Isopentenyladenine from mutants of the moss, Physcomitrella patens

The culture media from gametophore over-producing mutants of the moss Physcomitrella patens have been examined for their cytokinin content. Two cytokinins have been detected, one of which has been identified as N⁶-(Δ²-isopentenyl) adenine (2iP).     

Expression of the bacterial ipt gene in Physcomitrella rescues mutations in budding and in plastid division

Development of Physcomitrella patens (Hedw.) B.S.G. starts with a filamentous protonema growing by apical cell division. As a developmental switch, some subapical cells produce three-faced apical cells, the so-called buds, which grow to form leafy shoots, the gametophores. Application of cytokinins enhances bud formation but no subsequent gametophore development in several mosses. We used the ipt gene of Agrobacterium tumefaciens, encoding a protein which catalyzes the rate-limiting step in cytokinin biosynthesis, to transform two developmental Physcomitrella mutants. One mutant (P24) was defective in budding (bud) and thus did not produce three-faced cells, while the other one (PC22) was a double mutant, defective in plastid division (pdi), thus possessing at the most one giant chloroplast per cell, and in gametophore development (gad), resulting in malformed buds which could not differentiate into leafy gametophores. Expression of the ipt gene rescued the mutations in budding and in plastid division but not the one in gametophore development. By mutant rescue we provide evidence for a distinct physiological difference between externally applied and internally produced cytokinins. Levels of immunoreactive cytokinins and indole-3-acetic acid were determined in tissues and in culture media of the wild-type moss, both mutants and four of their stable ipt transformants. Isopentenyl-type cytokinins were the most abundant cytokinins in Physcomitrella, whereas zeatin-type cytokinins, the major native cytokinins of higher plants, were not detectable. Cytokinin as well as auxin levels were enhanced in ipt transgenics, demonstrating a cross-talk between both metabolic pathways. In all genotypes, most of the cytokinin and auxin was found extracellularly. These extracellular pools may be involved in hormone transport in the non-vascular mosses. We suggest that both mutants are defective in signal-transduction rather than in cytokinin metabolism. Received: 24 October 1997 / Accepted: 20 March 1998     

Fatty acid composition of mutants of the moss Physcomitrella patens

The fatty acid composition of various mutant strains of the moss Physcomitrella patens has been compared to the wild-type. These included strains defective in their responses to auxins and/or cytokinins, one which releases much more cytokinin into the medium than the wild-type, and two aphototropic strains. The lipids of the aphototropic mutants were also studied after culture in different light regimes. Although some differences in fatty acid composition have been found between strains, these alone are probably not responsible for their physiological differences. Considerable changes occur in many fatty acids in senescent or dark-grown material, including changes in the proportion of C₂₀ polyenoic fatty acids.     

Analysis of gametophytic development in the moss,Physcomitrella patens,using auxin and cytokinin resistant mutants

Mutants altered in their response to auxins and cytokinins have been isolated in the moss Physcomitrella patens either by screening clones from mutagenized spores for growth on high concentrations of cytokinin or auxin, in which case mutants showing altered sensitivities can be recognized 3–4 weeks later, or by non-selective isolation of morphologically abnormal mutants, some of which are found to have altered sensitivities. Most of the mutants obtained selectively are also morphologically abnormal. The mutants are heterogeneous in their responses to auxin and cytokinin, and the behaviour of some is consistent with their being unable to make auxin, while that of others may be due to their being unable to synthesize cytokinin. Physiological analysis of the mutants has shown that both endogenous auxin and cytokinin are likely to play important and interdependent roles in several steps of gametophytic development. Although their morphological abnormalities lead to sterility, genetic analysis of some of the mutants has been possible by polyethyleneglycol induced protoplast fusion.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - NAA 1-naphthalene acetic acid - 2,4D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - IAP 6-( ²isopentenyl) aminopurine - NAR NAA resistant mutants - BAR BAP resistant mutants     

Moss systems biology en route: phytohormones in Physcomitrella development

The moss Physcomitrella patens has become a powerful model system in modern plant biology. Highly standardized cell culture techniques, as well as the necessary tools for computational biology, functional genomics and proteomics have been established. Large EST collections are available and the complete moss genome will be released soon. A simple body plan and the small number of different cell types in Physcomitrella facilitate the study of developmental processes. In the filamentous juvenile moss tissue, developmental decisions rely on the differentiation of single cells. Developmental steps are controlled by distinct phytohormones and integration of environmental signals. Especially the phytohormones auxin, cytokinin, and abscisic acid have distinct effects on early moss development. In this article, we review current knowledge about phytohormone influences on early moss development in an attempt to fully unravel the complex regulatory signal transduction networks underlying the developmental decisions of single plant cells in a holistic systems biology approach.     

