Auxin-cytokinin interactions in higher plants: old problems and new tools

The plant hormones auxin and cytokinin interact in a complex manner to control many aspects of growth and differentiation. Recent advances in understanding their metabolism and the cell cycle are now helping to clarify the mechanisms by which these hormones act together to control various physiological and developmental responses. Molecular and genetic tools are being used to reveal interactions between auxin and cytokinin at multiple levels, including mutual regulation of active hormone availability, input into multiple signal transduction pathways, alteration of gene expression, post-translational modifications and direct modulation of enzyme activity.     

Genetic approach towards the identification of auxin-cytokinin crosstalk components involved in root development

Phytohormones are important plant growth regulators that control many developmental processes, such as cell division, cell differentiation, organogenesis and morphogenesis. They regulate a multitude of apparently unrelated physiological processes, often with overlapping roles, and they mutually modulate their effects. These features imply important synergistic and antagonistic interactions between the various plant hormones. Auxin and cytokinin are central hormones involved in the regulation of plant growth and development, including processes determining root architecture, such as root pole establishment during early embryogenesis, root meristem maintenance and lateral root organogenesis. Thus, to control root development both pathways put special demands on the mechanisms that balance their activities and mediate their interactions. Here, we summarize recent knowledge on the role of auxin and cytokinin in the regulation of root architecture with special focus on lateral root organogenesis, discuss the latest findings on the molecular mechanisms of their interactions, and present forward genetic screen as a tool to identify novel molecular components of the auxin and cytokinin crosstalk.     

Genetic approach towards the identification of auxin–cytokinin crosstalk components involved in root development

Phytohormones are important plant growth regulators that control many developmental processes, such as cell division, cell differentiation, organogenesis and morphogenesis. They regulate a multitude of apparently unrelated physiological processes, often with overlapping roles, and they mutually modulate their effects. These features imply important synergistic and antagonistic interactions between the various plant hormones. Auxin and cytokinin are central hormones involved in the regulation of plant growth and development, including processes determining root architecture, such as root pole establishment during early embryogenesis, root meristem maintenance and lateral root organogenesis. Thus, to control root development both pathways put special demands on the mechanisms that balance their activities and mediate their interactions. Here, we summarize recent knowledge on the role of auxin and cytokinin in the regulation of root architecture with special focus on lateral root organogenesis, discuss the latest findings on the molecular mechanisms of their interactions, and present forward genetic screen as a tool to identify novel molecular components of the auxin and cytokinin crosstalk.     

Auxin and cytokinin regulate each other's levels via a metabolic feedback loop

The hormones auxin and cytokinin are key regulators of plant growth and development. As they are active at minute concentrations and regulate dynamic processes, cell and tissue levels of the hormones are finely controlled developmentally, diurnally, and in response to environmental variables. This fine control, along with a regulation of the capacity to respond ensures that the appropriate type, duration and intensity of responses are elicited. We have recently discovered that cytokinin and auxin regulate the synthesis of each other, demonstrating a mechanism for mutual feed back and feed forward control of auxin and cytokinin levels. This regulatory loop could be important for many developmental processes in plants, i.e., in fine-tuning plant hormone levels in the developing meristems of the root and shoot apex. These findings could also give a molecular explanation for earlier observations of auxin and cytokinin effects on cell cultures,¹ where specific auxin and cytokinin ratios have been used to trigger different morphological events.Key words: auxin, cytokinin, biosynthesis, metabolism, signaling, root development, interactions     

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.     

The influence of cytokinin-auxin cross-regulation on cell-fate determination in Arabidopsis thaliana root development

Root growth and development in Arabidopsis thaliana are sustained by a specialised zone termed the meristem, which contains a population of dividing and differentiating cells that are functionally analogous to a stem cell niche in animals. The hormones auxin and cytokinin control meristem size antagonistically. Local accumulation of auxin promotes cell division and the initiation of a lateral root primordium. By contrast, high cytokinin concentrations disrupt the regular pattern of divisions that characterises lateral root development, and promote differentiation. The way in which the hormones interact is controlled by a genetic regulatory network. In this paper, we propose a deterministic mathematical model to describe this network and present model simulations that reproduce the experimentally observed effects of cytokinin on the expression of auxin regulated genes. We show how auxin response genes and auxin efflux transporters may be affected by the presence of cytokinin. We also analyse and compare the responses of the hormones auxin and cytokinin to changes in their supply with the responses obtained by genetic mutations of SHY2, which encodes a protein that plays a key role in balancing cytokinin and auxin regulation of meristem size. We show that although shy2 mutations can qualitatively reproduce the effect of varying auxin and cytokinin supply on their response genes, some elements of the network respond differently to changes in hormonal supply and to genetic mutations, implying a different, general response of the network. We conclude that an analysis based on the ratio between these two hormones may be misleading and that a mathematical model can serve as a useful tool for stimulate further experimental work by predicting the response of the network to changes in hormone levels and to other genetic mutations.     

