Agrobacterium tumefaciensTumor Morphology Root Plastid Localization and Preferential Usage of Hydroxylated Prenyl Donor Is Important for Efficient Gall Formation      

Abstract

Upon Agrobacterium tumefaciens infection of a host plant, Tumor morphology root (Tmr) a bacterial adenosine phosphateisopentenyltransferase (IPT), creates a metabolic bypass in the plastid for direct synthesis of trans-zeatin (tZ) with 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate as the prenyl donor. To understand the biological importance of Tmr function for gall formation, we compared Tmr and Trans-zeatin secretion (Tzs) another agrobacterial IPT that functions within the bacterial cell. Although there is no significant difference in their substrate specificities in vitro, ectopic overexpression of Tzs in Arabidopsis (Arabidopsis thaliana) resulted in the accumulation of comparable amounts of tZ-and N 6 -(Δ 2 -isopentenyl)adenine (iP)-type cytokinins, whereas overexpression of Tmr resulted exclusively in the accumulation of tZ-type cytokinins. Ectopic expression of Tzs in plant cells yields only small amounts of the polypeptide in plastid-enriched fractions. Obligatory localization of Tzs into Arabidopsis plastid stroma by translational fusions with ferredoxin transit peptide (TP-Tzs) increased the accumulation of both tZ-and iP-type cytokinins. Replacement of tmr on the Ti plasmid with tzs, TP-tzs, or an Arabidopsis plastidic IPT induced the formation of smaller galls than wild-type A. tumefaciens, and they were accompanied by the accumulation of iP-type cytokinins. Tmr is thus specialized for plastid localization and preferential usage of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate in vivo and is important for efficient gall formation.

Proteome and metabolome profiling of cytokinin action in Arabidopsis identifying both distinct and similar responses to cytokinin down- and up-regulation

“…Accordingly, native plant IPTs produce iP-type CKs in vivo (e.g. Ueda et al , 2012), t Z is the preferred substrate for HvCKX2 (Galuzska et al ., 2004), and CK degradation is a reaction concurrent with N 7 -glucosylation in CK base inactivation (Brzobohatý et al , 1994; Frébort et al , 2011). …”

“…Demands on the sucrose pool clearly rose following activation of both ipt and HvCKX2 , probably at the expense of starch biosynthesis via a mechanism that maintains the supply of pyruvate. IPT is reportedly specialized for plastid localization and preferential use of 1-hydroxy-2-methyl-2-( E )-butenyl 4-diphosphate (HMBDP) as a prenyl donor in vivo (Ueda et al , 2012). In plastids, HMBDP is generated via the methylerythritol phosphate (MEP) pathway starting from pyruvate.…”

De novo biosynthesis of cytokinins in the biotrophic fungus Claviceps purpurea

“…Interestingly, both LOG domains present within C. purpurea use c ZRMP as a substrate in vitro. As demonstrated for the IPT proteins of A. tumefaciens , the spatial distribution of substrate and enzyme in vivo can massively influence product formation (Ueda et al ., ). It is therefore possible that the two different LOG‐containing proteins expressed by C. purpurea function in different pathways.…”

“…The high potency of the novel bifunctional enzyme is further demonstrated by the measured kinetic constants (K m and k cat ) of the IPT domain, which are much better than those estimated for plant enzymes (Kakimoto, 2001; Takei et al ., ). However, in contrast to the Agrobacterial IPT, which preferentially uses HMBDP to directly produce t Z‐type CKs in planta (Sakakibara et al ., ; Ueda et al ., ), the transferase domain of the Claviceps enzyme preferentially uses DMAPP. The subsequent hydroxylation of iP to t Z is mediated via a CK‐specific cytochrome P450 monooxygenase encoded by a gene adjacent to cpipt‐log within the Cp 20.1 genome.…”

‘Real time’ genetic manipulation: a new tool for ecological field studies

“…The system was tested for heterologous gene expression as well as for RNA interference (RNAi)-mediated gene silencing (Craft et al, 2005;Samalova et al, 2005;Wielopolska et al, 2005), for example to regulate the expression of the Agrobacterium tumefaciens isopentenyl transferases (ipt) coding gene Tumor morphology root (Tmr), which catalyzes the rate-limiting step in the biosynthesis of the cytokinin (CK) trans-zeatin (tZ; Heidekamp et al, 1983;Craft et al, 2005;Samalova et al, 2005;Ueda et al, 2012). Since minor CK changes already affect plant development (Medford et al, 1989;Bohner and Gatz, 2001), ipt represents a sensitive visual marker for analyzing dose-dependent induction.…”