Cytokinins. New Insights into a Classic Phytohormone

Cytokinins were discovered in the search for factors that promoted division of plant cells in culture. Naturally occurring cytokinins areN 6-substituted adenine derivatives that generally contain an isoprenoid derivative side chain. These hormones influence numerous aspects of plant development and physiology, including seed germination, de-etiolation, chloroplast differentiation, apical dominance, plant-pathogen interactions, flower and fruit development, and leaf senescence. These processes are also influenced by various other stimuli (e.g. light and other phytohormones), and the physiological and developmental outcomes reflect a highly integrated response to these multiple stimuli. For example, the classical reports of Skoog and Miller (1957) revealed that undifferentiated callus cultures would form into roots or shoots depending on the relative amount of cytokinins and auxin in the medium; the ratio rather than the absolute amount of these two hormones is critical. A balanced ratio keeps the cells in an undifferentiated state, while high cytokinin to auxin ratios promote shoot and low ratios promote root development. In the last decade, genetic and molecular analysis of mutants has provided valuable insights into the molecular mechanisms underlying the action of other phytohormones. Cytokinin has lagged behind in such studies, but several exciting recent reports show that this is now changing. This review focuses on the dramatic recent progress in understanding cytokinin metabolism, perception, and signaling. The analysis of all three aspects is required to understand the different effects of cytokinin on plant physiology and its role as a developmental signal. Cytokinins occur as a bound form in the tRNA of most organisms, including plants, but plants also possess significant amounts of free cytokinins. Isoprenoid-type cytokinins are the most abundant, but several plant species contain adenine derivatives with aromatic substituents. In addition, there are synthetic cytokinins derived from diphenylurea (DPU) that are structurally unrelated to the adenine-type cytokinins. The structure and conformation of the side chain are critical to the activity of the respective cytokinin. For example, one of the most abundant cytokinins in higher plants, trans-zeatin, displays a high cytokinin activity in bioassays, but the cis-isomer possesses a significantly lower activity. In general, a given plant tissue contains several types of cytokinins and their modified forms. The distribution of the various cytokinins differs significantly between plant species. The phenotypic analysis of biosynthetic mutants could reveal the roles of specific cytokinin forms, but unfortunately neither general nor specific mutants with reduced cytokinin content have been isolated, and the overexpression of biosynthetic genes generally affects the whole pool of endogenous cytokinins. Furthermore, it is difficult to quantify cytokinins in vivo, especially with respect to their distribution within different plant tissues or organs. Our knowledge about the in vivo role of individual cytokinins and their modified forms is therefore very limited. The breakdown of tRNA was originally suggested as a possible mechanism for cytokinin synthesis. The released cis-zeatin could subsequently be converted to active trans-zeatin by the enzyme cis-trans-isomerase (Mok and Mok, 2001). However, the slow turnover rate of tRNA is not sufficient to account for the amount of cytokinins present in plants. Enzymatic activity that converts AMP and dimethylallyl-diphosphate (DMAPP) to the active cytokinin isopentenyladenosine-5′-monophosphate (iPMP) was firstly identified in Dictyostelium discoideum (Taya et al., 1978). Subsequently, the tmr gene (later designated asipt) from Agrobacterium tumefaciens, which results in root-like tumors when mutated, was shown to encode an enzyme with similar activity (Akiyoshi et al., 1984). ipt genes have also been identified in several other bacteria, and IPT activity was detected in crude extracts from a variety of plant tissues, but the plant enzymes were not purified and the corresponding genes were not cloned. Proposed biosynthetic and metabolic pathway for cytokinins. Left, The proposed biosynthesis of zeatin tri-/diphosphate in Arabidopsis. Both ADP and ATP are likely substrates for the plant IPT enzyme, and these and their di- and triphosphate derivatives are indicted together (e.g. ATP/ADP). The biosynthesis of cytokinins in bacteria (e.g. A. tumefaciens) is compared next to it. Right, Several possible