell wall structure ..................................................................................... 7 2.4.1 Cell wall remodelling in response to dehydration in resurrection plants ..... 8 2.4.2 Cell wall modifying proteins ........................................................................ 9 2.5 Glycine-rich proteins ....................................................................................... 12 2.6 Cell wall-associated protein kinases and their interaction with glycine-rich proteins ................................................................................................................. 13 2.7 Phosphatidic acid ............................................................................................ 14 2.8 Objectives of the study .................................................................................... 14 3. MATERIALS AND METHODS .......................................................................... 16 3.1 Cultivation of plants ......................................................................................... 16 3.2 Molecular biology techniques and DNA sequencing ....................................... 16 3.3 Scanning electron microscopy (SEM) ............................................................. 18 3.4 Protein analyses.............................................................................................. 19 3.5 Extraction of cell wall components .................................................................. 22 3.6 Enzyme-linked immunosorbent assays ........................................................... 22 3.7 Determination of galacturonic acid .................................................................. 24 3.8 Blue-native page gel-shift assays ................................................................... 24 3.9 Dot-blot pectin binding assays ........................................................................ 25 3.10 Protein-lipid overlay assays .......................................................................... 25 3.11 Liposome-binding assays .............................................................................. 25 3.12 Electrostatic surface modelling...................................................................... 26 TABLE OF CONTENTS 4. RESULTS .......................................................................................................... 27 4.1 Morphological characterisation of leaf structures ............................................ 27 4.2 Pectin and hemicellulose profiles determined in a desiccation/rehydration cycle .............................................................................................................................. 27 4.3 Interaction between CpGRP1 and pectin ........................................................ 29 4.3.1 Cloning of CpGRP1 fragments ................................................................. 30 4.3.2 Overexpression of CpGRP1 fragments .................................................... 31 4.3.3 Electrostatic surface modelling of CpGRP1 .............................................. 32 4.3.4 Blue-native page gel-shift pectin binding assays ...................................... 33 4.3.5 ELISA pectin binding assays .................................................................... 34 4.3.6 Quantification of galacturonic acid content ............................................... 35 4.3.7 Cell wall methylesterification status in L. brevidens and L. subracemosa 35 4.3.8 Dot-blot pectin binding assays .................................................................. 36 4.3.9 Effect of pectin de-methylesterification on CpGRP1-pectin interaction ..... 39 4.3.10 Effect of cysteine mutations on CpGRP1 migration behaviour in SDS- page .................................................................................................................. 40 4.4 Interaction between CpGRP1 and lipids ......................................................... 41 4.4.1 Protein-lipid overlay assays ...................................................................... 42 4.4.2 Liposome-binding assays ......................................................................... 43 4.5 Pectinmethylesterases in C. plantagineum ....................................................
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