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

Abstract

Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.

Control of cytokinin and auxin homeostasis in cyanobacteria and algae

“…iPR and cis ZR have also been reported previously as predominantly tRNA-bound as well as free CK forms in several cyanobacterial and microalgal species by Šimura et al (2014). The significance of cis Z-type CKs in the S cenedesmus obliquus growth cycle demonstrated in our observations and their potential origin from the tRNA pathway suggested by recent phylogenetic studies, in which higher similarity of IPTs from cyanobacteria and microalgae related to tRNA-IPTs was reported (Frébortová et al , 2015; Lu and Xu, 2015), is thus likely. The prevalence of tRNA-bound cis ZR and iPR, indicating an important involvement of a tRNA-dependent CK biosynthetic pathway, was also described in the moss Physcomitrella patens (Yevdakova et al , 2008).…”

“…This is in accordance with recently published data demonstrating only a very sporadic detection of homologous CKX sequences in algae (Lu et al , 2014) and no detectable enzymatic activity of NoCKX1 in cyanobacterium Nostoc sp. PCC 7120 (Frébortová et al , 2015). Notably, the occurrence of [ 3 H] trans Z metabolite peaks with retention times corresponding to DHZ and DHZR (Fig.…”

“…6B and data not shown). Moreover, the genes involved in CK conjugation ( UGT ) and degradation ( CKX ) have not been functionally characterized in algae yet (Lu et al , 2014; Frébortová et al , 2015). Thus, it can be hypothesized that CK homeostasis during algal growth is controlled basically by modulation of the pool of CK ribotides functioning at least partially as storage metabolites.…”

“…Recently, the function of gene encoded adenylate-IPT in the cyanobacterium Nostoc sp. PCC 7120 has been reported, although it clusters to plant tRNA-IPT (Frébortová et al , 2015). Interestingly, a putative CK oxidase/dehydrogenase (CKX) homologous gene sequence involved in CK degradation was discovered in Nostoc sp.…”

“…PCC 7120 ( NsCKX1 ), but the predicted NsCKX1 similarity to plant CKX proteins was very low (Schmülling et al , 2003). Moreover, functional analysis of the recombinant CKX protein named in the study as NoCKX1 revealed no detectable activity for CK downregulation (Frébortová et al , 2015). In addition, no matching CKX sequences were detected in Synechocystis sp.…”