Cytokinin: From autoclaved DNA to two-component signaling.

Since its first identification in the 1950s as a regulator of cell division, cytokinin has been linked to many physiological processes in plants, spanning growth and development and various responses to the environment. Studies from the last two and one-half decades have revealed the pathways underlying the biosynthesis and metabolism of cytokinin and have elucidated the mechanisms of its perception and signaling, which reflects an ancient signaling system evolved from two-component elements in bacteria. Mutants in the genes encoding elements involved in these processes have helped refine our understanding of cytokinin functions in plants. Further, recent advances have provided insight into the mechanisms of intracellular and long-distance cytokinin transport and the identification of several proteins that operate downstream of cytokinin signaling. Here, we review these processes through a historical lens, providing an overview of cytokinin metabolism, transport, signaling, and functions in higher plants.

More than growth: Phytohormone-regulated transcription factors controlling plant immunity, plant development and plant architecture.

Activation of immunity by exogenous signals or mutations leading to autoimmunity has long been associated with decreased plant growth, known as the growth-defense tradeoff. Originally thought to be a redirection of metabolic resources towards defense and away from growth, recent studies have demonstrated that growth and defense can be uncoupled, indicating that metabolic regulation is not solely responsible for the growth-defense tradeoff. Immunity activation has effects on plant development beyond the reduction of plant biomass, including changes in plant architecture. Phytohormone signaling pathways, and crosstalk between these pathways, are responsible for regulating plant growth and development, and plant defense responses. Here we review the hormonal regulation of transcription factors that play roles in both defense and development, with a focus on their effects on plant architecture, and suggest the targeting of these transcription factors to increase plant immunity and change plant growth and form for enhancement of agronomical traits.

Phytohormone Profile of Medicago in Response to Mycorrhizal Fungi, Aphids, and Gibberellic Acid.

Although gibberellic acid (GA) is widely used in agriculture, it is unclear whether exogenous GA makes aphid-infested, mycorrhizal plants more susceptible to herbivory. This study investigates the role of GA in modulating defenses in barrel medic plants (Medicago truncatula) that are infested with pea aphids (Acyrthosiphon pisum) and colonized by the beneficial symbiont Rhizophagus intraradices. Mock- and R. intraradices-inoculated potted plants were grown in a topsoil: sand mix for 42 days and were treated with GA or solvent. Subsequently, plants were exposed to herbivory or no aphid herbivory for 36 h and 7 days. Afterwards, plant growth parameters, aphid fitness, and foliar phytohormone concentrations were measured. The results revealed that GA regulates plant defenses during arbuscular mycorrhizal (AM) fungus-plant-aphid interactions as aphids that fed for 7 days on mycorrhizal, GA-untreated plants weighed more than those that fed on mycorrhizal, GA-treated plants. No major differences were detected in phytohormone levels at 36 h. Overall, mycorrhizal plants showed more shoot biomass compared to non-mycorrhizal controls. The arbuscule density and fungal biomass of R. intraradices were not altered by exogenous GA and aphid herbivory based on molecular markers. This study indicates that exogenous GA may help reduce aphid fitness when feeding on mycorrhizal plants.

Cytokinin Regulation of Source-Sink Relationships in Plant-Pathogen Interactions.

Cytokinins are plant hormones known for their role in mediating plant growth. First discovered for their ability to promote cell division, this class of hormones is now associated with many other cellular and physiological functions. One of these functions is the regulation of source-sink relationships, a tightly controlled process that is essential for proper plant growth and development. As discovered more recently, cytokinins are also important for the interaction of plants with pathogens, beneficial microbes and insects. Here, we review the importance of cytokinins in source-sink relationships in plants, with relation to both carbohydrates and amino acids, and highlight a possible function for this regulation in the context of plant biotic interactions.

A genome-wide association study reveals cytokinin as a major component in the root defense responses against Ralstonia solanacearum.

