Additional file 4: of The effects of repeated whole genome duplication events on the evolution of cytokinin signaling pathway

Supplemental alignment of #CHKs, #HPTs, #RRAs and #RRBs. (FASTA 770 kb

Additional file 1: of The effects of repeated whole genome duplication events on the evolution of cytokinin signaling pathway

List of analysed species and used data source, and list of the analysed CHK encoding sequences. (XLSX 46 kb

Additional file 2: of The effects of repeated whole genome duplication events on the evolution of cytokinin signaling pathway

Collinear regions, Ks distances. (XLSX 357 kb

List of vectors and <i>E</i>. <i>coli</i> expression strains used in <i>NoCKX1</i> study.

<p>^apCIOX vector is a modified version of the commercial pET SUMO vector of Invitrogen. It was a kind gift from prof. Andrea Mattevi.</p><p>List of vectors and <i>E</i>. <i>coli</i> expression strains used in <i>NoCKX1</i> study.</p

Phylogenetic tree for the CKX proteins shows a clear distinction between plants and bacterial sequences.

<p>Maximum likelihood phylogeny derived by the CKX domain sequences identified in both plants (green) and bacteria (orange). The robustness of the phylogenetic tree was assessed using 500 bootstrap repetitions. The sequence identifiers and species corresponding to the abbreviations are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138468#pone.0138468.s004" target="_blank">S2 Table</a>. The investigated sequence is marked by a red arrow.</p

SDS-PAGE analysis of purification of <i>NoCKX1</i> expressed from different vectors.

<p>A/ Purification of <i>NoCKX1</i> expressed from pMAL-c4X vector. Lane 1: molecular mass standard; lane 2: fusion protein (<i>NoCKX1</i> with maltose binding protein) after purification on amylose column; lane 3: same protein after cleavage by factor Xa protease. B/ Purification of <i>NoCKX1</i> expressed from pCIOX vector. Lane 1: molecular mass standard; lane 2: fusion protein (<i>NoCKX1</i> with SUMO and histidine tag) after purification on Ni-NTA column. Proteins were separated in 10% SDS-polyacrylamide gel.</p

Phylogenetic tree for the IPT proteins shows diverse pattern for the different subtypes.

<p>Maximum likelihood phylogeny derived by the full-protein sequences identified in both plants (green) and bacteria (orange). The robustness of the phylogenetic tree was assessed using 500 bootstrap repetitions. The sequence identifiers and species corresponding to the abbreviations are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138468#pone.0138468.s004" target="_blank">S2 Table</a>. The investigated sequence is marked with a red arrow. The tRNA IPTs clades were named as suggested before [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138468#pone.0138468.ref009" target="_blank">9</a>].</p

Concentration of cytokinins in <i>Nostoc</i> cells.

<p>^aAbbreviations: iP, <i>N</i>^<i>6</i>- (Δ²-isopentenyl) adenine; iPR, <i>N</i>^<i>6</i>- (Δ²-isopentenyl) adenine 9-riboside; iPNT, <i>N</i>^<i>6</i>- (Δ²-isopentenyl)adenine nucleotides; tZ, <i>trans</i>-zeatin; tZR, <i>trans</i>-zeatin 9-riboside; tZNT, <i>tran</i>s-zeatin nucleotides; tZOG, <i>trans</i>-zeatin-O-glucoside; tZROG, <i>trans</i>-zeatin 9-riboside-O-glucoside; cZ, <i>cis</i>-zeatin; cZR, <i>cis</i>-zeatin 9-riboside; cZNT, <i>ci</i>s-zeatin nucleotides; cZOG, <i>cis</i>-zeatin-O-glucoside; cZROG, <i>cis</i>-zeatin 9-riboside-O-glucoside; DHZ, dihydrozeatin; DHZR, dihydrozeatin 9-riboside; DHZNT, dihydrozeatin nucleotides; DHZOG, dihydrozeatin-O-glucoside; DHZROG, dihydrozeatin 9-riboside-O-glucoside.</p><p>Concentration of cytokinins in <i>Nostoc</i> cells.</p

Amino acid sequence alignment of ZmCKX1 and <i>NoCKX1</i>.

<p>Protein sequences were aligned using the ClustalW interface in BioEdit 7.0.5.3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138468#pone.0138468.ref055" target="_blank">55</a>]. Major differences in conserved regions are indicated by boxes. Amino acid residues which were mutated to match consensus sequence are marked by asterisk. Histidine in the motif ¹⁰⁴GHS is involved in FAD covalent linkage, ³⁹⁴PHPWLN and ⁵⁰⁴HFG are conserved C-terminal domains and the residues ¹⁶⁹D, ³⁷⁸V and ³⁸¹E are involved in substrate biding. FAD binding domain (pfam01565) and cytokinin binding domain (pfam09265) of <i>NoCKX1</i> identified by NCBI´s conserved domain search [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138468#pone.0138468.ref066" target="_blank">66</a>] are shown in red and underlined letters, respectively. Numbering refers to the sequence of ZmCKX1.</p

The CHASE domain of CRE1/AHK4 is necessary and sufficient for cytokinin binding

<p><b>Copyright information:</b></p><p>Taken from "Evolutionary proteomics identifies amino acids essential for ligand-binding of the cytokinin receptor CHASE domain"</p><p>http://www.biomedcentral.com/1471-2148/7/62</p><p>BMC Evolutionary Biology 2007;7():62-62.</p><p>Published online 17 Apr 2007</p><p>PMCID:PMC1863423.</p><p></p> binding of -[2-3H]zeatin to full length CRE1/AHK4 protein or different domains of the receptor overexpressed in BL21. Bacterial cells were assayed for specific -[2-3H]zeatin binding. Data are mean ± s.d.; n = 4