Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate:ATP/ADP isopentenyltransferases

It has been believed that the key step in cytokinin biosynthesis is the addition of a 5-carbon chain to the N(6) of AMP. To identify cytokinin biosynthesis enzymes that catalyze the formation of the isopentenyl side chain of cytokinins, the Arabidopsis genomic sequence was searched for genes that could code for isopentenyltransferases. This resulted in the identification of nine putative genes for isopentenyltransferases. One of these, AtIPT4, was subjected to detailed analysis. Overexpression of AtIPT4 caused cytokinin-independent shoot formation on calli. As shoot formation on calli normally occurs only when cytokinins are applied, it suggested that this gene product catalyzed cytokinin biosynthesis in plants. Recombinant AtIPT4 catalyzed the transfer of an isopentenyl group from dimethylallyl diphosphate to the N(6) of ATP and ADP, but not to that of AMP. AtIPT4 did not exhibit the DMAPP:tRNA isopentenyltransferase activity. These results indicate that cytokinins are, at least in part, synthesized from ATP and ADP in plants.     

Farnesylation directs AtIPT3 subcellular localization and modulates cytokinin biosynthesis in Arabidopsis

Cytokinins regulate cell division and differentiation as well as a number of other processes implicated in plant development. The first step of cytokinin biosynthesis in Arabidopsis (Arabidopsis thaliana) is catalyzed by adenosine phosphate-isopentenyltransferases (AtIPT). The enzymes are localized in plastids or the cytoplasm where they utilize the intermediate dimethylallyl-diphosphate from the methylerythritolphosphate or mevalonic acid pathways. However, the regulatory mechanisms linking AtIPT activity and cytokinin biosynthesis with cytokinin homeostasis and isoprenoid synthesis are not well understood. Here, we demonstrate that expression of AtIPT3, one member of the adenosine AtIPT protein family in Arabidopsis, increased the production of specific isopentenyl-type cytokinins. Moreover, AtIPT3 is a substrate of the protein farnesyl transferase, and AtIPT3 farnesylation directed the localization of the protein in the nucleus/cytoplasm, whereas the nonfarnesylated protein was located in the plastids. AtIPT3 gain-of-function mutant analysis indicated that the different subcellular localization of the farnesylated protein and the nonfarnesylated protein was closely correlated with either isopentenyl-type or zeatin-type cytokinin biosynthesis. In addition, mutation of the farnesyl acceptor cysteine-333 of AtIPT3 abolishes cytokinin production, suggesting that cysteine-333 has a dual and essential role for AtIPT3 farnesylation and catalytic activity.     

Identification of genes encoding adenylate isopentenyltransferase, a cytokinin biosynthesis enzyme, in Arabidopsis thaliana

The initial step in the de novo biosynthesis of cytokinin in higher plants is the formation of isopentenyladenosine 5'-monophosphate (iPMP) from AMP and dimethylallylpyrophosphate (DMAPP), which is catalyzed by adenylate isopentenyltransferase (IPT). Although cytokinin is an essential hormone for growth and development, the nature of the enzyme for its biosynthesis in higher plants has not been identified. Herein, we describe the molecular cloning and biochemical identification of IPTs from Arabidopsis thaliana. Eight cDNAs encoding putative IPT, designated as AtIPT1 to AtIPT8, were picked up from A. thaliana. The Escherichia coli transformants expressing the recombinant proteins excreted cytokinin species into the culture medium except for that expressing AtIPT2 that is a putative tRNA IPT. A purified recombinant AtIPT1 catalyzed the formation of iPMP from DMAPP and AMP. These results indicate that the small multigene family contains both types of isopentenyltransferase, which could synthesize cytokinin and mature tRNA.     

Two cytokinin receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, differ in their ligand specificity in a bacterial assay

Strains of Escherichia coli that express two different cytokinin receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, were used to study the relative sensitivity of these receptors to various cytokinins. Both receptors were most sensitive to the bases of the isoprenoid-type cytokinins trans-zeatin and isopentenyladenine but differed significantly in the recognition of other cytokinin compounds. In particular, CRE1/AHK4 recognized at 1 microm concentration only trans-zeatin while AHK3 recognized cis-zeatin and dihydrozeatin as well, although with a lower sensitivity. Similarly, CRE1/AHK4 was not activated by cytokinin ribosides and ribotides, but AHK3 was. Comparisons using the ARR5::GUS fusion gene as a cytokinin reporter in Arabidopsis showed similar relative degrees of responses in planta, except that cytokinins with aromatic side chains showed much higher activities than in the bacterial assay. These results indicate that the diverse cytokinin compounds might have specific functions in the numerous cytokinin-regulated processes, which may depend in turn on different receptors and their associated signalling pathways. The importance of precise control of local concentrations of defined cytokinin metabolites to regulate the respective downstream event is corroborated.     

