Predložená dizertačná práca je zameraná na mitogen-aktivovanú proteín kinázu 6 (MPK6) a proteín strihajúci mikrotubuli KATANIN 1 (KTN1) u modelovej rastliny Arabidopsis thaliana. V práci je prezentovaná genetická komplementácia mpk6-2 mutantného fenotypu chimérnym GFP-MPK6 proteínom exprimovaným pod natívnym promótorom, ktorý bol použitý na in vivo lokalizačné štúdie. Ďalší typ chimérneho proteínu MPK6-VenusN bol použitý na testovanie potenciálnej interakcie MPK6 kinázy a KTN1. Tiež boli vykonané fenotypické analýzy vybraných KTN1 mutantov a tieto mutanty boli geneticky komplementované chimérnym GFP-KTN1 proteínom exprimovaným pod natívnym promótorom.
Práca je rozdelení na dve časti, z toho prvá časť stručne sumarizuje súčasné poznatky o signalizácií sprostredkovanej MAP kinázami so zameraním na MPK6, ďalej na molekulárne klonovanie GFP-fúzovaného MPK6 proteínu exprimovaného pod natívnym MPK6 promótorom a jeho stabilnú transformáiu do mpk6-2 nulového mutanta za účelom získania "rescue" fenotypu. Dva konštrukty, pMPK6::GFP:MPK6 a pMPK6::MPK6:GFP, boli schopné zvrátiť alebo zachrániť (rescue) mpk6-2 fenotyp. Fúzny proteín GFP-MPK6 poskytoval lepší signál než MPK6-GFP a bol lokalizovaný v jadre, na plazmatickej membráne a cytosole v interfáznych bunkách. Počas mitózy bol GFP-MPK6 asociovaný s predprofáznym mikrotubulárnym pásom (PPB), fragmoplastom a tiež s novo sa tvoriacou bunkovou prepážkou. Tieto pozorovania spolu s inými výsledkami kde neaktívna forma MPK6 kinázy (kinase-dead) MPK6AEF transformovaná do mpk6-4 mutantného pozadia vykazovala podobný fenotyop koreňa ako yda1 mutant prispeli k záverom, že MPK6 je dôležitý hráč v signalizácií "downstream" od YODA MAPKKK4 kinázy. Oboje YODA aj MPK6 sú zapojené do signalizácie dôležitej pri post-embryonálnom vývine koreňa prostredníctvom orientácie roviny bunkového delenia.
Ďaľej boli testované potenciálne interakcie KTN1 s MPK6, ale aj s blízko príbuznými kinázami MPK3 a MPK4, pretože in silico štúdie zamerané na MAPK fosforyláciu odhalili peptid, ktorý obsahujúci SP motív (serín-prolín) v 92. pozícií KTN1 proteínu. Výsledky YFP-split experimentu (BiFC, Bimolecular Fluorescent Complementation) ukázali interakcie oboch MPK4 aj MPK6 s KTN1 a výsledky získané z kvasinkového dvojhybridného testu (yeast-two-hybrid,Y2H) ukázali interakciu MPK3 s KTN1. Tieto proteín-proteín interakčné štúdie podporujú potenciálnu reguláciu KTN1 proteínu MAP kinázami.
Druhá časť tejto práce sumarizuje súčasné poznatky o KTN1 proteíne, ktorý strihá mikrotubuly so zameraním na fenotypy jeho mutantov. Teda sa zaoberá fenotypovou analýzou vybraných KTN1 mutantov (fra2, lue1 a ktn1-2) so zameraním na plodnosť, vývin embrya a semien a tiež schopnosťou konštruktu pKTN1::GFP:KTN1 zvrátiť alebo zachrániť (rescue) mutantné fenotypy. Fenotypické analýzy KTN1 mutantov odhalili abnormálny vývin sporopfytických pletív a tiež aj samčích a samičích reprodčných orgánov. Stabilné transformácie mutantov fra2, lue1 a ktn1-2 s pKTN1::GFP:KTN1 konštruktom exprimovaným pod natívnym promótorm umožnili vzniknúť líniám T3 generácie, ktoré vykazovali záchranu "rescue" (genetickú komplementáciu) mutantných fenotypov. Z uvedených pozorovaní môžeme konštatovať, že KTN1 má esenciálnu úlohu v plodnosti a tvorbe semien u Arabidopsis thaliana.