Bacterial wilt caused by the soil-borne pathogen Ralstonia solancearum is economically devastating, with no effective methods to fight the disease. This pathogen invades plants through their roots and colonizes their xylem, clogging the vasculature and causing rapid wilting. Key to preventing colonization are the early defense responses triggered in the host's root upon infection, which remain mostly unknown. Here, we have taken advantage of a high-throughput in vitro infection system to screen natural variability associated with the root growth inhibition phenotype caused by R. solanacearum in Arabidopsis during the first hours of infection. To analyze the genetic determinants of this trait, we have performed a genome-wide association study, identifying allelic variation at several loci related to cytokinin metabolism, including genes responsible for biosynthesis and degradation of cytokinin. Further, our data clearly demonstrate that cytokinin signaling is induced early during the infection process and cytokinin contributes to immunity against R. solanacearum. This study highlights a new role for cytokinin in root immunity, paving the way for future research that will help in understanding the mechanisms underpinning root defenses.

Directions for research and training in plant omics: Big Questions and Big Data.

A key remit of the NSF-funded "Arabidopsis Research and Training for the 21st Century" (ART-21) Research Coordination Network has been to convene a series of workshops with community members to explore issues concerning research and training in plant biology, including the role that research using Arabidopsis thaliana can play in addressing those issues. A first workshop focused on training needs for bioinformatic and computational approaches in plant biology was held in 2016, and recommendations from that workshop have been published (Friesner et al., Plant Physiology, 175, 2017, 1499). In this white paper, we provide a summary of the discussions and insights arising from the second ART-21 workshop. The second workshop focused on experimental aspects of omics data acquisition and analysis and involved a broad spectrum of participants from academics and industry, ranging from graduate students through post-doctorates, early career and established investigators. Our hope is that this article will inspire beginning and established scientists, corporations, and funding agencies to pursue directions in research and training identified by this workshop, capitalizing on the reference species Arabidopsis thaliana and other valuable plant systems.

RNA-Seq analysis reveals insight into enhanced rice Xa7-mediated bacterial blight resistance at high temperature.

Plant disease is a major challenge to agriculture worldwide, and it is exacerbated by abiotic environmental factors. During some plant-pathogen interactions, heat stress allows pathogens to overcome host resistance, a phenomenon which could severely impact crop productivity considering the global warming trends associated with climate change. Despite the importance of this phenomenon, little is known about the underlying molecular mechanisms. To better understand host plant responses during simultaneous heat and pathogen stress, we conducted a transcriptomics experiment for rice plants (cultivar IRBB61) containing Xa7, a bacterial blight disease resistance (R) gene, that were infected with Xanthomonas oryzae, the bacterial blight pathogen of rice, during high temperature stress. Xa7-mediated resistance is unusual relative to resistance mediated by other R genes in that it functions better at high temperatures. Using RNA-Seq technology, we identified 8,499 differentially expressed genes as temperature responsive in rice cultivar IRBB61 experiencing susceptible and resistant interactions across three time points. Notably, genes in the plant hormone abscisic acid biosynthesis and response pathways were up-regulated by high temperature in both mock-treated plants and plants experiencing a susceptible interaction and were suppressed by high temperature in plants exhibiting Xa7-mediated resistance. Genes responsive to salicylic acid, an important plant hormone for disease resistance, were down-regulated by high temperature during both the susceptible and resistant interactions, suggesting that enhanced Xa7-mediated resistance at high temperature is not dependent on salicylic acid signaling. A DNA sequence motif similar to known abscisic acid-responsive cis-regulatory elements was identified in the promoter region upstream of genes up-regulated in susceptible but down-regulated in resistant interactions. The results of our study suggest that the plant hormone abscisic acid is an important node for cross-talk between plant transcriptional response pathways to high temperature stress and pathogen attack. Genes in this pathway represent an important focus for future study to determine how plants evolved to deal with simultaneous abiotic and biotic stresses.

Towards engineering of hormonal crosstalk in plant immunity.