The Diageotropica Gene Differentially Affects Auxin and Cytokinin Responses throughout Development in Tomato

The interactions between the plant hormones auxin and cytokinin throughout plant development are complex, and genetic investigations of the interdependency of auxin and cytokinin signaling have been limited. We have characterized the cytokinin sensitivity of the auxin-resistant diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) in a range of auxin- and cytokinin-regulated responses. Intact, etiolated dgt seedlings showed cross-resistance to cytokinin with respect to root elongation, but cytokinin effects on hypocotyl growth and ethylene synthesis in these seedlings were not impaired by the dgt mutation. Seven-week-old, green wild-type and dgt plants were also equally sensitive to cytokinin with respect to shoot growth and hypocotyl and internode elongation. The effects of cytokinin and the dgt mutation on these processes appeared additive. In tissue culture organ regeneration from dgt hypocotyl explants showed reduced sensitivity to auxin but normal sensitivity to cytokinin, and the effects of cytokinin and the mutation were again additive. However, although callus induction from dgt hypocotyl explants required auxin and cytokinin, dgt calli did not show the typical concentration-dependent stimulation of growth by either auxin or cytokinin observed in wild-type calli. Cross-resistance of the dgt mutant to cytokinin thus was found to be limited to a small subset of auxin- and cytokinin-regulated growth processes affected by the dgt mutation, indicating that auxin and cytokinin regulate plant growth through both shared and separate signaling pathways.     

Cytokinin inhibits a subset of diageotropica-dependent primary auxin responses in tomato

Many aspects of plant development are regulated by antagonistic interactions between the plant hormones auxin and cytokinin, but the molecular mechanisms of this interaction are not understood. To test whether cytokinin controls plant development through inhibiting an early step in the auxin response pathway, we compared the effects of cytokinin with those of the dgt (diageotropica) mutation, which is known to block rapid auxin reactions of tomato (Lycopersicon esculentum) hypocotyls. Long-term cytokinin treatment of wild-type seedlings phenocopied morphological traits of dgt plants such as stunting of root and shoot growth, reduced elongation of internodes, reduced apical dominance, and reduced leaf size and complexity. Cytokinin treatment also inhibited rapid auxin responses in hypocotyl segments: auxin-stimulated elongation, H(+) secretion, and ethylene synthesis were all inhibited by cytokinin in wild-type hypocotyl segments, and thus mimicked the impaired auxin responsiveness found in dgt hypocotyls. However, cytokinin failed to inhibit auxin-induced LeSAUR gene expression, an auxin response that is affected by the dgt mutation. In addition, cytokinin treatment inhibited the auxin induction of only one of two 1-aminocyclopropane-1-carboxylic acid synthase genes that exhibited impaired auxin inducibility in dgt hypocotyls. Thus, cytokinin inhibited a subset of the auxin responses impaired in dgt hypocotyls, suggesting that cytokinin blocks at least one branch of the DGT-dependent auxin response pathway.     

The role of local biosynthesis of auxin and cytokinin in plant development

Plant hormones are tightly regulated in response to environmental and developmental signals. It has long been speculated that biosynthesis of hormones occurs broadly in plant organs and that intricate, spatiotemporal regulation of hormones in developing organ primordia is achieved through transport and signal perception. However, recent identification of genes crucial for biosynthesis of auxin and cytokinin reveals that localized hormone biosynthesis also plays an important role in organ growth and patterning.     

Analysis of the protein expression profiling during rice callus differentiation under different plant hormone conditions

Plant hormones function to coordinate plant growth and development. While the plant hormones, mainly auxin and cytokinin, are exogenously added to various plant tissue cultures, their effects on the organogenesis are apparent, but little is known concerning the molecular mechanisms by which they function in cultured cells. Rice, as a model plant in monocots, is also suitable to tissue culture studies. Here, we used four types of regeneration mediums with different relative concentrations of cytokinin and auxin for rice callus differentiation, the calli at different differentiation stages were collected for proteomic analysis. 2-dimensional electrophoresis revealed that 213 protein spots significantly differentially expressed during callus differentiation under different hormone conditions. By using mass spectrometry, 183 differentially expressed protein spots were identified to match 157 unique proteins. Most of these differential proteins were cellular/metabolic process-related proteins, whose different expression patterns may be correlated with the cytokinin and auxin regulation. Several hormone-related proteins were prominently featured in differentiated calli as compared with the initiated calli, such as alpha-amylase isoforms, mannose-binding rice lectin, putative dehydration stress-induced protein, cysteine endopeptidase and cystatin. All these results provide a novel insight into how the two plant hormones effect the callus differentiation in rice on the proteomic level.     