Arabidopsis thaliana wild type, pho1, and pho2 mutant plants show different responses to exogenous cytokinins.

Despite the involvement of cytokinins in phosphate (Pi) signaling being highlighted, the physiological processes involved remain unclear. In this study, we have evaluated the effect of cytokinins on different physiological responses using wild type (wt) and two Arabidopsis mutants with altered shoot Pi content (pho1 and pho2). Physiological studies were related with those previously described as cytokinin-regulated: including hypocotyl elongation, root growth, anthocyanin accumulation, senescence and relative gene expression. Generally, pho1 mutants showed decreased sensitivity to cytokinin, whereas pho2 mutants showed increased sensitivity to the hormone. This observation applies to inhibition of hypocotyls and root growth and anthocyanin accumulation. However, this effect was not shown during senescence or in the expression of ARR6 (Arabidopsis response regulator, ARR). Interestingly, Pi content in shoot of pho1 mutants increased to wt levels after treatment with cytokinins. These results suggest that the interaction between phosphate signaling and cytokinin signaling may be bidirectional while the differential behavior in response to cytokinin is discussed further.     

Cytokinin biosynthesis and perception

Cytokinin has been considered to be a master regulator of plant growth and development, but only in the past several years has substantial progress been made uncovering the roles of cytokinins at various developmental stages. Recent studies on key metabolic enzymes and signaling components have contributed to understanding the basic mechanism of biosynthesis and perception of cytokinin within a whole plant body. The initial products of de novo cytokinin biosynthesis in higher plants and Agrobacterium are different, and the regulatory systems in biosynthesis and homeostasis are finely controlled and appear to be important in communicating nutrient signals to morphogenetic responses. The cytokinin receptors have largely overlapping, but still specific, functions in diverse cytokinin responses. In this review, we will specifically emphasize the biosynthesis of isoprenoid cytokinins and perception of cytokinin signals in Arabidopsis.     

Cytokinin biosynthesis and interconversion

To maintain hormone homeostasis, the rate of cytokinin biosynthesis, interconversion, and degradation is regulated by enzymes in plant cells. Cytokinins can be synthesized via direct (de novo) or indirect (tRNA) pathways. In the de novo pathway, a cytokinin nucleotide is synthesized from 5'-AMP and isopentenyl pyrophosphate; a key enzyme which catalyzes this synthesis has been isolated from plant tissues, slime mold, and some microorganisms. Studies on the in vitro synthesis of the isopentenyl side chain of cytokinin in tRNA demonstrated that the isopentenyl group was derived from mevalonate, and turnover of the cytokinin-containing tRNA may serve as a minor source of free cytokinins in plant cells. The interconversion of cytokinin bases, nucleosides and nucleotides is a major feature of cytokinin metabolism; and enzymes that regulate the interconversion have been identified. The N⁶-side chain and purine moiety of cytokinins are often modified and some of the enzymes involved in the modifications have been isolated. Most of the cytokinin metabolites have been characterized but very few enzymes regulating their metabolism have been purified to homogeneity. It remains a significant challenge to isolate plant genes involved in the regulation of cytokinin biosynthesis, interconversion and degradation.     

Cytokinins from the Moss Physcomitrella patens

Gametophore-over-producing mutants of the moss, Physcomitrella patens, when grown in liquid culture export high levels of cytokinin into their culture medium. The cytokinin produced by these mutants is postulated to account for their peculiar phenotype, that of mosses treated with exogenous cytokinin. N⁶-(Δ²-isopentenyl)adenine, the major cytokinin, has been identified previously in two of these mutants (Wang, Cove, Beutelmann, Hartmann 1980 Phytochemistry 19: 1103-1105) and now in additional representatives. A second cytokinin, zeatin, has been identified by its chromatographic behavior and mass spectrum including chemical ionization mass spectrometry of its permethyl derivative.     

