139
140
141 Suppl. Fig. 1 Alignment of PHY4 cDNAs cloned by Mittmann et al. (2004) and after genome sequencing (2007). CLUSTALW2-alignment. Point mutations are marked in purple.
142 12.1.2. Alignment of phytochrome sequences from higher and lower
plants
143 Suppl. Fig. 2 Alignment of Arabidopsis PHYA and PHYB with Physcomitrella PHY4 and PHY42004 protein sequences. CLUSTALW2-alignment. PHY42004 is designated Pp.PHY4*. Point mutations in conserved residues are marked in purple.
144 12.2. Y2H
12.2.1. Establishment of internal system controls
Suppl. Fig. 3 Dimerisation of BD:PHY4 and AD:PHY4 fusions in yeast strain AH109. Quantitative approach, nutritional selection as indicated (3DO: -Trp/-Leu/-His, 4DO: -Trp/-Leu/-His/-Ade).
12.3. In silico analysis of putative PHY4 interactors identified by Y2H screening 12.3.1. #16.1, p-loop containing nucleoside triphophaste hydrolase
(PLP)
Suppl. Table 1 Homologs of Pp.PLP used for alignment and phylogenetic analysis (in light yellow).
145 Suppl. Fig. 4: Phylogenetic tree of PLP homologs in different plant species.
PLP homologs are conserved throughout the plant kingdom, from lower plants (in light green) to higher plants (monocots in light red, dicots in purple). Homologs were identified by protein BLAST.
Distances and groupings were determined by Jukes-Cantor and Neighbour-Joining method and correspond to 500 replications. Bootstrap values are given at the branches. Scale bar represents protein distance as substitutions per site. Accession numbers together with a table of further information are given in the appendix.
12.3.2. #33.7, pleiotropic regulator locus (PRL)
Suppl. Table 2 Homologs of Pp.PRL used for alignment and phylogenetic analysis (in light yellow).
146 Suppl. Fig. 5: Phylogenetic tree of PRL1 homologs in different plant species.
PRL1 homologs are conserved throughout the plant kingdom, from algae (brown) to lower (in light green) and higher plants (monocots in light red, dicots in purple). Homologs were identified by protein BLAST. Distances and groupings were determined by Jukes-Cantor and Neighbour-Joining method and correspond to 500 replications. Bootstrap values are given at the branches. Scale bar represents protein distance as substitutions per site. Accession numbers together with a table of further information are given in the appendix.
147 12.3.3. #54.1, elongation factor 1 α (EF1α)
Suppl. Table 3 Homologs of Pp.EF1α used for alignment and phylogenetic analysis (in light yellow).
148 Suppl. Fig. 6 Consensus tree of EF1α homologs in different plant species.
EF1α homologs are conserved from archae to eukaryotes and are thus also found throughout the plant kingdom, from lower plants (in light green) to higher plants (gymnosperms in light blue, monocots in light red, dicots in purple). Homologs were identified by protein BLAST. Distances and groupings were determined by Jukes-Cantor and Neighbour-Joining method and correspond to 500 replications. Bootstrap values are given at the branches. Scale bar represents protein distance as substitutions per site. Accession numbers together with a table of further information are given in the appendix.
149 12.3.4. #61.4, Pirin-like protein (Pirin)
Suppl. Table 4 Homologs of Pp.Pirin used for alignment and phylogenetic analysis (in light yellow).
150 Suppl. Fig. 7 Consensus tree of pirin (-like) homologs in different plant species.
Pirin (-like) homologs are conserved throughout the plant kingdom, from lower plants (in light green) to higher plants (gymnosperms in light blue, monocots in light red, dicots in purple). Homologs were identified by protein BLAST. Distances and groupings were determined by Jukes-Cantor and Neighbour-Joining method and correspond to 500 replications. Bootstrap values are given at the branches. Scale bar represents protein distance as substitutions per site. Accession numbers together with a table of further information are given in the appendix.