modifications and the degradation of zeatin. The diagram only depicts reactions that are described in the text; cytokinin metabolism is more complex than the pathways shown (see Mok and Mok, 2001). See text for more details. Another study in Arabidopsis indicates that an alternative cytokinin biosynthesis pathway exists in plants (Åstot et al., 2000). The authors compared the biosynthetic rate of zeatin riboside-5′-monophosphate (ZMP) and iPMP in wild type and transgenic plants designed to inducibly overexpress the bacterial iptgene. iPMP is the direct product of the transfer of DMAPP to AMP, and it can be converted to ZMP by an endogenous hydroxylase activity. In vivo deuterium labeling revealed a 66-fold higher biosynthetic rate of ZMP than that of iPMP, the product of IPT. By a feeding experiment with two tracers, which allowed the simultaneous determination of iPMP-hydroxylase activity and the de novo synthesis of ZMP, it was shown that the major precursor for ZMP was not cytoplasmic iPMP. The authors suggested the presence of an iPMP-independent pathway, in which ZMP is directly synthesized by IPT from AMP and a yet unidentified side chain precursor. This pathway could be strongly inhibited by mevastatin, indicating a terpenoid origin of the hypothetical precursor. Almost all cytokinins are present in plants as both the free base and the corresponding nucleosides and nucleotides. The interconversions among these forms are likely to be performed by enzymes involved in the general purine metabolism, but their biological significance is unclear (Mok and Mok, 2001). The adenine ring system can be glucosylated at theN 3-, N 7-, andN 9-position. Glucosyl-conjugates at the N 7- and N 9- but not at the N 3-position are usually inactive in bioassays. It is assumed that the N 7- and N 9- modifications irreversibly inactivate cytokinins, but the precise in vivo function of theseN-glucosyl-conjugates is unknown (Mok and Mok, 2001). O-Glucosyl-conjugates of theN 6-side chain are a common modification in all plants. In addition, O-xylosyl-conjugates have been reported in Phaseolus spp. (Mok and Mok, 2001). The purified corresponding glycosyltransferases of Phaseolusspp. exhibited a high substrate specificity both to the cytokinins zeatin and dihydrozeatin and to the glycosyl donor (Dixon et al., 1989). The zeatin-O-glucosyltransferase (ZOG1) strongly preferred UDP-Glc while the zeatin-O-xylosyltransferase (ZOX1) exclusively utilized UDP-Xyl. The two genes,ZOG1 and ZOX1, have been isolated fromPhaseolus spp. and the binding site of the UDP-Glc has been mapped by domain swap experiments (Martin et al., 1999a, 1999b; Mok and Mok, 2001). Recently the isolation of a cis-zeatin-O-glucosyltransferase (cis-ZOG1) has been reported that specifically glucosylates cis-zeatin but not trans-zeatin or dihydrozeatin (Martin et al., 2001). The authors suggested that the high specificity of cis-ZOG1 to the cis-isomer may indicate an important role for cis-zeatin in plants. O-Glycosylated cytokinins are resistant to the cleavage of the N 6-side chain by cytokinin oxidases (see below). Additionally, these forms can easily be converted into active cytokinins by β-glucosidases (Brzobohaty et al., 1993). Thus, it is believed that O-glycosylated cytokinins are inactive, stable storage forms that play an important role in balancing cytokinin levels. Free cytokinin bases and nucleosides with an unsaturatedN 6-side chain are irreversibly degraded by cleavage of the side chain by cytokinin oxidases. Cytokinin oxidases are found in many plant species, and they represent a class of highly diverse proteins (Jones and Schreiber, 1997). The genes of two highly similar cytokinin oxidases have been isolated from maize (Zea mays; Houba-Hérin et al., 1999; Morris et al., 1999). Heterologous expression in Pichia pastoris andPhyscomitrella patens confirmed the predicted cytokinin oxidase activity. Maize cytokinin oxidase and its Arabidopsis homologs contain a signal sequence for the secretory pathway, and they may be therefore extracellular proteins, but no in vivo localization studies have yet been reported. Werner et al. (2001) have recently constructed transgenic tobacco (Nicotiana tabacum) expressing four Arabidopsis homologs of the maize cytokinin oxidase from the CaMV 35S promoter. For the first time, this allowed the phenotypic consequences of reduced endogenous cytokinin levels to be determined. Transgenic lines exhibited a higher cytokinin oxidase activity and significantly reduced amounts of iP and zeatin metabolites, including their glycosides as compared with wild type. The