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.     

Cytokinins induce sporulation in Dictyostelium

The social amoeba Dictyostelium discoideum diverged from the line leading to animals shortly after the separation of plants and animals but it retained characteristics of both kingdoms. A GABA(B)-like receptor and a peptide, SDF-2, with homologs found only in animals, control sporulation, while cytokinins, which act as hormones in plants, keep spores dormant. When SDF-2 binds its receptor DhkA, it reduces the activity of the cAMP phosphodiesterase RegA such that cAMP levels can increase. It has been proposed that the cytokinin discadenine also produces in an increase in cAMP but acts through a different histidine kinase, DhkB. We have found that discadenine and its precursor, isopentenyl adenine, not only maintain spore dormancy but also initiate rapid encapsulation independently of the SDF-2 signal transduction pathway. DhkB and the adenylyl cyclase of late development, AcrA, are members of two component signal transduction families and both are required to transduce the cytokinin signal. As expected, strains lacking the isopentenyl-transferase enzyme chiefly responsible for cytokinin synthesis are defective in sporulation. It appears that SDF-2 and cytokinins are secreted during late development to trigger signal transduction pathways that lead to an increase in the activity of the camp-dependent protein kinase, PKA, which triggers rapid encapsulation as well as ensuring spore dormancy.     

Cytokinins: Biosynthesis metabolism and perception

Summary Cytokinins are essential hormones for plant growth and development. They are also of vital importance for in vitro manipulations of plant cells and tissues. The biological activities and chemistry of cytokinins are well defined but very little is known about their mode of action and it is only recently that cytokinin genes have been identified in plants. This review summarizes the current status of knowledge on cytokinin biosynthesis, metabolism and signal transduction, with an emphasis on genes encoding metabolic enzymes and putative receptors, and genes rapidly induced by cytokinins.     

Moss biology and phytohormones--cytokinins in Physcomitrella

Mosses present several advantages for the analysis of phytohormone physiology. Their enormous regeneration capacity, the possibility of controlling their whole life cycle under in vitro culture conditions, as well as the small number of cell types facilitate studies of hormone homeostasis. This review focuses on the metabolism and biosynthesis of cytokinins, mostly summarising data obtained using the moss Physcomitrella patens (Hedw.) B.S.G. which has served as a model system for cytokinin research for many years. A comparison of metabolic differences with respect to seed plants is presented, pointing out an important role of adenosine kinase for the formation of nucleotides during cytokinin interconversion in Physcomitrella. Results on cytokinin biosynthesis in Physcomitrella are summarised with respect to the OVE mutants, which can be considered unique in the plant kingdom due to their strong overproduction of cytokinins. The OVE phenotype is correlated with both increased activity in early stages of cytokinin biosynthesis as well as increased conversion of cytokinin riboside to the base. Cytokinin interconverting reactions can contribute to the increased levels of cytokinins in OVE mutants. Further studies on hormone physiology in moss will help to complete our understanding of hormonal homeostasis by elucidating the situation in an evolutionary early embryophyte.     

The effect of plant cytokinin hormones on the production of ethylene, nitric oxide, and protein nitrotyrosine in ageing tobacco leaves

Transgenic plants with genetically increased or decreased levels of cytokinins were used to investigate the effect of cytokinin level on the production of ethylene, a plant hormone with suggested role in senescence, and the production of nitric oxide, potentially important signalling and regulatory molecule. The production of these gases was followed during the course of leaf development and senescence. The production of ethylene and nitric oxide is under genetic control of genes other than those involved in regulation of senescence. The difference in basic ethylene and NO levels in different tobacco cultivars was higher than their changes in senescence. The results of this study did not indicate a direct link between ethylene production and cytokinin levels. However, there was a decreased production of NO in senescent leaves. Low cytokinins level was associated with increased NO production during leaf development. Protein nitrotyrosine proved to be a better indicator of the reactive nitrogen species than measuring of the NO production. Higher nitrotyrosine concentrations were found in insoluble proteins than in the soluble ones, pointing to membrane proteins as the primary targets of the reactive nitrogen species. In plants with elevated cytokinin levels the content of nitrated proteins decreased both in soluble and insoluble fractions. This finding indicates an antioxidative function of cytokinins against reactive nitrogen species.     

Arabidopsis SOI33/AtENT8 Gene Encodes a Putative Equilibrative Nucleoside Transporter That Is Involved in Cytokinin Transport In Planta