Regulation of cytokinin biosynthesis, compartmentalization and translocation

Cytokinins, a group of mobile phytohormones, play an important role in plant growth and development, and their activity is finely controlled by environmental factors in the control of morphogenic and metabolic adaptations. Inorganic nitrogen sources, such as nitrate, are a major factor regulating gene expression of adenosine phosphate-isopentenyltransferase (IPT), a key enzyme of cytokinin biosynthesis. Modulation of IPT and macronutrient transporter gene expression in response to nitrate, sulphate and phosphate, and cytokinin-dependent repression of the transporter genes suggest that cytokinins play a critical role in balancing acquisition and distribution of macronutrients. Biased distribution of trans-zeatin (tZ)-type cytokinins in xylem and N(6)-(Delta(2)-isopentenyl)adenine (iP)-type cytokinins in phloem saps suggest that, in addition to acting as local signals, cytokinins communicate acropetal and systemic long-distance signals, and that structural side chain variations mediate different biological messages. The compartmentalization of tZ- and iP-type cytokinins implies the involvement of a selective transport system. Recent studies have raised the possibility of subsets of the purine permease family as a transporter of cytokinin nucleobases and equilibrative nucleoside transporters (ENT) for cytokinin nucleosides. These biochemical and transgenic data suggest that AtENT6, an Arabidopsis ENT, could also participate in cytokinin nucleoside transport with a preference for iP riboside in vascular tissue.     

The Arabidopsis AtIPT8/PGA22 gene encodes an isopentenyl transferase that is involved in de novo cytokinin biosynthesis

Cytokinin plays a critical role in plant growth and development by stimulating cell division and cell differentiation. Despite many years' research efforts, our current understanding of this hormone is still limited regarding both its biosynthesis and signaling. To genetically dissect the cytokinin pathway, we have used a functional screen to identify Arabidopsis gain-of-function mutations that enable shoot formation in the absence of exogenous cytokinins. By using a chemical-inducible activation tagging system, we have identified over 40 putative mutants, designated as pga (plant growth activators), which presumably were affected in key components of cytokinin biosynthesis and signaling pathway. Here, we report a detailed characterization of pga22, a representative mutant from this collection. A gain-of-function mutation in the PGA22 locus resulted in typical cytokinin responses. Molecular and genetic analyses indicated that PGA22 encodes an isopentenyl transferase (IPT) previously identified as AtIPT8. Plants of the pga22 mutant accumulated at remarkably higher levels of isopentenyladenosine-5'-monophosphate and isopentenyladenosine when analyzed by mass spectrometry, suggesting that AtIPT8/PGA22 is a functional IPT that may direct the biosynthesis of cytokinins in planta via an isopentenyladenosine-5'-monophosphate-dependent pathway.     

Cytokinins act directly on lateral root founder cells to inhibit root initiation

In Arabidopsis thaliana, lateral roots are formed from root pericycle cells adjacent to the xylem poles. Lateral root development is regulated antagonistically by the plant hormones auxin and cytokinin. While a great deal is known about how auxin promotes lateral root development, the mechanism of cytokinin repression is still unclear. Elevating cytokinin levels was observed to disrupt lateral root initiation and the regular pattern of divisions that characterizes lateral root development in Arabidopsis. To identify the stage of lateral root development that is sensitive to cytokinins, we targeted the expression of the Agrobacterium tumefaciens cytokinin biosynthesis enzyme isopentenyltransferase to either xylem-pole pericycle cells or young lateral root primordia using GAL4-GFP enhancer trap lines. Transactivation experiments revealed that xylem-pole pericycle cells are sensitive to cytokinins, whereas young lateral root primordia are not. This effect is physiologically significant because transactivation of the Arabidopsis cytokinin degrading enzyme cytokinin oxidase 1 in lateral root founder cells results in increased lateral root formation. We observed that cytokinins perturb the expression of PIN genes in lateral root founder cells and prevent the formation of an auxin gradient that is required to pattern lateral root primordia.     

