An alternative cytokinin biosynthesis pathway

Abstract

Studies of de novo cytokinin biosynthesis in isopentenyltransferase (ipt)-transformed Arabidopsis thaliana, involving in vivo deuterium labeling and mass spectrometry, showed that the biosynthetic rate of zeatinriboside-5 -monophosphate was around 66-fold higher than that of isopentenyladenosine-5 -monophosphate (iPMP), the proposed primary product of the Agrobacterium ipt. Double tracer analysis, using [ 2 H6] isopentenyladenosine and deuterium oxide, provided evidence for an alternative, iPMPindependent, biosynthetic pathway for zeatin-type cytokinins, present in both ipt-expressing and wild-type Arabidopsis thaliana. Reduction of the biosynthetic flux in the alternative pathway by use of mevastatin, an inhibitor for 3-hydroxy-3-methylglutaryl CoA reductase, indicated a terpenoid origin for the side-chain precursor of the iPMP independent pathway. C ytokinins are an important class of plant growth regulators, defined by their ability to promote cell division in tissue culture in the presence of auxins (1, 2). Virtually all naturally occurring cytokinins identified to date are adenine species substituted at N 6 with an isoprenoid or aromatic side chain. In this text, cytokinins will refer solely to the isoprenoid cytokinin bases and their sugar conjugates. Cytokinins affect many plant developmental processes including cell division, cell differentiation, chlorophyll senescence, and apical dominance (3).Efforts to elucidate the biosynthetic origin of cytokinins in plants have been inconclusive. Early suggestions that tRNA degradation could be the major source of free, active cytokinins (4) were disproved when calculations of tRNA turnover rates showed that a tRNA-independent de novo biosynthetic pathway also must be present in plants (5). A major breakthrough was the discovery of a cytokinin biosynthetic enzyme in the slime mold, Dictyostelium discoideum (6). Cell-free extracts from this organism can convert AMP and dimethylallyl-pyrophosphate (DMAPP) to the free cytokinins isopentenyladenosine-5Ј-monophosphate (iPMP) and the corresponding nucleoside (isopentenyladenosine, iPA). This finding, and studies on the metabolism of isopentenyl-type cytokinins (7,8), led to the proposal that iPMP is also the primary cytokinin intermediate in plants, and zeatin cytokinins are formed by hydroxylation of iPMP and its derivatives (9, 10) ( Fig. 1).Later, the product of the T-DNA gene 4 (ipt) of the crown gall-forming bacterium, Agrobacterium tumefaciens, also was described as a DMAPP:AMP isopentenyltransferase (ipt) (11,12). AMP was found to be the preferred adenylic substrate for the Agrobacterium ipt enzyme but searches for alternative side-chain donors have been limited. However, when de novo biosynthesis in crown gall tissue of Vinca rosea was traced with 14 C-adenine, in vivo production of iPMP was undetectable, whereas zeatinriboside-5Ј-monophospate (ZMP) production was strong (13). After studies of 14 C-isopententyladenine metabolism in this system, it was proposed that the very low levels of iPMP were caused by rapi...

Agrobacterium tumefaciens increases cytokinin production in plastids by modifying the biosynthetic pathway in the host plant

“…As reported, overexpression of Tmr in Arabidopsis resulted in accumulation of tZ-type CKs at extremely high levels without increasing the level of iP-type CKs substantially ( Fig. 2; see Tables 2 and 3, which are published as supporting information on the PNAS web site) (9,14). In contrast to the effect of Tmr overexpression, a large number of iP-type CKs accumulated in Arabidopsis seedlings when one of the native IPT genes (AtIPT1, -3, -4, -5, or -7) was overexpressed (Fig.…”

“…Therefore, this bacterial enzyme functions in plant cells upon infection. Previous reports indicated that engineered expression of Tmr in plants elevated the synthesis of tZ-type CKs without a substantial increase in the production of iP derivatives (9,13,14). These observations imply that Tmr may be involved in the iPRMP-independent synthesis of tZ by using HMBDP in infected plant cells.…”

“…1). The other proposed pathway is an iPRMPindependent bypass, in which an unidentified hydroxylated derivative of DMAPP is directly transferred to the adenine moiety (9). A candidate for this putative substrate is 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBDP) (10), which has recently been shown to occur as an intermediate in the MEP pathway (11) (Fig.…”

Adaptional evolution of trichome in Caragana korshinskii to natural drought stress on the Loess Plateau, China

“…GA is known to accelerate cell division and longitudinal elongation, while CTK can accelerate cell division and transverse elongation – increasing content of either accelerates the differentiation of epidermal cells, so they can be attributed to trichome formation (Blackwell and Horgan 1994; Åstot et al. 2000; Golovko et al. 2002).…”

Arabidopsis CYP735A1 and CYP735A2 Encode Cytokinin Hydroxylases That Catalyze the Biosynthesis of trans-Zeatin

“…In the iPRMP-independent pathway, tZR 5Ј-phosphates are proposed to be produced directly by IPT using an unknown hydroxylated side chain precursor that may be derived from the cytosolic mevalonate (MVA) pathway (12) (Fig. 1).…”