Topolins and Hydroxylated Thidiazuron Derivatives Are Substrates of Cytokinin O-Glucosyltransferase with Position Specificity Related to Receptor Recognition

Abstract

Glucosides of trans-zeatin occur widely in plant tissues, formed either by O-glucosylation of the hydroxylated side chain or N-glucosylation of the purine ring structure. O-Glucosylation is stereo-specific: the O-glucosyltransferase encoded by the Phaseolus lunatus ZOG1 gene has high affinity for trans-zeatin as the substrate, whereas the enzyme encoded by the maize (Zea mays) cisZOG1 gene prefers cis-zeatin. Here we show that hydroxylated derivatives of benzyladenine (topolins) are also substrates of ZOG1 and cisZOG1. The m-OH and o-OH derivatives are the preferred substrate of ZOG1 and cisZOG1, respectively. Among the hydroxylated derivatives of thidiazuron tested, the only enzyme/substrate combination resulting in conversion was cisZOG1/(o-OH) thidiazuron. The abilities of these cytokinins to serve as substrates to the glucosyltransferases were in a large part correlated with their biological activities in the P. lunatus callus bioassay, indicating that there may be similarities between cytokinin-binding sites on the enzymes and cytokinin receptors. Further support for this interpretation is provided by cytokinin recognition studies involving the Arabidopsis (Arabidopsis thaliana) CRE1/WOL/AHK4 and maize ZmHK1 receptors. The AHK4 receptor responded to trans-zeatin and m-topolin, while the ZmHK1 receptor responded also to cis-zeatin and o-topolin. Three-dimensional molecular models of the substrates were applied to explain the results.Cytokinins are a group of plant growth regulators with important functions at all phases of plant development, from seed germination to senescence. The natural cytokinins are adenine derivatives, of which trans-zeatin ( Fig. 1) is considered central due to its general occurrence and high activity in bioassays (Skoog and Armstrong, 1970). Also, cis-zeatin ( Fig. 1) and its derivatives have been found in many plant species (discussed in Mok and Mok, 2001), often at relatively high levels; however, cis-zeatin has much lower cytokinin activity than trans-zeatin (Schmitz et al., 1972). Dihydrozeatin, the saturated counterpart of zeatin, also occurs in plant tissues, has high cytokinin activity in bioassays, and is more stable than trans-zeatin due to its resistance to cytokinin oxidases/dehydrogenases (Skoog and Armstrong, 1970;Armstrong, 1994).Although the isoprenoid cytokinins are the major components of endogenous cytokinins, aromatic cytokinins have been known to occur since their isolation from poplar (Horgan et al., 1973). Benzyladenine (BAP; Fig. 1) and its derivatives have now been identified in a number of plant species as minor components of the total cytokinins (Strnad, 1997; Sáenz et al., 2003). The hydroxylated derivatives of BAP were named topolins (Strnad, 1997). Of the topolins, only the meta form exhibits high cytokinin activity (Strnad, 1997;Holub et al., 1998).Many cytokinin derivatives occur in plant tissues (for review, see Auer, 1997). The free bases may represent only a small portion of the total cytokinins, while the corresponding ribosides and especially nucle...

Cytokinin Activity of cis-Zeatin and Phenotypic Alterations Induced by Overexpression of Putative cis-Zeatin-O-glucosyltransferase in Rice

“…On the other hand, K m values in the micromolar range are not unusual for cytokinin metabolic enzymes, especially for glucosyltransferases. Examples are 50 mM in P. lunatus ZOG1 for tZ (Mok et al, 2005) and 70 to 240 mM in Arabidopsis N-glucosyltransferases UGT76C1 and UGT76C2 for iP and tZ (Hou et al, 2004). Moreover, substrate contents of cytokinin glucosyltransferases did not decrease in previously studied transgenic plants overexpressing P. lunatus ZOG1 (Pineda Rodó et al, 2008) and UGT76C2 (Wang et al, 2011), while glucosides increased.…”

“…The activities of rice cZOGT1 and cZOGT2 as well as maize cisZOG1 directed at the riboside form separate these enzymes from P. lunatus ZOG1, which does not accept tZR as a substrate (Dixon et al, 1989); the biological relevance of this difference, however, remains unclear. Maize cisZOG1 has been reported to catalyze oT-O-glucosylation (Mok et al, 2005); similarly, cZOGT1 and cZOGT2, but not cZOGT3, catalyzed oT-O-glucosylation in vitro (Fig. 4C).…”

Cytokinins - recent news and views of evolutionally old molecules

“…This is supported by identification of a maize gene encoding a cis-zeatin O-glucosyltransferase (cisZOG) that does not recognise either trans-zeatin or dihydrozeatin, and uses UPDG as the only sugar donor (Martin et al 2001). Besides trans-zeatin and cis-zeatin, ZOG and cisZOG also recognise topolins and hydroxylated phenylureas as substrates, and the substrate specificities correlate with CK recognition by the Arabidopsis thaliana and maize CK receptors (Mok et al 2005). O-glycosylation has been shown to preserve the CKs from N 6 -side chain cleavage by CK oxidase (Armstrong 1994).…”

“…1;Shantz and Steward 1955;Iwamura 1994). Despite their structural divergence, both the adenine-type and phenylurea-type CKs exhibit activity in different CK bioassays and are effectively recognised by CK receptors, as will be described later Spíchal et al 2004;Mok et al 2005).…”

Effect of Meta-topolin on in vitro propagation of Pelargonium × hortorum and Pelargonium × hederaefolium cultivars

“…Current knowledge suggests that responses to cytokinin are mediated by a family of receptors, which regulate a signal-transduction pathway (Heyl and Schmülling 2003). As reported by Mok et al (2005), BAP and mT have a different affinity in receptor recognition. The authors found that mT like trans-zeatin interacts with the Arabidopsis AHK4 receptor while BAP and TDZ with the maize ZmHK1 receptor.…”