Reproductive organs regulate leaf nitrogen metabolism mediated by cytokinin signal

The metabolism of vegetative organs in plants changes during the development of the reproductive organs. The regulation of this metabolism is important in the control of crop productivity. However, the complexity of the regulatory systems makes it difficult to elucidate their mechanisms. To examine these mechanisms, we constructed model experiments using Arabidopsis to analyze metabolic and gene expression changes during leaf-stage progression and after removal of the reproductive organs. Leaf gene expression levels and content of major amino acids, both of which decreased during leaf-stage progression, increased after removal of the reproductive organs. In particular, the levels of expression of cytokinin biosynthesis genes and cytokinin-responsive genes and the cytokinin content increased after removal of the reproductive organs. Analysis of plants with knockout of a cytokinin-biosynthetic gene (AtIPT3) and a cytokinin receptor gene (AHK3) indicated that glutamate dehydrogenase genes (GDH3) were regulated by cytokinin signaling. These data suggest that cytokinins regulate communication between reproductive and vegetative organs, and that GDH3 is one target of the cytokinin-mediated regulation of nitrogen metabolism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cytokinins play opposite roles in lateral root formation, and nematode and Rhizobial symbioses

We used the cytokinin-responsive Arabidopsis response regulator (ARR)5 gene promoter fused to a beta-glucuronidase (GUS) reporter gene, and cytokinin oxidase (CKX) genes from Arabidopsis thaliana (AtCKX3) and maize (ZmCKX1) to investigate the roles of cytokinins in lateral root formation and symbiosis in Lotus japonicus. ARR5 expression was undetectable in the dividing initial cells at early stages of lateral root formation, but later we observed high expression in the base of the lateral root primordium. The root tip continues to express ARR5 during subsequent development of the lateral root. These results suggest a dynamic role for cytokinin in lateral root development. We observed ARR5 expression in curled/deformed root hairs, and also in nodule primordia in response to Rhizobial inoculation. This expression declined once the nodule emerged from the parent root. Root penetration and migration of root-knot nematode (RKN) second-stage larvae (L2) did not elevate ARR5 expression, but a high level of expression was induced when L2 reached the differentiating vascular bundle and during early stages of the nematode-plant interaction. ARR5 expression was specifically absent in mature giant cells (GCs), although dividing cells around the GCs continued to express this reporter. The same pattern was observed using a green fluorescent protein (GFP) reporter driven by the ARR5 promoter in tomato. Overexpression of CKX genes rendered the transgenic hairy roots resistant to exogenous application of the cytokinin [N6-(Delta2 isopentenyl) adenine riboside] (iPR). CKX roots have significantly more lateral roots, but fewer nodules and nematode-induced root galls per plant, than control hairy roots.     

Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity

Cytokinins are hormones that regulate cell division and development. As a result of a lack of specific mutants and biochemical tools, it has not been possible to study the consequences of cytokinin deficiency. Cytokinin-deficient plants are expected to yield information about processes in which cytokinins are limiting and that, therefore, they might regulate. We have engineered transgenic Arabidopsis plants that overexpress individually six different members of the cytokinin oxidase/dehydrogenase (AtCKX) gene family and have undertaken a detailed phenotypic analysis. Transgenic plants had increased cytokinin breakdown (30 to 45% of wild-type cytokinin content) and reduced expression of the cytokinin reporter gene ARR5:GUS (beta-glucuronidase). Cytokinin deficiency resulted in diminished activity of the vegetative and floral shoot apical meristems and leaf primordia, indicating an absolute requirement for the hormone. By contrast, cytokinins are negative regulators of root growth and lateral root formation. We show that the increased growth of the primary root is linked to an enhanced meristematic cell number, suggesting that cytokinins control the exit of cells from the root meristem. Different AtCKX-green fluorescent protein fusion proteins were localized to the vacuoles or the endoplasmic reticulum and possibly to the extracellular space, indicating that subcellular compartmentation plays an important role in cytokinin biology. Analyses of promoter:GUS fusion genes showed differential expression of AtCKX genes during plant development, the activity being confined predominantly to zones of active growth. Our results are consistent with the hypothesis that cytokinins have central, but opposite, regulatory functions in root and shoot meristems and indicate that a fine-tuned control of catabolism plays an important role in ensuring the proper regulation of cytokinin functions.     

