Metabolism of Benzyladenine is Impaired in a Mutant of Arabidopsis thaliana Lacking Adenine Phosphoribosyltransferase Activity

Abstract

Formation of the riboside-5'-monophosphate is a general feature of the metabolism of cytokinins in plants. As part of a study of the biological significance of the nucleotide form of cytokinins, we analyzed a mutant of Arabidopsis thaliana deficient in adenine phosphoribosyltransferase (APRT) activity for its ability to metabolize N6-benzyladenine (BA). Formation of Nl-benzyladenosine-5'-monophosphate (BAMP) was assayed in vivo, by feeding tritiated BA to wild-type and mutant plantlets, and in crude plantlet extracts. Metabolites were separated by high performance liquid chromatography and quantitated by on-line liquid scintillation spectrometry. BA was rapidly absorbed by A. thaliana plantlets and pnmanly converted to BAMP and to BA 7-and 9-glucosides. BA was also rapidly absorbed by APRT-deficient plantlets, but its conversion to BAMP was strongly reduced. Formation of BAMP from N6-benzyladenosine was not affected in the mutant plantlets.In vitro conversion of BA to its nucleoside-5'-monophosphate was detected in crude extracts of wild-type plantlets, but not in extracts of APRT-deficient plantlets. Therefore, results of both assays indicate that APRT-deficient tissue does not convert BA to BAMP to a significant extent. Further, nondenaturing isoelectric focusing analysis of APRT activity in leaf extracts indicated that the enzyme activities which metabolize adenine and BA into their corresponding riboside-5'-monophosphate in extracts of wildtype plantlets have the same apparent isoelectric point. These activities were not detected in extracts prepared from APRTdeficient plantlets. Thus, these results demonstrate that APRT is the main enzyme which converts BA to its nucleotide form in young A. thaliana plants and that the ribophosphorylation of BA is not a prerequisite of its absorption by the plantlets. lism, but few of the enzyme activities effecting these steps in vivo have been identified. Furthermore, which modified form(s) constitutes the "active" cytokinin remains an open question. Elucidation of the enzymes involved in the synthesis and metabolism of cytokinins may provide valuable insight into the function of the various cytokinin metabolites, how their level in particular tissues is regulated and how these levels relate to physiology of plants.The interconversion of cytokinin bases, ribosides and nucleotides is a major feature of the metabolism of cytokinins (5,17,20,26), and it may be catalyzed by the enzyme system that metabolizes adenine. Several of these enzymes have been purified and shown to act on N-substituted substrates in vitro, albeit at significantly lower rates than those measured on the corresponding adenine substrates (3-6). Results of in vivo feeding experiments also support the notion that the purine metabolizing enzymes may be the main route by which cytokinins are interconverted (7).

SPINDLY inhibits class I TCP proteolysis to promote sensitivity to cytokinin

“…The reduced levels of active CKs might be the result of a negative feedback regulation, initiated by increased CK signaling (Kieber and Schaller, 2014). High CK activity induces the expression of CYTOKININ OXIDASE/DEHYDROGENASE genes for CK degradation (Brugière et al, 2003;Werner et al, 2003Werner et al, , 2006 and rapidly converts free active CKs to inactive derivatives (Singh et al, 1988;Moffatt et al, 1991;Yonekura-Sakakibara et al, 2004). Indeed, we found higher levels of inactive CKs in TCP14-overexpressing flowers.…”

Uridine-Ribohydrolase Is a Key Regulator in the Uridine Degradation Pathway of Arabidopsis

“…However, such a dual function has also been reported for other proteins of the salvage pathway, namely, adenosine kinase, adenine phosphoribosyl-transferase, and rice Equilibrative Nucleoside Transporter2 (ENT2). The latter protein transports cytokinin-ribosides in addition to nucleosides (von Schwartzenberg et al, 1998;Moffatt et al, 1991Moffatt et al, , 2000Hirose et al, 2005).…”

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

“…The cZ biosynthesis pathway has not been fully elucidated, but a major step in Arabidopsis (Arabidopsis thaliana) is the release of precursors in the course of tRNA degradation (Miyawaki et al, 2006). For inactivation, the free-base cytokinins may be degraded by cytokinin oxidase (Houba-Hérin et al, 1999;Werner et al, 2001;Bartrina et al, 2011) or conjugated by glucosyltransferases (Dixon et al, 1989;Martin et al, 1999Martin et al, , 2001Mok et al, 2000;Mok and Mok, 2001;Haberer and Kieber, 2002;Veach et al, 2003;Hou et al, 2004;Wang et al, 2011) and converted to cytokinin-ribotides in the purine salvage pathway (Moffatt et al, 1991;Allen et al, 2002). A pathway to convert cZ or cZ-riboside (cZR) into tZ or tZ-riboside (tZR) was also proposed following partial purification of a putative cis-trans-isomerase from endosperm of Phaseolus vulgaris (Bassil et al, 1993).…”