Anotace v angličtině
The present Ph.D. thesis is focused on Arabidopsis thaliana mitogen-activated protein kinase 6 (MPK6) and the microtubule severing protein KATANIN1 (KTN1). Herein, a chimeric GFP-MPK6 protein expressed under the native MPK6 promoter capable of rescuing the mpk6-2 mutant phenotype was used to perform in vivo localisation studies. Another chimeric protein MPK6-VenusN was used to test potential interactions of MPK6 with KTN1. Moreover phenotypic analyses of selected KTN1 mutants were performed and genetically rescued these mutants by chimeric GFP-KTN1 protein expressed under the native promoter. Therefore this thesis is divided into two parts.
The first part of thesis briefly summarises current knowledge on plant MAPK signalling with main focus on MPK6, and describes the molecular cloning of GFP- fused MPK6 expressed under the native MPK6 promoter and the stable transformation of the fusion construct into mpk6-2 null mutant to achieve rescue of the mutant phenotype. Two constructs, pMPK6::GFP:MPK6 and pMPK6::MPK6:GFP, were able to revert mpk6-2 phenotype. GFP-MPK6 provided better signal than MPK6-GFP and it was localised to the nucleus, the plasma membrane and the cytosol in non-dividing cells. During mitosis GFP-MPK6 was associated with the preprophase microtubule band (PPB), phragmoplast and the newly formed cell plates. These observations together with the finding that a kinase-dead form MPK6AEF transformed into mpk6-4 mutant background showed a similar root phenotype like yda1 mutant contributed to the conclusion that MPK6 is an important player downstream of YODA (MAPKKK4). Both YODA and MPK6 are involved in postembryonic root development by regulating cell division plane orientation.
Next, we searched for putative interactions between KTN1 and MPK6, its closely-related MPK3 and also MPK4, since in silico studies predicted the existence of a MAPK-targeted proline-directed serine at the 92nd position of KTN1. Results of YFP-split technology (BiFC, Bimolecular Fluorescent Complementation) experiments showed interactions of both MPK4 and MPK6 with KTN1 while preliminary results obtained by yeast-two-hybrid (Y2H) assay also showed interaction of MPK3 with KTN1. These protein-protein interaction data support the potential regulation of KTN1 by MAPKs.
The second part of thesis summarises current knowledge on microtubule severing protein KTN1 with main focus on mutant phenotypes. Thus, it is devoted to the phenotypic analysis of the selected KTN1 mutants used herein (fra2, lue1 and ktn1-2) as related to fertility, embryo and seed development, and to the ability of a pKTN1::GFP:KTN1 construct to rescue mutant phenotypes. The phenotypic analyses of KTN1 mutants revealed an abnormal development of sporophytic tissues and of male and female reproductive organs. Stable transformations of fra2, lue1 a ktn1-2 mutants with pKTN1::GFP:KTN1 construct (expressed under control of the native KTN1 promoter) were performed and T3 lines showed rescue (genetic complementation) of mutant phenotypes. Therefore, we propose a new role of KTN1 in plant fertility and seed formation of Arabidopsis thaliana.
Predložená dizertačná práca je zameraná na mitogen-aktivovanú proteín kinázu 6 (MPK6) a proteín strihajúci mikrotubuli KATANIN 1 (KTN1) u modelovej rastliny Arabidopsis thaliana. V práci je prezentovaná genetická komplementácia mpk6-2 mutantného fenotypu chimérnym GFP-MPK6 proteínom exprimovaným pod natívnym promótorom, ktorý bol použitý na in vivo lokalizačné štúdie. Ďalší typ chimérneho proteínu MPK6-VenusN bol použitý na testovanie potenciálnej interakcie MPK6 kinázy a KTN1. Tiež boli vykonané fenotypické analýzy vybraných KTN1 mutantov a tieto mutanty boli geneticky komplementované chimérnym GFP-KTN1 proteínom exprimovaným pod natívnym promótorom.