Transgenic studies on the involvement of cytokinin and gibberellin in male development

Numerous plant hormones interact during plant growth and development. Elucidating the role of these various hormones on particular tissue types or developmental stages has been difficult with exogenous applications or constitutive expression studies. Therefore, we used tissue-specific promoters expressing CKX1 and gai, genes involved in oxidative cytokinin degradation and gibberellin (GA) signal transduction, respectively, to study the roles of cytokinin and GA in male organ development. Accumulation of CKX1 in reproductive tissues of transgenic maize (Zea mays) resulted in male-sterile plants. The male development of these plants was restored by applications of kinetin and thidiazuron. Similarly, expression of gai specifically in anthers and pollen of tobacco (Nicotiana tabacum) and Arabidopsis resulted in the abortion of these respective tissues. The gai-induced male-sterile phenotype exhibited by the transgenic plants was reversible by exogenous applications of kinetin. Our results provide molecular evidence of the involvement of cytokinin and GA in male development and support the hypothesis that the male development is controlled in concert by multiple hormones. These studies also suggest a potential method for generating maintainable male sterility in plants by using existing agrochemicals that would reduce the expense of seed production for existing hybrid crops and provide a method to produce hybrid varieties of traditionally non-hybrid crops.     

Hormone interactions during lateral root formation

Lateral root (LR) formation, the production of new roots from parent roots, is a hormone- and environmentally-regulated developmental process in higher plants. Physiological and genetic studies using Arabidopsis thaliana and other plant species have revealed the roles of several plant hormones in LR formation, particularly the role of auxin in LR initiation and primordium development, resulting in much progress toward understanding the mechanisms of auxin-mediated LR formation. However, hormone interactions during LR formation have been relatively underexamined. Recent studies have shown that the plant hormones, cytokinin and abscisic acid negatively regulate LR formation whereas brassinosteroids positively regulate LR formation. On the other hand, ethylene has positive and negative roles during LR formation. This review summarizes recent findings on hormone-regulated LR formation in higher plants, focusing on auxin as a trigger and on the other hormones in LR formation, and discusses the possible interactions among plant hormones in this developmental process.     

The hormonal regulation of axillary bud growth in Arabidopsis

Apically derived auxin has long been known to inhibit lateral bud growth, but since it appears not to enter the bud, it has been proposed that its inhibitory effect is mediated by a second messenger. Candidates include the plant hormones ethylene, cytokinin and abscisic acid. We have developed a new assay to study this phenomenon using the model plant Arabidopsis. The assay allows study of the effects of both apical and basal hormone applications on the growth of buds on excised nodal sections. We have shown that apical auxin can inhibit the growth of small buds, but larger buds were found to have lost competence to respond. We have used the assay with nodes from wild-type and hormone-signalling mutants to test the role of ethylene, cytokinin and abscisic acid in bud inhibition by apical auxin. Our data eliminate ethylene as a second messenger for auxin-mediated bud inhibition. Similarly, abscisic acid signalling is not to be required for auxin action, although basally applied abscisic can enhance inhibition by apical auxin and apically applied abscisic acid can reduce it. By contrast, basally applied cytokinin was found to release lateral buds from inhibition by apical auxin, while apically applied cytokinin dramatically increased the duration of inhibition. These results are consistent with cytokinin acting independently to regulate bud growth, rather than as a second messenger for auxin. However, in the absence of cytokinin-signalling mutants, a role for cytokinin as a second messenger for auxin cannot be ruled out.     

The morphological and physiological properties of the genetic tumours from a Nicotiana interspecific hybrid

Many plant tumours have been loosely referred to as genetic tumours when no obvious external cause was identifiable. In this study, the morphology of genetic tumours was detected by scanning electron microscopy (SEM) and the influence of exogenous phytohormones on genetic tumour growth was examined. Genetic tumours in Nicotiana hybrid were first observed in the hypocotyl area and later appeared from stems as a cluster complex. In vitro, genetic tumours were produced 5-6 days after explant treatment near each of the cut ends. The morphology of genetic tumours was investigated using SEM. Genetic tumours are mainly composed of epidermal cells surrounded by a well developed extracellular matrix (ECM), that is organized in a network linking surface cells. Globular structures containing several cells were formed from these clusters of epidermal cells. Vigorous tumourous growth from the leaf discs of the Nicotiana hybrid was obtained on medium supplemented with auxin and cytokinin at 0 to 10 muM, but 100 muM of auxin and cytokinin inhibited genetic tumour growth. Leaf discs treated with 10 muM cytokinin or auxin alone produced genetic tumours, but 10 muM cytokinin supplemented with 100 muM auxin stopped growth. Therefore genetic tumours are tissues that are, clearly, disturbed in endogenous hormone balance or have altered sensitivity to hormones and response to exogenous hormones.     

Role of plant hormones in plant defence responses

Plant hormones play important roles in regulating developmental processes and signaling networks involved in plant responses to a wide range of biotic and abiotic stresses. Significant progress has been made in identifying the key components and understanding the role of salicylic acid (SA), jasmonates (JA) and ethylene (ET) in plant responses to biotic stresses. Recent studies indicate that other hormones such as abscisic acid (ABA), auxin, gibberellic acid (GA), cytokinin (CK), brassinosteroids (BR) and peptide hormones are also implicated in plant defence signaling pathways but their role in plant defence is less well studied. Here, we review recent advances made in understanding the role of these hormones in modulating plant defence responses against various diseases and pests.