The plant phytohormone cytokinin plays an important role in many facets of plant growth and development by regulating cell division and differentiation. Recent studies have shed significant light into the mechanisms of cytokinin metabolism and signaling. However, little is known about how the hormone is transported in planta, although it has been proposed that the hormone is presumably transported in nucleoside-conjugated forms. Here, we report the identification and characterization of cytokinin transport ers in Arabidopsis. We previously reported that a gain-of-function mutation in the PGA22/AtIPT8 gene caused overproduction of cytokinins in planta. In an effort to screen for suppressor of pga22/atipt8 (soi) mutants, we identified a mutant soi33-1. Molecular and genetic analyses indicated that SOI33 encodes a putative equilibrative nucleoside transporter (ENT), previously designated as AtENT8. Members of this small gene family are presumed to be involved in the transport of nucleosides in eukaryotic cells. Under conditions of nitrogen starvation, loss-of-function mutations in SOI33/AtENT8 or in a related gene AtENT3 cause a reduced sensitivity to the nucleoside-type cytokinins isopentenyladenine riboside (iPR) and trans zeatin riboside (tZR), but display a normal response to the free base-type cytokinins isopentenyladenine (iP) and trans-zeatin (tZ). Conversely, overexpression of SOI33/AtENT8 renders transgenic plants hyper sensitive to iPR but not to iP. An in planta measurement experiment indicated that uptake efficiency of 3H labeled iPR was reduced more than 40% in soi33 and atent3 mutants. However, a mutation inAtENT1 had no substantial effect on the cytokinin response and iPR uptake efficiency. Our results suggest that SOI33/ AtENT8 and AtENT3 are involved in the transport of nucleoside-type cytokinins in Arabidopsis.     

Arabidopsis SOI33／AtENT8 Gene Encodes a Putative Equilibrative Nucleoside Transporter That Is Involved in Cytokinin Transport In Planta

The plant phytohormone cytokinin plays an important role in many facets of plant growth and development by regulating cell division and differentiation. Recent studies have shed significant light into the mechanisms of cytokinin metabolism and signaling. However, little is known about how the hormone is transported in planta, although it has been proposed that the hormone is presumably transported in nucleoside-conjugated forms. Here, we report the identification and characterization of cytokinin transporters in Arabidopsis. We previously reported that a gain-of-function mutation in the PGA22/AtlPT8 gene caused overproduction of cytokinins in planta. In an effort to screen for suppressor of pga22/atipt8 (soi) mutants, we identified a mutant soi33-1. Molecular and genetic analyses indicated that S0133 encodes a putative equilibrative nucleoside transporter (ENT), previously designated as AtENT8. Members of this small gene family are presumed to be involved in the transport of nucleosides in eukaryodc cells. Under conditions of nitrogen starvation, loss-of-function mutations in SOI33/AtENT8 or in a related gene AtENT3 cause a reduced sensitivity to the nucleoside-type cytokinins isopentenyladenine riboside (iPR) and transzeatin riboside (tZR), but display a normal response to the free base-type cytokinins isopentenyladenine (iP) and trans-zeatin (tZ). Conversely, overexpression of SOI33/AtENT8 renders transgenic plants hypersensitive to iPR but not to iP. An in planta measurement experiment indicated that uptake efficiency of^3Hlabeled iPR was reduced more than 40% in soi33 and atent3 mutants. However, a mutation in AtENT1 had no substantial effect on the cytokinin response and iPR uptake efficiency. Our results suggest that SOI33/AtENT8 and AtENT3 are involved in the transport of nucleoside-type cytokinins in Arabidopsis.     

Evolution of cytokinin biosynthesis and degradation

Cytokinin hormones are important regulators of development and environmental responses of plants that execute their action via the molecular machinery of signal perception and transduction. The limiting step of the whole process is the availability of the hormone in suitable concentrations in the right place and at the right time to interact with the specific receptor. Hence, the hormone concentrations in individual tissues, cells, and organelles must be properly maintained by biosynthetic and metabolic enzymes. Although there are merely two active cytokinins, isopentenyladenine and its hydroxylated derivative zeatin, a variety of conjugates they may form and the number of enzymes/isozymes with varying substrate specificity involved in their biosynthesis and conversion gives the plant a variety of tools for fine tuning of the hormone level. Recent genome-wide studies revealed the existence of the respective coding genes and gene families in plants and in some bacteria. This review summarizes present knowledge on the enzymes that synthesize cytokinins, form cytokinin conjugates, and carry out irreversible elimination of the hormones, including their phylogenetic analysis and possible variations in different organisms.     

Cytokinins and plant immunity: old foes or new friends?

Cytokinins are plant growth promoting hormones involved in the specification of embryonic cells, maintenance of meristematic cells, shoot formation and development of vasculature. Cytokinins have also emerged as a major factor in plant-microbe interactions during nodule organogenesis and pathogenesis. Microbe-originated cytokinins confer abnormal hypersensitivity of cytokinins to plants, augmenting the sink activity of infected regions. However, recent findings have shed light on a distinct role of cytokinins in plant immune responses. Plant-borne cytokinins systemically induce resistance against pathogen infection. This resistance is orchestrated by endogenous cytokinin and salicylic acid signaling. Here, we discuss how plant- and pathogen-derived cytokinins inversely affect the plant defense response. In addition, we consider the molecular mechanisms underlying plant-derived cytokinin action in plant immunity.     