Práca je rozdelení na dve časti, z toho prvá časť stručne sumarizuje súčasné poznatky o signalizácií sprostredkovanej MAP kinázami so zameraním na MPK6, ďalej na molekulárne klonovanie GFP-fúzovaného MPK6 proteínu exprimovaného pod natívnym MPK6 promótorom a jeho stabilnú transformáiu do mpk6-2 nulového mutanta za účelom získania "rescue" fenotypu. Dva konštrukty, pMPK6::GFP:MPK6 a pMPK6::MPK6:GFP, boli schopné zvrátiť alebo zachrániť (rescue) mpk6-2 fenotyp. Fúzny proteín GFP-MPK6 poskytoval lepší signál než MPK6-GFP a bol lokalizovaný v jadre, na plazmatickej membráne a cytosole v interfáznych bunkách. Počas mitózy bol GFP-MPK6 asociovaný s predprofáznym mikrotubulárnym pásom (PPB), fragmoplastom a tiež s novo sa tvoriacou bunkovou prepážkou. Tieto pozorovania spolu s inými výsledkami kde neaktívna forma MPK6 kinázy (kinase-dead) MPK6AEF transformovaná do mpk6-4 mutantného pozadia vykazovala podobný fenotyop koreňa ako yda1 mutant prispeli k záverom, že MPK6 je dôležitý hráč v signalizácií "downstream" od YODA MAPKKK4 kinázy. Oboje YODA aj MPK6 sú zapojené do signalizácie dôležitej pri post-embryonálnom vývine koreňa prostredníctvom orientácie roviny bunkového delenia.
Ďaľej boli testované potenciálne interakcie KTN1 s MPK6, ale aj s blízko príbuznými kinázami MPK3 a MPK4, pretože in silico štúdie zamerané na MAPK fosforyláciu odhalili peptid, ktorý obsahujúci SP motív (serín-prolín) v 92. pozícií KTN1 proteínu. Výsledky YFP-split experimentu (BiFC, Bimolecular Fluorescent Complementation) ukázali interakcie oboch MPK4 aj MPK6 s KTN1 a výsledky získané z kvasinkového dvojhybridného testu (yeast-two-hybrid,Y2H) ukázali interakciu MPK3 s KTN1. Tieto proteín-proteín interakčné štúdie podporujú potenciálnu reguláciu KTN1 proteínu MAP kinázami.
Druhá časť tejto práce sumarizuje súčasné poznatky o KTN1 proteíne, ktorý strihá mikrotubuly so zameraním na fenotypy jeho mutantov. Teda sa zaoberá fenotypovou analýzou vybraných KTN1 mutantov (fra2, lue1 a ktn1-2) so zameraním na plodnosť, vývin embrya a semien a tiež schopnosťou konštruktu pKTN1::GFP:KTN1 zvrátiť alebo zachrániť (rescue) mutantné fenotypy. Fenotypické analýzy KTN1 mutantov odhalili abnormálny vývin sporopfytických pletív a tiež aj samčích a samičích reprodčných orgánov. Stabilné transformácie mutantov fra2, lue1 a ktn1-2 s pKTN1::GFP:KTN1 konštruktom exprimovaným pod natívnym promótorm umožnili vzniknúť líniám T3 generácie, ktoré vykazovali záchranu "rescue" (genetickú komplementáciu) mutantných fenotypov. Z uvedených pozorovaní môžeme konštatovať, že KTN1 má esenciálnu úlohu v plodnosti a tvorbe semien u Arabidopsis thaliana.
