Phytohormone metabolism in human cells: Cytokinins are taken up and interconverted in HeLa cell culture

Abstract

Cytokinins (CKs) encompass a group of phytohormones, known to orchestrate many critical processes in plant development. Excluding Archaea, CKs are pervasive among all kingdoms, but much less is reported about their metabolism beyond plants. Recent evidence from mammalian tissues indicates the presence of six additional CK forms beyond the previously identified, single mammalian CK, N6‐isopentenyladenosine (i6A). There is limited understanding of CK biosynthesis pathways in mammalian systems; therefore, human cervical cancer (HeLa) cells were used to further characterize CK processing by tracking the interconversion of CKs into their various structural derivatives in mammalian cells in a time‐course study. Through high‐performance liquid chromatography‐positive electrospray ionization‐tandem mass spectrometry (HPLC‐(+ESI)‐MS/MS), we document changes in the functional profiles of endogenous CKs in a human cell line following metabolism by HeLa cell cultures. The nucleotide CK fraction (iPRP) was found exclusively within the cell pellet (0.34 pmol/106 cells), and the active free base (FB) form (iP) and riboside fraction (iPR) were found in greater abundance extracellularly (1.67 and 0.10 nmol/L respectively). For further confirmation, we demonstrate that HeLa cells metabolize an exogenously supplied CK, N6‐benzyladenosine (BAR). In the HeLa culture supernatant, a 12‐fold decrease in BAR concentration was observed within the first 24 hours of incubation accompanied by a fivefold increase in the FB form, N6‐benzyladenine (BA). These findings support the hypothesis that HeLa cells have the enzymatic pathways required for the metabolism of both endogenous and exogenous CKs.

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

“…Since then, more recent studies on CK profiling by High Performance Liquid Chromatography Tandem Mass Spectrometry (HPLC-MS/MS) revealed a presence of seven types of CKs in a range of canine tissues (Seegobin et al, 2018). The presence of unbound CK derivatives in HeLa cell cultures were detected, strengthening the evidence of TRIT1 activity in CK biosynthesis in mammalian cells (Aoki et al, 2019b).…”

“…Cytokinin presence and metabolism has been reported in canine tissues (Seegobin et al, 2018) and human cell cultures (Aoki et al, 2019b), and the CK profiles indicate that tRNA degradation is the only source of unbound mammalian CKs. tRNA-isopentenyltransferase 1 (TRIT1), a homolog of the prokaryotic miaA, facilitates production of tRNA-bound iP-type CKs in human cytosol and mitochondria (Lamichhane et al, 2013;Smaldino et al, 2015).…”

“…AT4G36390 is predicted to localize mainly to the mitochondria and carry an [Fe-S]; however, much of its role remains unknown (Dabravolski, 2020). 1,000-5,000 pmol/mL 5,000-10,000 pmol/mL Aoki et al, 2019b Using HPLC-MS/MS analysis at the Water Quality Centre, Trent University, example organisms of the Prokaryotae (bacteria) and the Eukaryotae (protists, fungi, plants, and animals) were scanned for presence of 2MeSZ, 2MeSZR, 2MeSiP, and 2MeSiPR. 2MeS-CKs were detected in the representatives of all the analyzed kingdoms except for fungi.…”

Cytokinins Are Abundant and Widespread among Insect Species

“…CKs are not exclusively plant-derived compounds. CKs have been reported from a variety of organisms including bacteria [4][5][6][7][8][9], fungi [10][11][12], protists [13], nematodes [14], insects [15][16][17][18][19], and mammals [20], including humans [21]. CKs can be seen as inter-kingdom signaling molecules that orchestrate cross-talk with plants but may also display bioactive functions not related to interactions with plants.…”

Integrated structural and functional analysis of the protective effects of kinetin against oxidative stress in mammalian cellular systems

“…In this regard, Seegobin et al 16 unequivocally detected the presence of seven CK forms in a wide selection of canine tissues: iPRP (mono-, di-, and triphosphate), cis-zeatin riboside (cZR), cis-zeatin nucleotide (cZRP; mono, di-, and triphosphate), 2-methylthio-isopentenyladenine (2MeSiP), 2-methylthio-isopentenyl-adenosine (2MeSiPR) and 2-methylthio-zeatin riboside (2MeSZR) by mass spectrometry. Another interesting development is the metabolism of benzylaminopurine (BAP) in HeLa cells; a 12-fold decrease in BAPR concentration was observed within the first 24-h of incubation 17 of BAP with HeLa cells. These findings point to the notion that mammalian cells possess the enzymatic pathways for the metabolism of both endogenous and exogenous CKs.…”