Cytokinin Detection during the Dictyostelium discoideum Life Cycle: Profiles Are Dynamic and Affect Cell Growth and Spore Germination

Abstract

Cytokinins (CKs) are a family of evolutionarily conserved growth regulating hormones. While CKs are well-characterized in plant systems, these N6-substituted adenine derivatives are found in a variety of organisms beyond plants, including bacteria, fungi, mammals, and the social amoeba, Dictyostelium discoideum. Within Dictyostelium, CKs have only been studied in the late developmental stages of the life cycle, where they promote spore encapsulation and dormancy. In this study, we used ultra high-performance liquid chromatography-positive electrospray ionization-high resolution tandem mass spectrometry (UHPLC-(ESI+)-HRMS/MS) to profile CKs during the Dictyostelium life cycle: growth, aggregation, mound, slug, fruiting body, and germination. Comprehensive profiling revealed that Dictyostelium produces 6 CK forms (cis-Zeatin (cZ), discadenine (DA), N6-isopentenyladenine (iP), N6-isopentenyladenine-9-riboside (iPR), N6-isopentenyladenine-9-riboside-5′ phosphate (iPRP), and 2-methylthio-N6-isopentenyladenine (2MeSiP)) in varying abundance across the sampled life cycle stages, thus laying the foundation for the CK biosynthesis pathway to be defined in this organism. Interestingly, iP-type CKs were the most dominant CK analytes detected during growth and aggregation. Exogenous treatment of AX3 cells with various CK types revealed that iP was the only CK to promote the proliferation of cells in culture. In support of previous studies, metabolomics data revealed that DA is one of the most significantly upregulated small molecules during Dictyostelium development, and our data indicates that total CK levels are highest during germination. While much remains to be explored in Dictyostelium, this research offers new insight into the nature of CK biosynthesis, secretion, and function during Dictyostelium growth, development, and spore germination.

Cytokinins in Dictyostelia – A Unique Model for Studying the Functions of Signaling Agents From Species to Kingdoms

“…Interestingly, iP-type CKs were the dominant CK forms during single-celled growth and aggregation, and DA was not present during these early life cycle stages. We detected high levels of DA following the onset of culmination, which is consistent with previous research (Ihara et al, 1980;Aoki et al, 2019). Exogenous application of iP affected single-celled growth by prolonging the stationary phase of cultures (Aoki et al, 2019).…”

“…This early evidence of the developmental role of CK in D. discoideum was later confirmed and expanded upon by Anjard and Loomis (2008). More recently, a comprehensive scan of 30 potential CKs in D. discoideum revealed that 6 different CKs are synthesized and secreted during growth, development, and germination (Aoki et al, 2019). Total levels of CK production were highest in the fruiting body and during germination, followed by aggregation and single-celled growth.…”

“…Exogenous application of CKs on mammalian cancer cell lines show that CKs have anticancer activity (Voller et al, 2010). Moreover, CKs affect growth and germination in the social amoeba, Dictyostelium discoideum and in the biotrophic fungus, Claviceps purpurea (Hinsch et al, 2015;Aoki et al, 2019). In the coming years, it is likely that we will continue to see increasing roles of CKs beyond the plant kingdom as research continues to use organisms from all kingdoms of life.…”

The Origins and Roles of Methylthiolated Cytokinins: Evidence From Among Life Kingdoms

“…Three groups of enzymes (red font) involved in the production of 2MeS-CKs have been previously identified across kingdoms of life including: tRNA-isopentenyltransferases (e.g., miaA, MOD5, IPT2 and 9, TRIT1), tRNA-methylthiotransferases (e.g., miaB + C, CDK5RAP1), and cis-hydroxylases (e.g., miaE) (Dabravolski, 2020). The inferred enzymes involved in further modification of tRNA-bound and free 2MeS-CKs (blue font, dashed arrows) were predicted based on the information available from other CK producing pathways (Sakakibara, 2006;Spíchal, 2012;Morrison et al, 2017;Aoki et al, 2019a). Mevalonate (MVA) pathway is the main source of the isoprenoid substrate for tRNA-isopentenyltranserases; however, a small pool of the prenyl chain molecules can originate from the methylerythritol phosphate (MEP) pathway.…”

Cytokinin Sensing in Bacteria

“…Another interesting example of a pivotal role of CKs in biotic interactions is the ability of a plant-parasitic nematode to synthesize CK derivatives to manipulate the host system and establish long-term parasitic interactions [6,7]. Regarding the roles of CKs in amoebae, it was reported recently that the slime mold Dictyostelium discoideum produces six different CKs (notably cis-zeatin, isopentenyladenine, and discadenine) that coordinately orchestrate the different developmental stages of this protist [8][9][10]. However, studies of the occurrence and roles of CKs in non-plant organisms have been largely focused on bacteria [11].…”