151 12.4. Light dependent interaction of phy4:BD with putative interactors in yeast
12.4.1. Quantitative growth assay on PCB-complemented medium:
full-length phy4:BD
Suppl. Fig. 8 Analysis of light dependent phy4:BD interaction with putative interacting proteins. No interaction with the given putative interacting proteins was observed.
12.4.2. Quantitative growth assay on PCB-complemented medium:
phy4_N:BD
Suppl. Fig. 9 Analysis of light dependent phy4_N:BD interaction with putative interacting proteins.
No interaction with the given putative interacting proteins was observed.
152 12.4.3. Quantitative growth assay on PCB-complemented medium:
PHY4_C:BD
Suppl. Fig. 10 Analysis of light dependent interaction of PHY4_C:BD with putative interactors. Apo-PHY4_C interacted only weakly with PLP and Pirin prey-proteins under most stringent selection conditions. No interaction was seen for EF1α and PRL1.
12.5. sYFP-based in vivo interaction studies of phy4 with its putative interactors
Suppl. Fig. 11 Expression of YFPN:[empty] and YFPC:[empty] did not lead to emission of sYFP fluorescence signals. Scale bars 50 µm.
153 12.6. Light dependent localisation studies of putative phy4 interactors in
Physcomitrella
These localisation studies were carried out by Rabea Krikor in course of her master thesis project under my supervision.
Suppl. Fig. 12 Localisation of N-terminally tagged CFP-fusions of EF1α and PRL1 in darkness and after red light incubation. EF1α was strictly localised to the cytoplasm, as seen from the clear exclusion of the signal from the nucleus (b). PRL1 was clearly located to the nucleus in both darkness and after red light treatment. Scale bars 50 µm.
Suppl. Fig. 13 Localisation of N-terminally tagged CFP-fusions of PLP and Pirin in darkness and after red light incubation. PLP showed homogenous fluorescence in both darkness and after red illumination, whereas Pirin exhibited clear accumulation within the nucleus under both light conditions. A considerable cytoplasmic signal remained, however. Scale bars 50 µm.
154 12.7. Studies on Physcomitrella phytochrome 4-phototropin interaction
12.7.1. Quantitative growth assay on PCB-complemented medium: full- length phy4:BD
Suppl. Fig. 14 Analysis of light dependent interaction of C-terminally fused phy4 with any of the four phototropins by Y2H. No interaction was observed.
12.7.2. Quantitative growth assay on PCB-complemented medium:
phy4_N:BD
Suppl. Fig. 15 Analysis of light dependent interaction of C-terminally fused phy4_N with any of the four phototropins by Y2H. No interaction was observed.
155 12.7.3. Quantitative growth assay on PCB-complemented medium:
PHY4_C:BD
Suppl. Fig. 16 Analysis of light dependent interaction of C-terminally fused PHY4_C with any of the four phototropins by Y2H. Interaction only with photA1 was observed, whereas no other phototropin was bound by PHY4_C.
12.7.4. sYFP-based analysis of phototropin homodimerisation
Suppl. Fig. 17 N-terminal fusions of YFPN/C to photA2 form homodimers independent from light.
Scale bars 50 µm.
156 Suppl. Fig. 18 N-terminal fusions of YFPN/C to photB2 form homodimers independent from light.
Scale bars 50 µm.
12.8. Y2H-based studies on phyA-phototropin interaction
12.8.1. Quantitative Y2H growth assay on PCB-complemented medium
To test for direct phyA-phot1/2 interaction N-terminal BD/AD:fusions of phyA (BD:phyA) and phot1/2 (AD:phot1/2) were cloned, following the example of Y2H experiments carried out with Physcomitrella phy4 and phototropin fusions. phyA_pGBKT7 was cloned by Anna Lena Lichtenthäler, phot1_pGADT and phot2_pGADT7 were cloned by Melanie Bingel.