Anotace v angličtině
The present Ph.D. thesis is focused on Arabidopsis thaliana mitogen-activated protein kinase 6 (MPK6) and the microtubule severing protein KATANIN1 (KTN1). Herein, a chimeric GFP-MPK6 protein expressed under the native MPK6 promoter capable of rescuing the mpk6-2 mutant phenotype was used to perform in vivo localisation studies. Another chimeric protein MPK6-VenusN was used to test potential interactions of MPK6 with KTN1. Moreover phenotypic analyses of selected KTN1 mutants were performed and genetically rescued these mutants by chimeric GFP-KTN1 protein expressed under the native promoter. Therefore this thesis is divided into two parts.
The first part of thesis briefly summarises current knowledge on plant MAPK signalling with main focus on MPK6, and describes the molecular cloning of GFP- fused MPK6 expressed under the native MPK6 promoter and the stable transformation of the fusion construct into mpk6-2 null mutant to achieve rescue of the mutant phenotype. Two constructs, pMPK6::GFP:MPK6 and pMPK6::MPK6:GFP, were able to revert mpk6-2 phenotype. GFP-MPK6 provided better signal than MPK6-GFP and it was localised to the nucleus, the plasma membrane and the cytosol in non-dividing cells. During mitosis GFP-MPK6 was associated with the preprophase microtubule band (PPB), phragmoplast and the newly formed cell plates. These observations together with the finding that a kinase-dead form MPK6AEF transformed into mpk6-4 mutant background showed a similar root phenotype like yda1 mutant contributed to the conclusion that MPK6 is an important player downstream of YODA (MAPKKK4). Both YODA and MPK6 are involved in postembryonic root development by regulating cell division plane orientation.
Next, we searched for putative interactions between KTN1 and MPK6, its closely-related MPK3 and also MPK4, since in silico studies predicted the existence of a MAPK-targeted proline-directed serine at the 92nd position of KTN1. Results of YFP-split technology (BiFC, Bimolecular Fluorescent Complementation) experiments showed interactions of both MPK4 and MPK6 with KTN1 while preliminary results obtained by yeast-two-hybrid (Y2H) assay also showed interaction of MPK3 with KTN1. These protein-protein interaction data support the potential regulation of KTN1 by MAPKs.
The second part of thesis summarises current knowledge on microtubule severing protein KTN1 with main focus on mutant phenotypes. Thus, it is devoted to the phenotypic analysis of the selected KTN1 mutants used herein (fra2, lue1 and ktn1-2) as related to fertility, embryo and seed development, and to the ability of a pKTN1::GFP:KTN1 construct to rescue mutant phenotypes. The phenotypic analyses of KTN1 mutants revealed an abnormal development of sporophytic tissues and of male and female reproductive organs. Stable transformations of fra2, lue1 a ktn1-2 mutants with pKTN1::GFP:KTN1 construct (expressed under control of the native KTN1 promoter) were performed and T3 lines showed rescue (genetic complementation) of mutant phenotypes. Therefore, we propose a new role of KTN1 in plant fertility and seed formation of Arabidopsis thaliana.
Objectives
1. Summary of the recent knowledge on plant mitogen-activated protein kinases (MAPKs) focused on MPK6.
2. Preparation of proMPK6::GFP:MPK6 and proMPK6::MPK6:GFP constructs and their transformation to mpk6-2 mutant background to gain rescue of the mutant phenotype.
3. Subcellular localisation of GFP-fused MPK6 driven under native promoter using confocal scanning microscopy.
4. Characterization of potential new MAPK substrate, namely microtubule severing protein KATANIN 1.
5. Summary of the recent knowledge on plant KATANIN 1.
6. Phenotypical characterization of fra2, lue1 and ktn1-2 mutants and preparation of mutant lines genetically rescued with proKTN1::GFP:KTN1 construct.
Zásady pro vypracování
Objectives
1. Summary of the recent knowledge on plant mitogen-activated protein kinases (MAPKs) focused on MPK6.
2. Preparation of proMPK6::GFP:MPK6 and proMPK6::MPK6:GFP constructs and their transformation to mpk6-2 mutant background to gain rescue of the mutant phenotype.