Plant hormones regulate physiological responses in plants, including responses to pathogens and beneficial microbes. The last decades have provided a vast amount of evidence about the contribution of different plant hormones to plant immunity, and also of how they cooperate to orchestrate immunity activation, in a process known as hormone crosstalk. In this review we highlight the complexity of hormonal crosstalk in immunity and approaches currently being used to further understand this process, as well as perspectives to engineer hormone crosstalk for enhanced pathogen resistance and overall plant fitness.

Evolution of Hormone Signaling Networks in Plant Defense.

Studies with model plants such as Arabidopsis thaliana have revealed that phytohormones are central regulators of plant defense. The intricate network of phytohormone signaling pathways enables plants to activate appropriate and effective defense responses against pathogens as well as to balance defense and growth. The timing of the evolution of most phytohormone signaling pathways seems to coincide with the colonization of land, a likely requirement for plant adaptations to the more variable terrestrial environments, which included the presence of pathogens. In this review, we explore the evolution of defense hormone signaling networks by combining the model plant-based knowledge about molecular components mediating phytohormone signaling and cross talk with available genome information of other plant species. We highlight conserved hubs in hormone cross talk and discuss evolutionary advantages of defense hormone cross talk. Finally, we examine possibilities of engineering hormone cross talk for improvement of plant fitness and crop production.

Communication in the Phytobiome.

The phytobiome is composed of plants, their environment, and diverse interacting microscopic and macroscopic organisms, which together influence plant health and productivity. These organisms form complex networks that are established and regulated through nutrient cycling, competition, antagonism, and chemical communication mediated by a diverse array of signaling molecules. Integration of knowledge of signaling mechanisms with that of phytobiome members and their networks will lead to a new understanding of the fate and significance of these signals at the ecosystem level. Such an understanding could lead to new biological, chemical, and breeding strategies to improve crop health and productivity.

Assessment of Cytokinin-Induced Immunity Through Quantification of Hyaloperonospora arabidopsidis Infection in Arabidopsis thaliana.

Cytokinins have been shown to regulate plant immunity. Application of high levels of cytokinin to plants leads to decreased susceptibility to pathogens. In this chapter, we describe a fast and accurate protocol for assessment of cytokinin-induced immunity in Arabidopsis plants against an oomycete plant pathogen.

Should I fight or should I grow now? The role of cytokinins in plant growth and immunity and in the growth-defence trade-off.

BACKGROUND: Perception and activation of plant immunity require a remarkable level of signalling plasticity and control. In Arabidopsis and other plant species, constitutive defence activation leads to resistance to a broad spectrum of biotrophic pathogens, but also frequently to stunted growth and reduced seed set. Plant hormones are important integrators of the physiological responses that influence the outcome of plant-pathogen interactions. SCOPE: We review the mechanisms by which the plant hormone cytokinin regulates both plant growth and response to pathogens, and how cytokinins may connect these two processes, ultimately affecting the growth trade-offs observed in plant immunity.

Pathogen Tactics to Manipulate Plant Cell Death.

Cell death is a vital process for multicellular organisms. Programmed cell death (PCD) functions in a variety of processes including growth, development, and immune responses for homeostasis maintenance. In particular, plants and animals utilize PCD to control pathogen invasion and infected cell populations. Despite some similarity, there are a number of key differences between how these organisms initiate and regulate cell death. In contrast to animals, plants are sessile, lack a circulatory system, and have additional cellular structures, including cell walls and chloroplasts. Plant cells have the autonomous ability to induce localized cell death using conserved eukaryotic pathways as well as unique plant-specific pathways. Thus, in order to successfully infect host cells, pathogens must subvert immune responses and avoid detection to prevent PCD and allow infection. Here we discuss the roles of cell death in plant immune responses and the tactics pathogens utilize to avert cell death.

No hormone to rule them all: Interactions of plant hormones during the responses of plants to pathogens.

Plant hormones are essential regulators of plant growth and immunity. In the last few decades, a vast amount of information has been obtained detailing the role of different plant hormones in immunity, and how they work together to ultimately shape the outcomes of plant pathogen interactions. Here we provide an overview on the roles of the main classes of plant hormones in the regulation of plant immunity, highlighting their metabolic and signaling pathways and how plants and pathogens utilize these pathways to activate or suppress defence.

