Since their discovery as cell-division factors in plant tissue culture about five decades ago, cytokinins have been hypothesized to play a central role in the regulation of cell division and differentiation in plants. To test this hypothesis in planta, we isolated Arabidopsis plants lacking one, two, or three of the genes encoding a subfamily of histidine kinases (CRE1, AHK2, and AHK3) that function as cytokinin receptors. Seeds were obtained for homozygous plants containing mutations in all seven genotypes, namely single, double, and triple mutants, and the responses of germinated seedlings in various cytokinin assays were compared. Both redundant and specific functions for the three different cytokinin receptors were observed. Plants carrying mutations in all three genes did not show cytokinin responses, including inhibition of root elongation, inhibition of root formation, cell proliferation in and greening of calli, and induction of cytokinin primary-response genes. The triple mutants were small and infertile, with a reduction in meristem size and activity, yet they possessed basic organs: roots, stems, and leaves. These results confirm that cytokinins are a pivotal class of plant growth regulators but provide no evidence that cytokinins are required for the processes of gametogenesis and embryogenesis.S ince the discovery of kinetin in 1956 as a degradation product of DNA that promotes cell division in plants (1), a considerable amount of biochemical, physiological, and, most recently, genetic research has focused on elucidating the diverse roles that cytokinins play in plant growth and development. Perturbations of cytokinin levels in plants via over-expression of bacterial cytokinin synthesis genes (2-4), recovery of mutant plants with a higher-than-normal cytokinin content (5), and characterization of loss-of-function mutants of the cytokinin receptor CYTOKININ RESPONSE 1 (CRE1) (6-9) have implicated cytokinins in a wide variety of processes, including cell division, organ formation and regeneration, senescence, apical dominance, vascular development, response to pathogens, and nutrient mobility. These numerous roles for cytokinins, coupled with the failure of mutant screens to yield plants with nondetectable cytokinin levels, led to the longstanding belief that cytokinins are essential for plant growth and development.Plants respond to cytokinin through a multistep phosphorelay system, consisting of sensor histidine kinase (HK) proteins, histidine phosphotransfer (HPt) proteins, and effector response regulator (RR) proteins. Over-expression and loss-of-function analyses of particular HK, HPt, and RR proteins in Arabidopsis (8-13), combined with transient expression assays in protoplasts (14), have led to a model for cytokinin signaling (for a review, see refs. 15 and 16), beginning with perception of cytokinins by HK proteins.The Arabidopsis genome encodes six nonethylene receptor HKs: CRE1͞WOL͞AHK4, AHK2, AHK3, AtHK1, CKI1, and CKI2͞AHK5. Among them, CRE1, Arabidopsis HK2 (AHK2), and Arabidopsis HK3 (A...

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...

SummaryThe rate-limiting step of cytokinin biosynthesis in Arabidopsis thaliana Heynh. is catalyzed by ATP/ADP isopentenyltransferases, A. thaliana IsoPentenyl Transferase (AtIPT)1, and AtIPT4, and by their homologs AtIPT3, AtIPT5, AtIPT6, AtIPT7, and AtIPT8. To understand the dynamics of cytokinins in plant development, we comprehensively analyzed the expression of isopentenyltransferase genes of Arabidopsis. Examination of their mRNA levels and the expression patterns of the beta-glucuronidase (GUS) gene fused to the regulatory sequence of each AtIPT gene revealed a speci®c expression pattern of each gene. The predominant expression patterns were as follows: AtIPT1::GUS, xylem precursor cell ®les in the root tip, leaf axils, ovules, and immature seeds; AtIPT3::GUS, phloem tissues; AtIPT4::GUS and AtIPT8::GUS, immature seeds with highest expression in the chalazal endosperm (CZE); AtIPT5::GUS, root primordia, columella root caps, upper part of young in¯orescences, and fruit abscission zones; AtIPT7::GUS, endodermis of the root elongation zone, trichomes on young leaves, and some pollen tubes. AtIPT1, AtIPT3, AtIPT5, and AtIPT7 were downregulated by cytokinins within 4 h. AtIPT5 and AtIPT7 was upregulated by auxin within 4 h in roots. AtIPT3 was upregulated within 1 h after an application of nitrate to mineral-starved Arabidopsis plants. The upregulation by nitrate did not require de novo protein synthesis. We also examined the expression of two genes for tRNA isopentenyltransferases, AtIPT2 and AtIPT9, which can also be involved in cytokinin biosynthesis. They were expressed ubiquitously, with highest expression in proliferating tissues. These ®nd-ings are discussed in relation to the role of cytokinins in plant development.