In search of the phytohormone functions in Fungi:Cytokinins

Cytokinins are phytohormones that participate in regulation of all aspects of plant growth and development, including response to biotic agents. Fungi of different taxonomic and trophic groups synthesize cytokinins, employing them for interaction with plants, both friendly and hostile. They also appear to be able to manipulate the host plant genes of cytokinin biosynthesis and metabolism to their own benefit. In this review, we analyzed the data about changes in the level and composition of cytokinin pool under fungi influence, the effect of exogenous hormones on the growth of fungi in culture and in situ, changes in the physiology and metabolism of fungi due to genetic transformations related to cytokinins. The possible role of cytokinins in the regulation of macromycete development is discussed as well. The pattern of cytokinin dynamics allow us to consider the hormones of this class as potential regulators of fungi growth. Further explore of fungal cytokinins is essential to improve biotechnology using fungi as raw materials for medicine and agricultural production.     

New Insights into the Functions of Cytokinins in Plant Development

Recent breakthroughs in cytokinin research have shed new light on the role of cytokinin in plant development. Loss-of-function mutants of a cytokinin receptor reveal a role for the hormone in establishment of the vasculature during embryonic development. Cytokinin controls the number of early cell divisions via a two-component signaling system. Genetically engineered plants that have a reduced cytokinin content demonstrate the regulatory role of the hormone in control of meristem activity and organ growth during postembryonic development, with opposite roles in roots and shoots. There is increasing evidence from work with transgenic plants and mutant analysis that cytokinins do not perform the previously proposed function as a root-derived signal for the regulation of shoot branching. Root-borne cytokinins might serve as a long-range signal controling other processes at distant sites, such as responding to nutritional status, particularly nitrogen availability.     

Monoclonal antibodies for the analysis and purification of isopentenyladenine cytokinins

A series of monoclonal antibodies (McAb) have been generated which show a high affinity for the cytokinin, isopentenyladenosine (2iPA). The McAb show different specificities to a range of cytokinins in a radioimmunoassay and are suitable for the analysis of isopentenyladenine (2iP) and related compounds in plant extracts. One McAb, MAC 160, has a high cross-reactivity with several cytokinins and is a good candidate for use in group separation or the analysis of a number of cytokinins simultaneously after separation. When coupled to Sepharose 4B affinity support, the McAb can also be used as immunoaffinity matrices to purify and resolve cytokinins from plant extracts. The McAb have been used to demonstrate the presence of concentrations of 2iP in tissues of the moss, Physcomitrella patens which exceed those in its culture medium.     

The interaction of cytokinin with other signals

Cytokinins are important signalling molecules in plants, and recent studies have begun to shed light on the molecular mechanisms underlying their biosynthesis and response pathways. However, from the time of their discovery, it has been clear that cytokinins interact with other signals to regulate plant growth and development. Herein the interaction of cytokinin with three other signals: light, ethylene, and auxin is discussed. The interaction between light and cytokinin signalling, highlighted by recent analysis of cytokinin signalling mutants is reviewed. A discussion of another aspect of cytokinin cross-talk, its induction of ethylene biosynthesis in etiolated Arabidopsis seedlings, and recent studies that have begun to elucidate the mechanism underlying this regulation is also presented. Finally, there is a brief review of the interaction of auxin and cytokinin, and present novel expression profiling data of Arabidopsis seedlings treated with combinations of these two hormones, which provide insight into this interaction.     

Regulation of cytokinin content in plant cells

Cytokinin levels in plant cells are dependent on cytokinin biosynthesis and/or uptake from extracellular sources, metabolic interconversions, inactivation and degradation. Cytokinin conversion to compounds differing in polarity seems to be decisive for their entrapment within the cell and intracellular compartmentation, which affects their metabolic stability. Increase in cytokinin levels, resulting either from their uptake or intracellular biosynthesis, may promote further autoinductive accumulation of cytokinins which may function in the induction of cytokinin-initiated physiological processes. Accumulated cytokinins are capable of inducing cytokinin oxidase which consequently decreases cytokinin levels. This seems to be the mechanism of re-establishment and maintenance of cytokinin homeostasis required for further development of physiological events induced by transient cytokinin accumulation. Auxin may influence cytokinin levels by down regulation of cytokinin biosynthesis and/or by promotion of cytokinin degradation. A model of the regulation of cytokinin levels in plant cells based on these phenomena is presented and its physiological role(s) is discussed.     

