Keywords

Plant signaling systems that integrate and control cellular responses are, at least partially, based on active chemical substances called phytohormones. Cytokinins represent a group of phytohormones defined as the compounds that in the presence of auxin (another phytohormone) induce cell division in a suitable assay material grown on a defined medium (Shaw 1994). Among other functions, they also regulate morphogenesis and cambial development; modulate the activity of root, shoot, and reproductive meristems; and inhibit leaf senescence. All native cytokinins are derivatives of adenine (free bases, sugar conjugates, or nucleotides) with at least one substituent at the exocyclic N6 position. Depending on the side chain structure, two main subclasses of cytokinins are recognized – isoprenoid and aromatic. Isoprenoid cytokinins, comprising N6-isopentenyladenine (iP), trans-zeatin (tZ), cis-zeatin (cZ) and dihydrozeatin, are ubiquitous in plants, whereas their aromatic counterparts, which consist of N6-benzyladenine (BA) and ortho- and meta-topolin (oT and mT), are very rare. For this reason, much is known about the function, metabolism, and perception of isoprenoid cytokinins, while the origin and fate of aromatic cytokinins remain largely unexplained (Frébort et al. 2011).

Several aromatic cytokinins have been identified in crown gall tumors of tomato (Nandi et al. 1989a, b), calla (Chaves dasNeves and Pais 1980), palm oil (Jones et al. 1996), and red goosefoot (Doležal et al. 2002). However, poplar (Populus × canadensis cv. Robusta) appears to be the best model plant for studying aromatic cytokinins. Mainly hydroxylated derivatives of BAP, oT, and mT as well as their sugar conjugates have been identified in extracts of poplar leaves (Horgan et al. 1975; Strnad et al. 1992, 1994, 1997; Tarkowská et al. 2003). Recently, we performed screening for aromatic cytokinins in a collection of poplar genetic resources (Forestry and Game Management Research Institute, Kunovice, Czech Republic). UHPLC-MS/MS analyses of leaf extracts from 12 gene bank accessions revealed no BAP-, methoxy-oT-, mT-, or para-topolin-type cytokinins (Jaworek et al. 2019). On the other hand, ortho-topolin riboside (oTR) was present in all accessions and especially abundant in P. × canadensis, P. × berolinensis, P. laurifolia, P. maximovici, and P. × oxford. Surprisingly, apart from P. × canadensis, oT was present in low quantities or not at all in the accessions tested. A concentration of aromatic cytokinins is reported to vary according to the length of exposure to light and the quality of light (Hewett and Wareing 1973). Time-dependent accumulation of aromatic cytokinins was observed in P. × canadensis, with the highest levels of oT (253 pmol g−1 FW) and oTR (156 pmol g−1 FW) occurring 4 h after daybreak. Interestingly, only negligible fluctuations of tZ and its riboside (tZR) were observed, with average concentrations of 2.2 pmol g−1 FW and 1.2 pmol g−1 FW, respectively (Jaworek et al. 2019). Annual and seasonal fluctuations were also observed in P. tremula (Edlund et al. 2017).

Three cytokinin moieties were identified in tRNA hydrolysates of P. × canadensis and P. × deltoides—N6-isopentenyladenosine (iPR) (50 pmol g−1 FW), cis-zeatin riboside (cZR) (19 pmol g−1 FW) and oTR (4 pmol g−1 FW) (Jaworek et al. 2019). While the dynamics of free oTR was observed in both P. × canadensis and P. × deltoides, there were no significant changes in levels of tRNA-bound oTR, either in a the short time (day) or throughout the season. In addition, tissues of mature P. × canadensis and P. × deltoides trees contained much higher free aromatic cytokinin levels than those detected in leaves from young suckers. It seems to be likely that hybrid P. × canadensis inherited the trait to synthesize aromatic cytokinins from P. × deltoides (Jaworek et al. 2019, 2020).

The presence of oTR and oT in various poplar cultivars raises the question on their mutual interconversion. Nucleosides can be synthesized or de novo or transported from the vacuole or apoplast into the cytosol after RNA degradation by members of the equilibrative nucleoside transporter (ENT) family exhibiting broad substrate specificity toward purine, pyrimidine, and cytokinin ribosides with affinities in the micromolar range (Wormit et al. 2004; Hirose et al. 2008; Girke et al. 2014). In the cytosol, purine ribosides can be hydrolyzed by calcium-dependent nucleoside N-ribohydrolases (NRHs, E.C. 3.2.2.-) to corresponding nitrogenous bases and ribose (Jung et al. 2009, 2011; Kopečná et al. 2013) or phosphorylated to monophosphates by adenosine kinases (ADK, E.C. 2.7.1.20, Moffatt et al. 2000; Schoor et al. 2011). Purine nucleoside phosphorylase in plants (PNP, E.C. 2.4.2.1, Chen and Petschow 1978; Bromley et al. 2014) preferentially catalyzes a ribosylation reaction of adenine/isoprenoid cytokinin with ribose-1-phosphate to release adenosine/cytokinin riboside and phosphate moiety. Although phosphorolytic reaction can also appear in the presence of phosphate, lower Km values for bases favor the ribosylation reaction as shown for potato enzyme (Bromley et al. 2014). ADKs found in Arabidopsis and tobacco are known to catalyze phosphorylation of adenosine and isoprenoid cytokinins with Km values in low micromolar range (Moffatt et al. 2000; Kwade et al. 2005). NRHs hydrolyze various ribosides including isoprenoid cytokinin ribosides. NRHs contain calcium ions in the active site which is coordinated by aspartate residues from the conserved N-terminal DXDXXXDD motif. Calcium ions are essential for the ribose moiety binding. Plant NRHs belong to nonspecific inosine-uridine containing NRHs as they are able to act on a wide range of ribosides. Km values of Arabidopsis and maize NRHs for iPR and tZR are in high micromolar range (Jung et al. 2009; Kopečná et al. 2013).

