Roles of Arabidopsis ATP/ADP isopentenyltransferases and tRNA isopentenyltransferases in cytokinin biosynthesis

Abstract

Cytokinins, which are central regulators of cell division and differentiation in plants, are adenine derivatives carrying an isopentenyl side chain that may be hydroxylated. Plants have two classes of isopentenyltransferases (IPTs) acting on the adenine moiety: ATP͞ ADP isopentenyltransferases (in Arabidopsis thaliana, AtIPT1, 3, 4 -8) and tRNA IPTs (in Arabidopsis, AtIPT2 and 9). ATP͞ADP IPTs are likely to be responsible for the bulk of cytokinin synthesis, whereas it is thought that cis-zeatin (cZ)-type cytokinins are produced possibly by degradation of cis-hydroxy isopentenyl tRNAs, which are formed by tRNA IPTs. However, these routes are largely hypothetical because of lack of in vivo evidence, because the critical experiment necessary to verify these routes, namely the production and analysis of mutants lacking AtIPTs, has not yet been described. We isolated null mutants for all members of the ATP͞ADP IPT and tRNA IPT gene families in Arabidopsis. Notably, our work demonstrates that the atipt1 3 5 7 quadruple mutant possesses severely decreased levels of isopentenyladenine and trans-zeatin (tZ), and their corresponding ribosides, ribotides, and glucosides, and is retarded in its growth. In contrast, these mutants possessed increased levels of cZ-type cytokinins. The atipt2 9 double mutant, on the other hand, lacked isopentenyl-and cishydroxy isopentenyl-tRNA, and cZ-type cytokinins. These results indicate that whereas ATP͞ADP IPTs are responsible for the bulk of isopentenyladenine-and tZ-type cytokinin synthesis, tRNA IPTs are required for cZ-type cytokinin production. This work clarifies the long-standing questions of the biosynthetic routes for isopentenyladenine-, tZ-, and cZ-type cytokinin production.S ince the discovery of cytokinins as inducers of plant cell division (1) and differentiation (2), they have been recognized as central regulators of plant development (3). Cytokinins also increase nutrient sink strength, delay senescence, stimulate outgrowth from lateral buds, and inhibit cell elongation (4). The important roles for cytokinins in cell division were verified by overexpression of genes for cytokinin-degrading enzymes (cytokinin oxidases, CKXs) (5-7) and by examination of single and higher-order cytokinin-receptor null mutants (8-10).Most naturally occurring cytokinins are N 6 -isopentenyladenine (iP) derivatives. iP carries an unmodified isopentenyl side chain, whereas trans-zeatin (tZ) and cis-zeatin (cZ) carry hydroxylated side chains. Cytokinins exist in free-base, riboside, and ribotide forms, with varying degrees of biological activity. Cytokinins also may be modified in several ways. For example, the N7 and N9 positions of the adenine moiety of cytokinins may be glucosylated to form N-glucosides. Alternatively, the hydroxyl group of tZ and cZ may be glucosylated or xylosylated to form zeatin-O-glucosides or zeatin-O-xylosides. N-and O-glycosides are biologically inactive (3).Experimental evidence demonstrates that free-base cytokinins are biologically active. For example, iP (11) and t...

The ArabidopsisGRF-INTERACTING FACTORGene Family Performs an Overlapping Function in Determining Organ Size as Well as Multiple Developmental Properties

“…Therefore, it is conceivable that the small size of leaf primordia in the gif mutants may be, in part, attributable to the small size of the SAM cell pool, probably because the SAM tissues do not have sufficient numbers of founder cells. This notion is in line with the fact that all of the cytokinin-signaling mutants and transgenic plants with reduced cytokinin activities have both the smaller SAM cell pool and leaf organs with fewer cells compared with the wild type (Werner et al, 2001(Werner et al, , 2003Higuchi et al, 2004;Nishimura et al, 2004;Miyawaki et al, 2006). However, precise data on the causal relationship between altered numbers of the SAM cells and leaf cells are scarce in Arabidopsis.…”

“…The cytokinin mutants and transgenic plants mentioned above have much smaller SAM and fewer leaves compared with the wild type, which is indicative of a longer plastochron (Werner et al, 2001(Werner et al, , 2003Higuchi et al, 2004;Nishimura et al, 2004;Miyawaki et al, 2006). Upregulation of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE9 expression results in both a smaller size of the meristem and a longer plastochron (Wang et al, 2008).…”

Cytokinins - recent news and views of evolutionally old molecules

“…Transgenic tobacco and Arabidopsis thaliana plants constitutively expressing the CKX genes from the 35S promoter exhibited reduced CK contents, leading to the CK deficiency phenotype, which is characterised by formation of slow-growing, stunted shoots with small leaves; an enlarged root system; and distinct changes in plant sink and source parameters (Werner et al 2001Yang et al 2003). The developmental consequences of CK deficiency were later confirmed by studies of Arabidopsis thaliana ipt (CK biosynthetic) loss-of-function mutants (Miyawaki et al 2006). Construction of Arabidopsis thaliana CK receptor mutants revealed a high level of redundancy of CRE1/AHK4, AHK3 and AHK2; however, analyses of the multiple mutants has assigned functions to CKs in the control of shoot growth, leaf senescence, seed size and germination, root elongation and branching, and CK metabolism (Higuchi et al 2004;Nishimura et al 2004;Riefler et al 2006).…”

“…Plant tRNAs have typically been reported to contain cis-zeatin ribosides (cZR) as the most abundant cytokinin (Taller 1994) and indeed cis-zeatin CKs have been found to be dominant in representatives of liverworts, mosses and ferns (Gajdošová et al 2011). Although the tRNA pathway is generally considered to be insufficient to establish the significant amount of CKs required by seed plants (Barnes et al 1980), analysis of atipt2 and atipt9 (both encoding tRNA-IPTs) mutants of Arabidopsis thaliana suggested that tRNA may still represent a significant source of cis-zeatin in higher plants (Miyawaki et al 2006). A similar situation can be described for CKX genes.…”

Root system architecture: insights fromArabidopsisand cereal crops

“…Cytokinin is primarily synthesized in the root cap [69] and has a largely antagonistic effect to auxin in terms of root architecture: cytokinin has an inhibitory effect on lateral root branching, with mutants for cytokinin biosynthesis and sensitivity demonstrating increased numbers of lateral roots [70][71][72][73]. Auxin is synthesized primarily in young aerial tissues and undergoes polar transport towards the root tissues [74].…”