Cytokinins inhibit epiphyllous plantlet development on leaves of Bryophyllum (Kalanchoë) marnierianum

When leaves of Bryophyllum marnierianum are detached from the plant, plantlets develop from primordia located at their margins. Leaves excised with a piece of stem attached do not produce plantlets. Severing the major leaf veins overcomes the inhibitory effect of the attached stem, indicating that the control agent is transmitted through the vascular system. A possible mechanism is that an inhibitory substance, possibly a known plant hormone, transported from the stem to the leaf, suppresses plantlet development. A number of hormones were tested for their ability to inhibit plantlet primordium development in whole isolated leaves. Auxins had no effect, indicating that apical dominance is not involved. The cytokinins zeatin, kinetin, and benzylaminopurine (BAP) strongly inhibited plantlet development, suggesting that they may be the or a factor involved in maintenance of plantlet primordium dormancy when the leaf is attached to the plant. This hypothesis was strongly supported by the finding that treatment of leaves attached to stems with a cytokinin antagonist (purine riboside) released the primordia from inhibition. In contrast to whole leaves, plantlet primordium development on leaf explants incubated on Murashige Skoog medium containing 3% sucrose was strongly stimulated by cytokinins. A possible explanation of these observations is that in whole leaves the cytokinin signal is transduced into an inhibitory signal whereas in the isolated primordium cytokinin has a direct stimulatory effect. The inhibitory cytokinin pathway must be dominant as long as the leaf is attached to the plant. A model is proposed which could explain these findings. This study points to a novel role of cytokinins in the maintenance of foliar plantlet primordium dormancy.     

Characterization of cytokinin and adenine transport in Arabidopsis cell cultures

Cytokinins are distributed through the vascular system and trigger responses of target cells via receptor-mediated signal transduction. Perception and transduction of the signal can occur at the plasma membrane or in the cytosol. The signal is terminated by the action of extra- or intracellular cytokinin oxidases. While radiotracer studies have been used to study transport and metabolism of cytokinins in plants, little is known about the kinetic properties of cytokinin transport. To provide a reference dataset, radiolabeled trans-zeatin (tZ) was used for uptake studies in Arabidopsis (Arabidopsis thaliana) cell culture. Uptake kinetics of tZ are multiphasic, indicating the presence of both low- and high-affinity transport systems. The protonophore carbonyl cyanide m-chlorophenylhydrazone is an effective inhibitor of cytokinin uptake, consistent with H(+)-mediated uptake. Other physiological cytokinins, such as isopentenyl adenine and benzylaminopurine, are effective competitors of tZ uptake, whereas allantoin has no inhibitory effect. Adenine competes for zeatin uptake, indicating that the degradation product of cytokinin oxidases is transported by the same systems. Comparison of adenine and tZ uptake in Arabidopsis seedlings reveals similar uptake kinetics. Kinetic properties, as well as substrate specificity determined in cell cultures, are compatible with the hypothesis that members of the plant-specific purine permease family play a role in adenine transport for scavenging extracellular adenine and may, in addition, be involved in low-affinity cytokinin uptake.     

CREam of cytokinin signalling: receptor identified.

Cytokinins play a central role in the regulation of plant cell division and numerous developmental processes. Pleiotropic effects have made studies of this hormone difficult, and cytokinin signalling pathways have long remained elusive. The recent identification of CRE1 (a histidine kinase identical to AHK4 and WOL) as the cytokinin receptor of Arabidopsis thaliana is a landmark in cytokinin research. Mutations have been identified in CRE1, and the phenotype of loss-of-function mutations sheds new light on the role of cytokinins in plant development. This article describes the experimental paths leading to receptor identification and the current interpretation of its function.     

Effects of Root Cutting on Cytokinin Content in the Shoot Apex Cells of Arabidopsis Plants

The dependence of cytokinin accumulation in the shoot apexes of Arabidopsis plants on the delivery of these hormones from the roots was studied. For the estimation of cytokinin content in the cells, the immunohistochemical localization method using antibodies against zeatin riboside was used. Differential conjugation of free cytokinin bases and their ribosides was used to prevent washing of cytokinins during the dehydration process. Root cutting decreased the immunostaining of zeatin in the cells of the shoot apical meristem, thereby supporting the hypothesis about dependence of cytokinin accumulation in these cells on the hormone delivery from the roots. The level of cytokinins in the cells of the shoot apex decreased under the effect of protonophore, indicating the important role of the secondary-active transmembrane transport process of cytokinins in the maintenance of their level in the cells of the shoot apex.     

Cytokinins in plant senescence: From spray and pray to clone and play

Three approaches have been used to investigate the inhibitory role of the cytokinin class of phytohormones in plant senescence: external application of cytokinins, measurement of endogenous cytokinin levels before and during senescence, and manipulation of endogenous cytokinin production in transgenic plants. In transgenic plant studies, endogenous cytokinin levels are manipulated by expression of IPT, a gene encoding isopentenyl transferase. Transgenic plants expressing IPT from a variety of promoters exhibit developmental and morphological alterations and often display retarded leaf senescence. A recently developed autoregulatory senescence-inhibition system targets cytokinin production quantitatively, spatially and temporally, and results in transgenic plants that exhibit significantly delayed senescence without abnormalities. These transgenic studies not only confirm the regulatory role of cytokinins in plant senescence, but also provide a way to manipulate senescence for potential agricultural applications.