From biosynthesis and beyond-Loss or overexpression of the cytokinin synthesis gene, iptA, alters cytokinesis and mitochondrial and amino acid metabolism in Dictyostelium discoideum.

Cytokinins (CKs) are a class of growth-promoting signaling molecules that affect multiple cellular and developmental processes. These phytohormones are well studied in plants, but their presence continues to be uncovered in organisms spanning all kingdoms, which poses new questions about their roles and functions outside of plant systems. Cytokinin production can be initiated by one of two different biosynthetic enzymes, adenylate isopentenyltransfases (IPTs) or tRNA isopentenyltransferases (tRNA-IPTs). In this study, the social amoeba, Dictyostelium discoideum, was used to study the role of CKs by generating deletion and overexpression strains of its single adenylate-IPT gene, iptA. The life cycle of D. discoideum is unique and possesses both single- and multicellular stages. Vegetative amoebae grow and divide while food resources are plentiful, and multicellular development is initiated upon starvation, which includes distinct life cycle stages. CKs are produced in D. discoideum throughout its life cycle and their functions have been well studied during the later stages of multicellular development of D. discoideum. To investigate potential expanded roles of CKs, this study focused on vegetative growth and early developmental stages. We found that iptA-deficiency results in cytokinesis defects, and both iptA-deficiency and overexpression results in dysregulated tricarboxylic acid (TCA) cycle and amino acid metabolism, as well as increased levels of adenosine monophosphate (AMP). Collectively, these findings extend our understanding of CK function in amoebae, indicating that iptA loss and overexpression alter biological processes during vegetative growth that are distinct from those reported during later development.

Biochemical characterization of a unique cytokinin and nucleotide phosphoribohydrolase Lonely Guy protein from Dictyostelium discoideum.

Lonely guy (LOG) proteins are phosphoribohydrolases (PRHs) that are key cytokinin (CK)-activating enzymes in plant and non-plant CK-producing organisms. During CK biosynthesis, LOGs catalyze the conversion of precursor CK-nucleotides (CK-NTs) to biologically active free base forms. LOG/PRH activity has been detected in bacteria, archaea, algae, and fungi. However, in these organisms, the LOG/PRH activity for CK-NTs and non-CK-NTs (e.g., adenine-NTs) has not been assessed simultaneously, which leaves limited knowledge about the substrate specificity of LOGs. Thus, we performed bioinformatic analyses and a biochemical characterization of a LOG ortholog from Dictyostelium discoideum, a soil-dwelling amoeba, which produces CKs during unicellular growth and multicellular development. We show that DdLog exhibits LOG/PRH activity on two CK-NTs, N 6 -isopentenyladenosine-5'-monophosphate (iPMP) and N 6 -benzyladenosine-5'-monophosphate (BAMP), and on adenosine 5'-monophosphate (AMP) but not on 3', 5'-cyclic adenosine-monophosphate (cAMP). Additionally, there were higher turnover rates for CK-NTs over AMP. Together, these findings confirm that DdLog acts as a CK-activating enzyme; however, in contrast to plant LOGs, it maintains a wider specificity for other substrates (e.g., AMP) reflecting it has maintained its original, non-CK related role even after diversifying into a CK-activating enzyme.