3. Subcellular localisation of GFP-fused MPK6 driven under native promoter using confocal scanning microscopy.
4. Characterization of potential new MAPK substrate, namely microtubule severing protein KATANIN 1.
5. Summary of the recent knowledge on plant KATANIN 1.
6. Phenotypical characterization of fra2, lue1 and ktn1-2 mutants and preparation of mutant lines genetically rescued with proKTN1::GFP:KTN1 construct.
Seznam doporučené literatury
Colcombet J, Hirt H (2008) Arabidopsis MAPKs: a complex signalling network involved in multiple biological processes. Biochem J. 413:217-226; Rodriguez MC, Petersen M, Mundy J (2010) Mitogen-activated protein kinase signaling in plants. Annu Rev Plant Biol. 61:621-49; Šamajová O, Plíhal O, Al-Yousif M, Hirt H, Šamaj J. (2012) Improvement of stress tolerance in plants by genetic manipulation of mitogen-activated protein kinases. Biotechnol Adv. (in press, published on line); Rasmussen MW, Roux M, Petersen M, Mundy J (2012) MAP Kinase Cascades in Arabidopsis Innate Immunity. Front Plant Sci.;3:169; Beck M, Komis G, Müller J, Menzel D, Šamaj J (2010) Arabidopsis Homologs of Nucleus- and Phragmoplast-Localized Kinase 2 and 3 and Mitogen-Activated Protein Kinase 4 Are Essential for Microtubule Organization. Plant Cell 22:755-771; Beck M, Komis G, Ziemann A, Menzel D, Šamaj J (2010) Mitogen-activated protein kinase 4 is involved in the regulation of mitotic and cytokinetic microtubule transitions in Arabidopsis thaliana. New Phytologist 189: 1069-1083; Müller J, Beck M, Mettbach U, Komis G, Hause G, Menzel D, Šamaj J (2010) Arabidopsis MPK6 is involved in cell division plane control during early root development, and localizes to the pre-prophase band, phragmoplast, trans-Golgi network and plasma membrane. Plant J 61: 234-248. Panteris E, Komis G, Adamakis I-D S, Šamaj J, Bosabalidis AM (2010) MAP65 in Tubulin/Colchicine Paracrystals of Vigna sinensis Root Cells: Possible Role in the Assembly and Stabilization of Atypical Tubulin Polymers. Cytoskeleton 67:152-160. Šamaj J, Ovecka M, Hlavacka A, Lecourieux F, Meskiene I, Lichtscheidl I, Lenart P, Salaj J, Volkmann D, Borge L, Baluška F, Hirt H (2002) Involvement of the mitogen-activated protein kinase SIMK in regulation of root hair tip growth. EMBO J. 21: 3296-3306. Wang H, Ngwenyama N, Liu Y, Walker JC, Zhang S (2007) Stomatal Development and Patterning Are Regulated by Environmentally Responsive Mitogen-Activated Protein Kinases in Arabidopsis. Plant Cell 19: 63-73. Bouquin T, Mattsson O, Naested H, Foster R, Mundy J (2003) The Arabidopsis lue1 mutant defines a katanin p60 ortholog involved in hormonal control of microtubule orientation during cell growth. J Cell Sci 116: 791-801. Burk DH, Ye ZH (2002) Alteration of oriented deposition of cellulose microfibrils by mutation of a katanin-like microtubule-severing protein. Plant Cell 14: 2145-2160. Nakamura M, Ehrhardt DW, Hashimoto T (2010) Microtubule and katanin-dependent dynamics of microtubule nucleation complexes in the acentrosomal Arabidopsis cortical array. Nat Cell Biol 12: 1064-1070.