The cytokinin response factors modulate root and shoot growth and promote leaf senescence in Arabidopsis.

The cytokinin response factors (CRFs) are a group of related AP2/ERF transcription factors that are transcriptionally induced by cytokinin. Here we explore the role of the CRFs in Arabidopsis thaliana growth and development by analyzing lines with decreased and increased CRF function. While single crf mutations have no appreciable phenotypes, disruption of multiple CRFs results in larger rosettes, delayed leaf senescence, a smaller root apical meristem (RAM), reduced primary and lateral root growth, and, in etiolated seedlings, shorter hypocotyls. In contrast, overexpression of CRFs generally results in the opposite phenotypes. The crf1,2,5,6 quadruple mutant is embryo lethal, indicating that CRF function is essential for embryo development. Disruption of the CRFs results in partially insensitivity to cytokinin in a root elongation assay and affects the basal expression of a significant number of cytokinin-regulated genes, including the type-A ARRs, although it does not impair the cytokinin induction of the type-A ARRs. Genes encoding homeobox transcription factors are mis-expressed in the crf1,3,5,6 mutant, including STIMPY/WOX9 that is required for root and shoot apical meristem maintenance roots and which has previously been linked to cytokinin. These results indicate that the CRF transcription factors play important roles in multiple aspects of plant growth and development, in part through a complex interaction with cytokinin signaling.

Two-component elements mediate interactions between cytokinin and salicylic acid in plant immunity.

Recent studies have revealed an important role for hormones in plant immunity. We are now beginning to understand the contribution of crosstalk among different hormone signaling networks to the outcome of plant-pathogen interactions. Cytokinins are plant hormones that regulate development and responses to the environment. Cytokinin signaling involves a phosphorelay circuitry similar to two-component systems used by bacteria and fungi to perceive and react to various environmental stimuli. In this study, we asked whether cytokinin and components of cytokinin signaling contribute to plant immunity. We demonstrate that cytokinin levels in Arabidopsis are important in determining the amplitude of immune responses, ultimately influencing the outcome of plant-pathogen interactions. We show that high concentrations of cytokinin lead to increased defense responses to a virulent oomycete pathogen, through a process that is dependent on salicylic acid (SA) accumulation and activation of defense gene expression. Surprisingly, treatment with lower concentrations of cytokinin results in increased susceptibility. These functions for cytokinin in plant immunity require a host phosphorelay system and are mediated in part by type-A response regulators, which act as negative regulators of basal and pathogen-induced SA-dependent gene expression. Our results support a model in which cytokinin up-regulates plant immunity via an elevation of SA-dependent defense responses and in which SA in turn feedback-inhibits cytokinin signaling. The crosstalk between cytokinin and SA signaling networks may help plants fine-tune defense responses against pathogens.

Cytokinin signaling and transcriptional networks.

The cytokinin signaling pathway consists of a phosphorelay mechanism that is initiated by binding of cytokinin to histidine kinase receptors and culminates with the transcription of cytokinin-responsive genes in the nucleus. Type-B response regulators (ARR) encode transcription factors that act as major players in the transcriptional activation of cytokinin-responsive genes, among which are many transcription factors. In this review, we highlight the transcriptional networks regulated by cytokinin that have been identified and their roles in the regulation of a subset of the many developmental and physiological processes regulated by this plant hormone.

Environmental perception avenues: the interaction of cytokinin and environmental response pathways.

Cytokinins were discovered in the 1950s by their ability to promote cell division in cultured plant cells. Recently, there have been significant breakthroughs in our understanding of the biosynthesis, metabolism, perception and signal transduction of this phytohormone. These advances, coupled with physiological and other approaches, have enabled remarkable progress to be made in our understanding of the interactions between cytokinin function and environmental inputs. In this review, we first highlight the most recent advances in our understanding of cytokinin biosynthesis, metabolism and signalling. We then discuss how various environmental signals interact with these pathways to modulate plant growth, development and physiology.