transgenic lines showed a dwarfed growth as a consequence of a severely retarded shoot development. In contrast, the growth of the root system was enhanced, indicating that cytokinin may have opposing roles in shoot and root development. These defects could be traced back to alterations in the cell number and the rate of cell division in the apical meristems. Shoot apical meristems consisted of fewer cells with sizes comparable to wild type. Leaves were formed from a significantly decreased number of cells, which was partly compensated for by an increased cell size. Nevertheless, the transgenic leaves were about 15% of the size of their wild-type counterparts. The reduced endogenous cytokinin levels resulted in alterations in cell proliferation in the apical meristems, consistent with an in vivo role for cytokinins in the regulation of cell division. In contrast, there was an increased number and size of cells in the root apical meristem, which contributed to a general enlargement of the root. Based on the occurrence of cytokinins in the xylem sap and the identification of the root tip as a major site of cytokinin biosynthesis, it is generally assumed that cytokinins are transported in the xylem to exert their effects on the aerial parts of plants. A purine transporter, AtPUP1, has been isolated in Arabidopsis by the functional complementation of a yeast mutant deficient in adenine uptake (Gillissen et al., 2000). Adenine uptake was competitively inhibited by free cytokinin bases. Although no direct evidence for cytokinin transport was presented, this competitive inhibition suggests that AtPUP1 represents a possible cytokinin/purine transporter involved in the translocation of root-derived cytokinins to the aerial plant parts. Insight into the biological role of cytokinin transport was provided by feeding roots of nitrogen-depleted maize with nitrate (Takei et al., 2001b). In response to the applied nitrate, cytokinin accumulated first in the roots, subsequently in the xylem sap, and finally in leaves. This suggests that in response to nitrate, these plants made cytokinins in the root, which was subsequently transported into the aerial portion of the plant through the xylem. Thus, cytokinin may represent a long-distance signal for nitrogen availability from the root to the shoot, presumably to coordinate shoot and root development. Somewhat conflicting results with respect to the biological significance of transported cytokinin were obtained from reciprocal grafting experiments with wild-type and cytokinin-overproducing tobacco plants (Faiss et al., 1997). The phenotypic effects of elevated cytokinin were restricted to the part of the plant that was derived from the ipt overexpressing mutant. Elevated level of cytokinins in the root led to only a slight increase in cytokinin levels in the xylem and had no phenotypic consequences in the scion. Thus, it was concluded that cytokinin may act as paracrine signal, at least with respect to apical dominance and leaf senescence. The concept of cytokinin as a phytohormone implies that the site of synthesis and the site of action can spatially be separated. Thus, the search for receptors for cytokinin is almost as old as its discovery. Biochemical approaches led to the isolation of cytokinin binding proteins, but none has been unequivocally shown to be a receptor. The report of a G-protein-coupled receptor, GCR1, as a candidate for a cytokinin receptor has recently been retracted (Kanyuka et al., 2001). In an activation-tagging mutagenesis, a His kinase (CKI1) was identified by its ability to confer cytokinin-independent callus growth when overexpressed in Arabidopsis (Kakimoto, 1996). It was proposed that the elevated basal level of CKI1 overcomes the cytokinin requirement for callus growth and greening, and that CKI1 represents a putative cytokinin receptor. Transient expression of CKI1 in Arabidopsis protoplasts indeed showed activation of the promoter of cytokinin primary response gene (Hwang and Sheen, 2001). However, this expression of ARR6 was constitutive, not responsive to exogenous cytokinin. The constitutive induction of ARR6 by CKI1 in this system might reflect cross talk between CKI1 and the cytokinin signaling pathway due to the overexpression of CKI1, and may not reflect the situation in vivo. Although the of overexpressing mutant an important role for CKI1 or a similar His kinase in the regulation of cell proliferation the of the not a the wild-type function of the The of a His kinase in cytokinin signaling has recently been confirmed by two have reported the identification of a cytokinin