Functional expression and purification of cytokinin dehydrogenase from Arabidopsis thaliana (AtCKX2) in Saccharomyces cerevisiae

Abstract

Cytokinin dehydrogenase (CKX) is responsible for regulating the endogenous cytokinin content by oxidative removal of the side chain and seven distinct genes, AtCKX1 to AtCKX7, code for the enzyme in Arabidopsis thaliana. The recombinant enzyme AtCKX2 was produced in Saccharomyces cerevisiae after expressing the corresponding gene from a plasmid (pDR197) or following chromosomal integration, under either the constitutive promoter PMA1 or the inducible promoter GAL1. The recombinant protein was purified from yeast culture media using a sequence of chromatographic steps. The purified enzyme had a molecular mass of 61 kDa and a typical flavoprotein spectrum. The specific activity of the enzyme was 87.8 kat g -1 , with isopentenyladenine as a substrate and 2,3-dimethoxy-5-methyl-pbenzoquinone as an electron acceptor. The pH optimum lay between 7.0 and 8.0, depending on the electron acceptor used. AtCKX2 reacts both with isoprenoid and aromatic cytokinins, the activity with isoprenoid cytokinins being two to three orders of magnitude higher. AtCKX2 prefers p-quinones and the synthetic dye 2,6-dichlorophenol indophenol as electron acceptors, although low reactivity with oxygen can also be observed. This study presents the first purification and characterization of the enzyme from Arabidopsis thaliana.Additional key words: cytokinin metabolism, cytokinin oxidase.

Methylated Cytokinins from the Phytopathogen Rhodococcus fascians Mimic Plant Hormone Activity

“…The recombinant MT proteins were assayed for methyltransferase activity as follows: MT buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10 mM MgCl 2 , 1 mg mL -1 bovine serum albumin, and 0.5 mM dithiothreitol), 2.5 mM SAM (Wako Chemicals), 100 mM IDP, DMAPP, or 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate, and 40 mg of recombinant enzyme for a 100-mL reaction mixture. Arabidopsis CKX2 recombinant enzyme was prepared as described (Frébortová et al, 2007). The CKX assay was conducted as described (Laskey et al, 2003) with two independent assays.…”

Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120

“…The NoCKX1 gene from previously prepared plasmid constructs was inserted into the pBin-HYG-TX vector downstream of the 35S promoter. In addition, NoCKX1 gene was fused with an oligonucleotide coding for apoplastic N-terminal signal sequence from AtCKX2 protein [ 43 ] or a vacuolar signal sequence from AtCKX1 [ 37 ]. The final vectors were electro-transformed into Agrobacterium tumefaciens GUS3101 and all three constructs were used for further transformation of tobacco plants.…”

Remodeling the isoprenoid pathway in tobacco by expressing the cytoplasmic mevalonate pathway in chloroplasts

“…The freeze-dried samples of wildtype and transplastomic leaves were used for separation of isoprenoid compounds (Frébortová et al, 2007). Before use, 5000-Da 4 mL filter units (Amicon Ultra, Millipore) were washed with 1 mL sterile water by centrifuging at 4000g for 2 h at 30 °C to remove glycerol and sodium salts.…”