Molecular cloning, expression, and characterization of adenylate isopentenyltransferase from hop (Humulus lupulus L.)

A cDNA encoding adenylate isopentenyltransferase (AIPT) was cloned and sequenced from cones of hop (Humulus lupulus L.) by RT-PCR using oligonucleotide primers based on the conserved sequences of Arabidopsis thaliana AIPT isozymes (AtIPT1, AtIPT3, AtIPT4, AtIPT5, AtIPT6, AtIPT7 and AtIPT8). A full-length cDNA contained a 990-bp open reading frame encoding a molecular mass of 36,603 Da protein with 329 amino acids. Further, DNA sequencing of genomic DNA revealed absence of introns in the frame. On Southern blot analysis, a single AIPT gene was detected in H. lupulus, while RT-PCR analyses demonstrated that the gene was equally expressed in almost all tissues in the plant including roots, stems, leaves and cones. The deduced amino acid sequence shares 38-51% identity to those of A. thaliana AtIPTs. A recombinant enzyme expressed in Escherichia coli catalyzed isopentenyl transfer reaction from dimethylallyldiphosphate (DMAPP) to the N6 amino group of adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP), respectively. In contrast, other nucleotides; guanosine monophosphate (GMP), inosine monophosphate (IMP), cytosine monophosphate (CMP), uridine monophosphate (UMP), were not accepted as a substrate. Interestingly, steady-state kinetic analyses revealed that the isopentenylation of ADP and ATP were more efficient than that of AMP as previously reported for A. thaliana AtIPT4. Finally, H. lupulus AIPT contains the putative ATP/GTP binding motif at the N-terminal as in the case of other known isopentenyltransferases. Site-directed mutagenesis of a conserved Asp62, located right after the ATP/GTP binding motif, with Ala resulted in complete loss of enzyme activity.     

Arabidopsis KNOXI proteins activate cytokinin biosynthesis

Plant architecture is shaped through the continuous formation of organs by meristems. Class I KNOTTED1-like homeobox (KNOXI) genes are expressed in the shoot apical meristem (SAM) and are required for SAM maintenance. KNOXI proteins and cytokinin, a plant hormone intimately associated with the regulation of cell division, share overlapping roles, such as meristem maintenance and repression of senescence, but their mechanistic and hierarchical relationship have yet to be defined. Here, we show that activation of three different KNOXI proteins using an inducible system resulted in a rapid increase in mRNA levels of the cytokinin biosynthesis gene isopentenyl transferase 7 (AtIPT7) and in the activation of ARR5, a cytokinin response factor. We further demonstrate a rapid and dramatic increase in cytokinin levels following activation of the KNOXI protein SHOOT MERISTEMLESS (STM). Application of exogenous cytokinin or expression of a cytokinin biosynthesis gene through the STM promoter partially rescued the stm mutant. We conclude that activation of cytokinin biosynthesis mediates KNOXI function in meristem maintenance. KNOXI proteins emerge as central regulators of hormone levels in plant meristems.     

Expression of three <Emphasis Type="Italic">Arabidopsis</Emphasis> cytokinin oxidase/dehydrogenase promoter::GUS chimeric constructs in tobacco: response to developmental and biotic factors

Expression patterns of three Arabidopsis thaliana cytokinin oxidase/dehydrogenase promoter::GUS reporter fusions were investigated in tobacco plants. While cytokinin oxidase/dehydrogenase promoter 2 showed no expression in tobacco, the cytokinin oxidase/dehydrogenase promoters 3 and 4 were active in various tissues throughout development of the tobacco. Recently, the 1452 bp promoter region of AtCKX3 was reported as almost inactive in Arabidopsis. In contrast, the 1627 bp DNA fragment preceding the AtCKX3 coding region drove expression of the reporter GUS gene in various tobacco tissues. The promoter was mainly expressed in tobacco leaves and roots during early stages of development but also later in young flower buds as well as in pollen grains. The construct was particularly active before (hypocotyl region) and during (vascular system) lateral root initiation, supporting the idea of an inhibitory role of active cytokinins in the process of root initiation. The cytokinin oxidase/dehydrogenase promoter 4::GUS fusion in tobacco was shown to share some common (but weaker) expression patterns with promoter 3, namely in the leaves and pollen, but also conferred specific expression in tobacco root cap cells and trichomes. In addition, the response of cytokinin oxidase/dehydrogenase promoter::GUS reporter fusions to infection with the leafy gall-forming bacteria Rhodococcus fascians was examined. While an avirulent strain of R. fascians did not induce expression of any of the cytokinin oxidase/dehydrogenase promoters, the cytokinin oxidase/dehydrogenase promoter 3::GUS fusion was specifically induced at the site of infection when plants were challenged with a virulent strain of R. fascians, providing a possible explanation for the lack of significantly elevated cytokinin concentrations in tissues infected with virulent strains of R. fascians.This revised version was published online in August 2005 with some black and white figures replaced by coloured figures.     