Suppl. Fig. 19 Analysis of light dependent phyA - phototropin interaction by conventional Y2H approach. Scheme in the upper left corner indicates bait and prey molecules used. N-terminal fused BD:phyA fusion proteins exhibited red light dependent growth only upon interaction with FHY1 under medium (middle panel). No interaction was observed for phyA with phot1 or phot2.
157
Danksagung
Jon Hughes möchte ich von Herzen danken für ein offenes Gespräch zur Weihnachtszeit 2006, das eine Kehrtwende meiner Pläne bedeutete und mich in ein brandneues und unwahrscheinlich faszinierendes Physcomitrella Projekt brachte. Ich bin dankbar für alle Unterstützung und das Vertrauen, das mir die Gelegenheit gab, zu lernen, dass man meist doch immer noch einen Schritt weiter gehen kann. Und noch einen. Und noch einen… Komm nach Jülich!
Ich bin dankbar und froh Mathias Zeidler als Moos-Veteranen begleitend an meiner Seite gewusst zu haben. Seine bewundernswerte Eigenschaft Motivation eimerweise über mich zu schütten hat mehr als einmal meine frühzeitige Kapitulation abgewendet und dazu geführt, dass mein Ehrgeiz wieder entfacht wurde. Er war Netz und doppelter Boden - ohne ihn hätte es Knochenbrüche gegeben.
Sehr dankbar bin ich TanJA! Gans und Andrea Weisert - unser gemeinsamer Weg begann schon früher als diese Arbeit und hat nun zu diesem kleinen Meilenstein geführt. Nicht nur die exzellente technische Assistenz, sondern auch Unterstützung, Motivation und Hilfestellungen im Großen wie im Kleinen hat dazu geführt, dass ich diese Arbeit zu einem Ende bringen konnte. Von ganzem Herzen Danke auch an Melanie Bingel, die unzählige DNA-Präparationen für mich übernommen habt. Tina Lang und Spirulina danke ich für PCB-Präparationen im mg-Bereich – ohne Euch wäre es bloß grau in grau! Für alle pflegerische Unterstützung danke ich Roland Kürschner bei der Versorgung mehrerer Generationen von Gänseblümchen und Zwiebeln. Es tut mir im Nachhinein Leid um die vielen schönen Blumen, die unter mir so schwer zu leiden hatten. Anette Münndelein danke ich für all die Herzlichkeit, die staunenden Augen und den frischen Wind.
Anna Lena Lichtenthäler und Rabea Krikor möchte ich von Herzen danken für einige experimentelle Unterstützung während der letzten Züge meiner Arbeit und der aufregenden Zeit des „großen Manuskript“ Schreibens.
Adriaan Dorresteijn und Anne Holz danke ich für hilfsbereite Unterstützung und Einführung in die hohe Kunst der konfokalen Laser Scanning Microscopy.
Jutta Rösler bin ich für so viele verschiedene Dinge dankbar, dass ich sie hier unmöglich aufzählen kann. Es gibt kaum einen Menschen, der mich und meine Arbeit schon seit dem allerersten Moment derart beeinflusst hat, von dem ich soviel Unterstützung bekommen habe und der mich, bis zuletzt!, gleichzeitig so häufig in den Wahnsinn getrieben hat.
Einen großen Anteil am Gelingen meiner Doktorarbeit haben meine Freunde, allen voran Ilse Klein, Silke Krüger, Marika Midon und Ines Winkler – einfach dafür, dass sie mit Phytochrom nichts (mehr) am Hut haben.
Ich bin froh, von meiner Familie soviel Kraft und Verständnis bekommen zu haben, jedes Mal wenn ich nicht vom Phytochrom los kam. Jo danke ich für das gemeinsame Forschen, Diskutieren, Leben, Arbeiten, Schimpfen, Herzrasen, Kochen, Doktorarbeit schreiben, Köpfe schütteln und Lachen, Lachen, Lachen!
(In Ge-)danke(n) an Gottfried Wagner, der sicher seine große Freude mit dieser Arbeit gehäbt hätte.