Seznam doporučené literatury
Colcombet J, Hirt H (2008) Arabidopsis MAPKs: a complex signalling network involved in multiple biological processes. Biochem J. 413:217-226; Rodriguez MC, Petersen M, Mundy J (2010) Mitogen-activated protein kinase signaling in plants. Annu Rev Plant Biol. 61:621-49; Šamajová O, Plíhal O, Al-Yousif M, Hirt H, Šamaj J. (2012) Improvement of stress tolerance in plants by genetic manipulation of mitogen-activated protein kinases. Biotechnol Adv. (in press, published on line); Rasmussen MW, Roux M, Petersen M, Mundy J (2012) MAP Kinase Cascades in Arabidopsis Innate Immunity. Front Plant Sci.;3:169; Beck M, Komis G, Müller J, Menzel D, Šamaj J (2010) Arabidopsis Homologs of Nucleus- and Phragmoplast-Localized Kinase 2 and 3 and Mitogen-Activated Protein Kinase 4 Are Essential for Microtubule Organization. Plant Cell 22:755-771; Beck M, Komis G, Ziemann A, Menzel D, Šamaj J (2010) Mitogen-activated protein kinase 4 is involved in the regulation of mitotic and cytokinetic microtubule transitions in Arabidopsis thaliana. New Phytologist 189: 1069-1083; Müller J, Beck M, Mettbach U, Komis G, Hause G, Menzel D, Šamaj J (2010) Arabidopsis MPK6 is involved in cell division plane control during early root development, and localizes to the pre-prophase band, phragmoplast, trans-Golgi network and plasma membrane. Plant J 61: 234-248. Panteris E, Komis G, Adamakis I-D S, Šamaj J, Bosabalidis AM (2010) MAP65 in Tubulin/Colchicine Paracrystals of Vigna sinensis Root Cells: Possible Role in the Assembly and Stabilization of Atypical Tubulin Polymers. Cytoskeleton 67:152-160. Šamaj J, Ovecka M, Hlavacka A, Lecourieux F, Meskiene I, Lichtscheidl I, Lenart P, Salaj J, Volkmann D, Borge L, Baluška F, Hirt H (2002) Involvement of the mitogen-activated protein kinase SIMK in regulation of root hair tip growth. EMBO J. 21: 3296-3306. Wang H, Ngwenyama N, Liu Y, Walker JC, Zhang S (2007) Stomatal Development and Patterning Are Regulated by Environmentally Responsive Mitogen-Activated Protein Kinases in Arabidopsis. Plant Cell 19: 63-73. Bouquin T, Mattsson O, Naested H, Foster R, Mundy J (2003) The Arabidopsis lue1 mutant defines a katanin p60 ortholog involved in hormonal control of microtubule orientation during cell growth. J Cell Sci 116: 791-801. Burk DH, Ye ZH (2002) Alteration of oriented deposition of cellulose microfibrils by mutation of a katanin-like microtubule-severing protein. Plant Cell 14: 2145-2160. Nakamura M, Ehrhardt DW, Hashimoto T (2010) Microtubule and katanin-dependent dynamics of microtubule nucleation complexes in the acentrosomal Arabidopsis cortical array. Nat Cell Biol 12: 1064-1070.
Přílohy volně vložené
Supplement I
Research article
KATANIN 1 is essential for embryogenesis and seed formation in Arabidopsis
Luptovčiak I*, Samakovli D*, Komis G, Šamaj J (2017)
Frontiers in Plant Science 2017; 8:728, * joined first authors.
Supplement I
Research article
KATANIN 1 is essential for embryogenesis and seed formation in Arabidopsis
Luptovčiak I*, Samakovli D*, Komis G, Šamaj J (2017)
Frontiers in Plant Science 2017; 8:728, * joined first authors.
Supplement III
Research article
Katanin effects on dynamics of cortical microtubules and mitotic arrays in Arabidopsis thaliana revealed by advanced live-cell imaging
Komis G*, Luptovčiak I*, Ovečka M*, Samakovli D, Šamajová O, Šamaj J
Frontiers in Plant Science 2017; 8:866, * joined first authors.
Supplement IV
Review article
Transient plant transformation mediated by Agrobacterium tumefaciens: Principles, methods and applications
Křenek P, Šamajová O, Luptovčiak I, Doskočilová A, Komis G, Šamaj J
Biotechnology Advances 2015; 33(6), 1024-1042.
Supplement V
Ph.D. Thesis summary
Supplement VI
1x DVD-RW - PhD Thesis
Supplement VII
1x CD-RW - Ph.D. Thesis summary