Immunoassay and ultrastructural localization of isopentenyladenine and related cytokinins using monoclonal antibodies

Two hybridoma cell lines, J40-IV-A1 and J40-IV-C4 were obtained from a fusion of spleen cells of Balb/c mice immunized against an isopentenyladenosine-bovine serum albumin conjugate with X63. Ag 8.653 myeloma cells. These hybrids secrete monoclonal antibodies of the immunoglobulin G (IgG) class and share high affinities and specificities to isopentenyladenine and isopentenyladenosine suitable for the detection of femtomole amounts of these cytokinins in plant extracts by enzyme-linked immunosorbent assay (ELISA). One of the monoclonal antibodies (J40-IV-C4) has been employed to localize isopentenyladenine immunoreactivity in a cytokinin-over-producing mutant of the moss, Physcomitrella patens. After fixation and embedding at low temperature, immunoreactivity was visualized in protonemal filaments of the moss mutant by the use of indirect immunogold labelling. In the mutant, the labelling was predominantly in the wall of the protonemal cells. Neither the wild-type nor control treatments showed any labelling. The signficance of these observations is discussed with respect to the applicability of immunocytochemical techniques for the localization of low-molecular-weight compounds in plant tissue.Abbreviations ELISA enzyme linked immunosorbent assay - HPLC high-performance liquid chromatography - IP isopentenyladenine - IPA isopentenyladenosine - mAB monoclonal antibody - OVE cytokinin-over-producing mutant - RIA radioimmunoassay     

On the Significance of Cytokinin Incorporation into RNA

The clarification of the following 2 questions was attempted: (a) are cytokinins precursors in the formation of sRNA, (b) is the observed incorporation of cytokinins into sRNA related to the action of the hormone? Although Escherichia coli contains cytokinins in its sRNA, no cytokinin auxotroph mutants of E. coli could be found and the statistical probability for the existence of such mutants is extremely low. This suggests that cytokinins are not precursors in the synthesis of sRNA. A radioactive cytokinin, 6-benzylamino-9-methyl-purine was synthesized and it was tested whether or not it is incorporated into sRNA of soybean callus tissue. Masking the 9-position of the purine inhibited the incorporation of this cytokinin into RNA while not affecting its biological activity. This is taken as an indication that the observed incorporation of cytokinins such as benzyladenine into sRNA is not related to the action of this hormone.     

Cytokinins in the bryophyte Physcomitrella patens: analyses of activity, distribution, and cytokinin oxidase/dehydrogenase overexpression reveal the role of extracellular cytokinins

Ultra-performance liquid chromatography-tandem mass spectrometry was used to establish the cytokinin profile of the bryophyte Physcomitrella patens (Hedw.) B.S.G.; of 40 analyzed cytokinins, 20 were detected. cis-Zeatin-riboside-O-glucoside, N(6)-(Delta(2)-isopentenyl)adenosine-5'-monophosphate (iPRMP), and trans-zeatin-riboside-O-glucoside were the most abundant intracellular cytokinins. In addition, the aromatic cytokinins N(6)-benzyladenosine (BAR), N(6)-benzyladenine, meta-, and ortho-topolin were detected. Unexpectedly, the most abundant extracellular cytokinin was the nucleotide iPRMP, and its identity was confirmed by quadrupole time-of-flight mass spectrometry. The effects of overexpressing a heterologous cytokinin oxidase/dehydrogenase (CKX; EC 1.4.3.18/1.5.99.12) gene (AtCKX2 from Arabidopsis [Arabidopsis thaliana]) on the intracellular and extracellular distribution of cytokinins was assessed. In cultures of CKX-transformed plants, ultra-performance liquid chromatography-tandem mass spectrometry measurements showed that there were pronounced reductions in the extracellular concentrations of N(6)-(Delta(2)-isopentenyl)adenine (iP) and N(6)-(Delta(2)-isopentenyl)adenosine (iPR), but their intracellular cytokinin concentrations were only slightly affected. In vitro and in vivo measured CKX activity was shown to be strongly increased in the transformants. Major phenotypic changes observed in the CKX-overexpressing plants included reduced and retarded budding, absence of sexual reproduction, and abnormal protonema cells. In bud-induction bioassays with wild-type Physcomitrella, the nucleotides iPRMP, trans-zeatin-riboside-5'-monophosphate, BAR monophosphate, and the cis-zeatin forms cZ and cZR had no detectable effects, while the activities displayed by other selected cytokinins were in the following order: iP > tZ > N(6)-benzyladenine > BAR > iPR > tZR > meta-topolin > dihydrozeatin > ortho-topolin. The results on wild type and CKX transgenics suggest that extracellular iP and iPR are the main cytokinins responsible for inducing buds in the bryophyte Physcomitrella. Cytokinin profile is discussed regarding the evolution of cytokinin biosynthetic pathways.     

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.