N-glucosyltransferase UGT76C2 is involved in cytokinin homeostasis and cytokinin response in Arabidopsis thaliana

Cytokinins are a class of phytohormones that play a crucial role in plant growth and development. The gene UGT76C2 encoding cytokinin N-glucosyltransferase of Arabidopsis thaliana has been previously identified. To determine the in planta role of UGT76C2 in cytokinin metabolism and response, we analyzed the phenotypes of its loss-of-function mutant (ugt76c2) and its overexpressors. The accumulation level of the cytokinin N-glucosides was significantly decreased in ugt76c2, but substantially increased in UGT76C2 overexpressors compared with the wild type. When treated with exogenously applied cytokinin, ugt76c2 showed more sensitivity and UGT76C2 overexpressors showed less sensitivity to cytokinin in primary root elongation, lateral root formation, Chl retention and anthocyanin accumulation. Under normal growth conditions ugt76c2 had smaller seeds than the wild type, with accompanying lowered levels of active and N-glucosylated cytokinin forms. The expression levels of cytokinin-related genes such as AHK2, AHK3, ARR1, IPT5 and CKX3 were changed in ugt76c2, suggesting homeostatic control of cytokinin activity. Studies of spatiotemporal expression patterns showed that UGT76C2 was expressed at a relatively higher level in the seedling and developing seed. In their entirety, our data, based mainly on this comparison and opposite phenotypes of knockout and overexpressors, strongly suggest that UGT76C2 is involved in cytokinin homeostasis and cytokinin response in planta through cytokinin N-glucosylation.     

AtMYB2 regulates whole plant senescence by inhibiting cytokinin-mediated branching at late stages of development in Arabidopsis

Whole plant senescence of monocarpic plants consists of three major processes: arrest of shoot apical meristem, organ senescence, and permanent suppression of axillary buds. At early stages of development, axillary buds are inhibited by shoot apex-produced auxin, a mechanism known as apical dominance. How the buds are suppressed as an essential part of whole plant senescence, especially when the shoot apexes are senescent, is not clear. Here, we report an AtMYB2-regulated post apical dominance mechanism by which Arabidopsis (Arabidopsis thaliana) inhibits the outgrowth of axillary buds as part of the whole plant senescence program. AtMYB2 is expressed in the compressed basal internode region of Arabidopsis at late stages of development to suppress the production of cytokinins, the group of hormones that are required for axillary bud outgrowth. atmyb2 T-DNA insertion lines have enhanced expression of cytokinin-synthesizing isopentenyltransferases genes, contain higher levels of cytokinins, and display a bushy phenotype at late stages of development. As a result of the continuous generation of new shoots, atmyb2 plants have a prolonged life span. The AtMYB2 promoter-directed cytokinin oxidase 1 gene in the T-DNA insertion lines reduces the endogenous cytokinin levels and restores the bushy phenotype to the wild type.     

Design and synthesis of photolabile caged cytokinin

Cytokinins are phytohormones that regulate diverse developmental processes throughout the life of a plant. trans-Zeatin, kinetin, benzyladenine and dihydrozeatin are adenine-type cytokinins that are perceived by the AHK cytokinin receptors. Endogenous cytokinin levels are critical for regulating plant development. To manipulate intracellular cytokinin levels, caged cytokinins were designed on the basis of the crystal structure of the AHK4 cytokinin receptor. The caged cytokinin was photolyzed to release the cytokinin molecule inside the cells and induce cytokinin-responsive gene expression. The uncaging of intracellular caged cytokinins demonstrated that cytokinin-induced root growth inhibition can be manipulated with photo-irradiation. This caged cytokinin system could be a powerful tool for cytokinin biology.