receptor in Arabidopsis. This a His kinase that to the sequence of CKI1 et al., et al., A of Arabidopsis plants identified a mutant that reduced to cytokinin in tissue including shoot and proliferation of callus et al., 2001). mutant to cytokinin in a root isolated showed a similar Both to be by molecular analysis (see also but are in the root This of a implies indicating a action of the receptor in this an type of kinase with two response In addition, contains a His kinase domain and two a extracellular identified the part of the receptor, and the functional of the two response is that is a cytokinin receptor. The pathway depicts the pathway in wild-type the His kinase which the activity of kinase kinase activity a of and response A mutant of is due to of can the growth in an only in the presence of cytokinins of cytokinin signaling in Arabidopsis. Cytokinin to the domain of likely other similar and its His kinase activity. the which in transfer the to the domain of presumably to other their other primary are by the The also with the and are also likely The in in with the proteins, with various to including the a inhibition of their The indicate See text for details. is the biological role of has been shown to be to the which defects in the development of tissue et al., 2000). have a reduced number of cells in the of the root and the lower part of the and these almost of xylem cells et al., Thus, and are in these parts of The defects in can be traced back to the of the which has a reduced number of due to the of specific cell et al., et al., 2000). The of and cells is presumably by reduced cell division rather than cell the mutant can be by which has cell et al., It was proposed that the determination of xylem cells the of cells and that the reduced number of are as xylem no to et al., This of the together the role of as a cytokinin receptor and the effects of cytokinin on cell division. However, may also have roles in the of has a than the other et al., et al., it is unknown it is a the is a the extracellular domain of the which is the putative site et al., 2000). In binding have recently shown that the binding of cytokinin to et al., 2001). is in the root, and in the it is restricted to the tissue from the root to the of the et al., 2000). This expression may the rather of a in the aerial of mutant and In to the of a shoot in plants, mutants show a in the of shoots from tissue cells in response to cytokinin. It may be that the of a shoot in tissue the of a In this it is that roots from callus phenotypic roots of et al., may be with other genes in the shoots of plants, but not with respect to shoot in there are two His and in Arabidopsis that are in the aerial parts of wild-type plants et al., 2001). Both His are highly to the including the putative binding and also with the presence of two response in their Furthermore, in a expression both His may the promoter of a primary cytokinin response gene in a (Hwang and Sheen, also below). These results strongly that and indeed act as cytokinin receptors in Arabidopsis. The of a His kinase in cytokinin signaling suggests that homologs of bacterial may be involved in the cytokinin signal homologs of these other transfer proteins and response have been identified in Arabidopsis. The possible role of several of these genes in cytokinin signaling has been by numerous for example, the induction of the promoter of a cytokinin primary response the (see was by a in Arabidopsis protoplasts that wild-type and forms of these genes (Hwang and Sheen, 2001). The with numerous of other important and valuable insights into the putative of cytokinin signaling The for of the three types of of the receptors first be the about these into a of cytokinin signal are Arabidopsis genes putative His proteins which function as a in the primary between the present in the domain of the kinase and the domain of the response et al., 2000). et al. a system in to the yeast system to that could act as a cytokinin receptor, and to the as of The His in in this analysis was and the response was The activation of by could be by a promoter to the of four different inhibited the It was proposed that the with the endogenous for by consistent with an between that and these The different of specific might reflect specificity of the proposed However, the individual expression levels of the four in this system were not and be localization studies also the that at least of the are involved in cytokinin signaling (Hwang and Sheen, 2001). It was shown that and but not from the to the of Arabidopsis protoplasts in response to applied several