The genome of poplar (https://phytozome.jgi.doe.gov/pz/portal.html) contains at least two NRH genes, two ADK genes, and one PNP gene (Table 4.1). Two most active NRHs from maize, namely, ZmNRH2b and ZmNRH3, hydrolyze not only isoprenoid cytokinin ribosides (Kopečná et al. 2013) but also aromatic cytokinins including oTR to oT (Fig. 4.1a, b). Both isoforms display specific activities of 0.3 and 0.5 nkat mg−1 with iPR. Their activities with oTR are in similar range and attain values of 0.5 and 0.4 nkat mg−1, respectively. For this reason, it is very likely that ADK and PNP reactions will also catalyze the conversion of oT and oTR in addition to isoprenoid cytokinins.

Table 4.1 A list of genes coding for a nucleosidase (NRH), adenosine kinase (ADK), and purine nucleoside phosphorylase (PNP) in Populus trichocarpa
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Fig. 4.1
figure 1

(a) A scheme of the catalytic conversion of oTR to oT catalyzed by plant NRHs. (b) Confirmation of oTR conversion by ZmNRH2b using UHPLC-MS/MS with m/z transitions indicated for each peak. (c) Competitive binding curves for oTR, oT, and mT versus [3H]-tZ using five poplar cytokinin receptors in live cell assay. Binding was studied at pH 7.0 (adapted from Jaworek et al. 2020)

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The poplar genome contains five genes coding for CHASE-containing histidine kinases (HKs), which are known to function as cytokinin receptors. The abbreviation “CHASE” denotes a cyclase/histidine kinase-associated sensory extracellular domain, which comprises a region of ~270 amino acids and represents the cytokinin binding site located at the N-terminus of HK (Heyl et al. 2007). HKs from Populus × canadensis cv. Robusta, namely, PcHK2, PcHK3a, PcHK3b, PcHK4a and PcHK4b, display kinase activity and are able to bind both isoprenoid and aromatic cytokinins (Jaworek et al. 2020). All five PcHKs display strong tZ binding affinities ranging from 1.8 nM to 5.5 nM concentrations.

While both PcHK3a and PcHK3b display the strongest binding at pH 7.5, binding to both PcHK4 increases steadily toward pH 5.5 in line with their putative membrane localization in the endoplasmic reticulum and plasma membrane. Eventual intra- and intercellular transport of oT to the receptor can be mediated by several transporter families including purine permeases, ureide permeases, and nucleobase: cation symporter families 1 and 2 (reviewed in Girke et al. 2014). Of the tested aromatic cytokinins, mT binds more strongly than BAP. However, it is unlikely that these appear in vivo in poplar. Ki values for mT range between 23 nM and 300 nM (Jaworek et al. 2020). Conversely, oT, the metabolite found in poplar, is a much weaker ligand for PcHKs. The lowest Ki value of 1.1 μM and thus the highest sensitivity for oT are displayed by PcHK2 (Fig. 4.1c) (Jaworek et al. 2020). Its riboside is inactive and not bound below 10 μM concentrations. As it has been shown that leaves of mature P. × canadensis trees exhibit diurnally fluctuating levels of oT under physiological conditions and the concentration can peak at around 250 nM, it is possible that the local concentration may be even higher and trigger cytokinin signaling via PcHK2 (Jaworek et al. 2019).

A chlorophyll retention bioassay was used to assess the biological activity of oT and mT in poplar. The leaf discs floated on the micromolar cytokinin solution in the dark for 3 weeks at 25 °C. Data are presented in Fig. 4.2 as chlorophyll content relative to the initial value. Unlike the results outlined by Holub et al. (1998), the assay does not permit satisfactory determination of biological activity, mainly due to extremely high biological variability. While winter wheat plants were cultivated under controlled conditions in the original assay, poplar leaves were harvested from adult trees in an urban environment. The activity of mTR is higher and delays chlorophyll degradation. The effect of the oT is comparable with other three cytokinins mT, tZ, and BAP, and the values range between 60% and 80%. However, the differences are not significant enough from the control. Interestingly, chlorophyll degradation in poplar leaf discs is very slow compared with winter wheat. Bioassay with winter wheat requires 96 h, while bioassay with poplar needs 3 weeks or more.

Fig. 4.2
figure 2

A biological activity of topolins in poplar analyzed by a chlorophyll retention bioassay. The chlorophyll content was measured in leaf discs left in the dark for 3 weeks at 25 °C. It is shown relative to the initial value. The content in the absence of cytokinin is shown by a dashed line and corresponds to 51.5%

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To conclude, levels of aromatic cytokinins found in P. × canadensis and measured in leaves from the same tree are very variable between years and during the season (Jaworek et al. 2019, 2020). While oT derivatives are found, those of mT are not. Moreover, mT is more active in bioassays and activates cytokinin receptors at nanomolar concentrations. Currently, we cannot exclude the possibility that topolins are not products of poplar metabolism but indeed endophyte products (Wang et al. 2019). Observed fluctuations could account for the changes in the endophyte growth. The origin of topolins in poplar remains to be identified.