have reported yeast between His and these reports indicate that at least a of may be involved in cytokinin signaling. These likely the signal from its site of perception, the to the in the results indicate that these at least in of the class of of the response The response in Arabidopsis form a gene that the and the and 1999; et al., 1999). The are of a the have an domain to the This domain has been to in yeast and in and to to in a The have been shown to be proteins et al., 1999; et al., and Sheen, 2001). The levels of of most of the but not the are and specifically elevated in response to cytokinin and et al., et al., 1999; et al., 2000). response in maize very similar induction indicating that the function of is highly in plants et al., The induction by cytokinin of several of the is at least in to the activation of these genes and is not by which indicates that these are primary response genes and et al., 2000). The to bacterial signaling predicted to act of His with the induction by that these may play a role in cytokinin signaling. with the gene has been found to be in the shoot and root apical meristems, likely of cytokinin action et al., 2000). studies in protoplasts have shown that the overexpression of their that may a regulation of cytokinin signaling (Hwang and Sheen, 2001). mutant forms of these genes a the predicted were in their Thus, wild-type and mutant proteins may with cytokinin signaling such as the these to the may also with and could have activation by cytokinin may into cytokinin but would also yet to cytokinin. This may also there are in Arabidopsis. In two recent the of the was shown to be at least by et al., and Sheen, 2001). mutant was found to be resistant to cytokinin in shoot and root and an overexpressing transgenic increased to cytokinin in these These results that this gene a role in cytokinin of the expression of the genes in the mutants revealed that the exhibited reduced expression of several and that overexpression of resulted in an induction of the in the presence of These with the presence of multiple binding in the promoter of several that directly the of these primary of cytokinin signaling et al., 2001). The activity of the domain is inhibited by the response domain et al., 2000). Thus, it was proposed that the inhibition of the domain by the response domain was by cytokinin. results were obtained by expression studies in Arabidopsis protoplasts (Hwang and Sheen, 2001). of three and highly increased both the basal level and the induction of the promoter of a exhibited the induction and was in of the binding domain of the and domain the cytokinin of the ARR6 presumably as a The overexpression of in promoted cell proliferation and shoot and leaf in tissue cultures in the of exogenous cytokinin. The transgenic plants also showed leaf as for cytokinin These results at least several of the as of cytokinin proteins in the in which the the site of was to were in the induction of the by cytokinin (Hwang and Sheen, 2001). This the of cytokinin signaling as a classical at least with to the induction of the The authors that an endogenous may be by the overexpression of the Several the The of the within all and about in Arabidopsis as as the of these between and Arabidopsis homologs strongly suggests the functional of these least at the level of the receptor, the is also The complementation in yeast and an of with endogenous His transfer proteins similar to their with their His In addition, mutant forms of in which these have been are to in protoplasts by of cytokinin. Thus, it is highly likely that both in the expression system and in the complementation in yeast and as a His Biochemical studies and mutants of the and to the significance of their These results are consistent with the of cytokinin of cytokinin to other His a that an in the and activation of a of the These the of the which in into the The together in with the with various to the in cell function to cytokinin The significant of this as the are now in to the among these The of cytokinin has dramatic the last Several of the of cytokinin action and metabolism have been but many to be The in vivo role of most of the of the in Arabidopsis including their putative function in cytokinin signaling. The function and specificity of the among the various in vivo represents one of the The analysis of expression and the identification of mutants in these genes to understand the numerous effects of cytokinin on plant physiology and development at the molecular Furthermore, these genes can now be to and which the complex of cytokinin metabolism and signaling.