Cytokinin metabolite distribution at tissue- and cell-specific levels

Cytokinins are pivotal plant hormones regulating the cell cycle and many components of development. They also transduce environmental signals such as nutrient deficiency and drought. The significance of cytokinin genes in agriculture has been highlighted by several reports, which associate them with improved crop performance. The aim of this study is to gain a better understanding of cytokinin metabolite distribution, in order to shed light on cytokinin transport, biochemistry and its function. Cytokinin metabolite levels were shown to vary amongst pea tissues and vascular saps, and across developmental stages. Detection was pushed to new limits by performing cytokinin measurements in distinct cell populations. For the first time, heterogeneous distribution was demonstrated within the Arabidopsis root apex and between the intra- and extra-cellular compartments. A cytokinin gradient was revealed within the Arabidopsis root tip with maximal concentration in the columella, root cap, initials and QC cells. Cell-specific analysis of the TCSn:GFP cytokinin signaling reporter line indicated tZ as the only bioactive cytokinin related to intra-cellular cytokinin signaling. This result, coupled with identification of active cytokinins in the apoplast, indicates a significant role for cytokinin receptors at the plasma membrane. Cytokinin glucosides were largely intra-cellular, with their prevalence in cytokinin responsive cells indicating a role in cytokinin signaling. The riboside cZR was identified as a major transported cytokinin form while tZ-cytokinins were predominantly shoot compounds. The lateral root zone was also identified as a candidate site for cytokinin loading into the xylem. Comprehensive analysis of cytokinin’s distribution was also examined, particularly in response to strigolactone effects, another plant hormone which regulates branching. Strigolactone inhibited cytokinin degradative enzymes in root apices, thus acting as a local positive regulator of cytokinins. Furthermore, an RMS2-dependent mechanism regulating cytokinin homeostasis in the shoots was identified. In conclusion, this research provides new insights into the importance of cytokinin spatial distribution for understanding its roles in cell and developmental biology.Open Acces

Best practices in plant cytometry

Editorialinfo:eu-repo/semantics/publishedVersio

Fluorescence activated cell sorting-A selective tool for plant cell isolation and analysis

Instrumentation for flow cytometry and sorting is designed around the assumption that samples are single-cell suspensions. However, with few exceptions, higher plants comprise complex multicellular tissues and organs, in which the individual cells are held together by shared cell walls. Single-cell suspensions can be obtained through digestion of the cells walls and release of the so-called protoplasts (plants without their cell wall). Here we describe best practices for protoplast preparation, and for analysis through flow cytometry and cell sorting. Finally, the numerous downstream applications involving sorted protoplasts are discussed

IPT9, a cis-zeatin cytokinin biosynthesis gene, promotes root growth

Cytokinin and auxin are plant hormones that coordinate many aspects of plant development. Their interactions in plant underground growth are well established, occurring at the levels of metabolism, signaling, and transport. Unlike many plant hormone classes, cytokinins are represented by more than one active molecule. Multiple mutant lines, blocking specific parts of cytokinin biosynthetic pathways, have enabled research in plants with deficiencies in specific cytokinin-types. While most of these mutants have confirmed the impeding effect of cytokinin on root growth, the ipt29 double mutant instead surprisingly exhibits reduced primary root length compared to the wild type. This mutant is impaired in cis-zeatin (cZ) production, a cytokinin-type that had been considered inactive in the past. Here we have further investigated the intriguing ipt29 root phenotype, opposite to known cytokinin functions, and the (bio)activity of cZ. Our data suggest that despite the ipt29 short-root phenotype, cZ application has a negative impact on primary root growth and can activate a cytokinin response in the stele. Grafting experiments revealed that the root phenotype of ipt29 depends mainly on local signaling which does not relate directly to cytokinin levels. Notably, ipt29 displayed increased auxin levels in the root tissue. Moreover, analyses of the differential contributions of ipt2 and ipt9 to the ipt29 short-root phenotype demonstrated that, despite its deficiency on cZ levels, ipt2 does not show any root phenotype or auxin homeostasis variation, while ipt9 mutants were indistinguishable from ipt29. We conclude that IPT9 functions may go beyond cZ biosynthesis, directly or indirectly, implicating effects on auxin homeostasis and therefore influencing plant growth

Dynamics of Auxin and Cytokinin Metabolism during Early Root and Hypocotyl Growth in Theobroma cacao

The spatial location and timing of plant developmental events are largely regulated by the well balanced effects of auxin and cytokinin phytohormone interplay. Together with transport, localized metabolism regulates the concentration gradients of their bioactive forms, ultimately eliciting growth responses. In order to explore the dynamics of auxin and cytokinin metabolism during early seedling growth in Theobroma cacao (cacao), we have performed auxin and cytokinin metabolite profiling in hypocotyls and root developmental sections at different times by using ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry (UHPLC-MS/MS). Our work provides quantitative characterization of auxin and cytokinin metabolites throughout early root and hypocotyl development and identifies common and distinctive features of auxin and cytokinin metabolism during cacao seedling development

Reaction Wood Anatomical Traits and Hormonal Profiles in Poplar Bent Stem and Root

Reaction wood (RW) formation is an innate physiological response of woody plants to counteract mechanical constraints in nature, reinforce structure and redirect growth toward the vertical direction. Differences and/or similarities between stem and root response to mechanical constraints remain almost unknown especially in relation to phytohormones distribution and RW characteristics. Thus, Populus nigra stem and root subjected to static non-destructive mid-term bending treatment were analyzed. The distribution of tension and compression forces was firstly modeled along the main bent stem and root axis; then, anatomical features, chemical composition, and a complete auxin and cytokinin metabolite profiles of the stretched convex and compressed concave side of three different bent stem and root sectors were analyzed. The results showed that in bent stems RW was produced on the upper stretched convex side whereas in bent roots it was produced on the lower compressed concave side. Anatomical features and chemical analysis showed that bent stem RW was characterized by a low number of vessel, poor lignification, and high carbohydrate, and thus gelatinous layer in fiber cell wall. Conversely, in bent root, RW was characterized by high vessel number and area, without any significant variation in carbohydrate and lignin content. An antagonistic interaction of auxins and different cytokinin forms/conjugates seems to regulate critical aspects of RW formation/development in stem and root to facilitate upward/downward organ bending. The observed differences between the response stem and root to bending highlight how hormonal signaling is highly organ-dependent

iP & OEIP - Cytokinin Micro Application Modulates Root Development with High Spatial Resolution

State-of-the-art technology based on organic electronics can be used as a flow-free delivery method for organic substances with high spatial resolution. Such highly targeted drug micro applications can be used in plant research for the regulation of physiological processes on tissue and cellular levels. Here, for the first time, an organic electronic ion pump (OEIP) is reported that can transport an isoprenoid-type cytokinin, N-6-isopentenyladenine (iP), to intact plants. Cytokinins (CKs) are plant hormones involved in many essential physiological processes, including primary root (PR) and lateral root (LR) development. Using the Arabidopsis thaliana root as a model system, efficient iP delivery is demonstrated with a biological output - cytokinin-related PR and LR growth inhibition. The spatial resolution of iP delivery, defined for the first time for an organic compound, is shown to be less than 1 mm, exclusively affecting the OEIP-targeted LR. Results from the application of the high-resolution OIEP treatment method confirm previously published findings showing that the influence of CKs may vary at different stages of LR development. Thus, OEIP-based technologies offer a novel, electronically controlled method for phytohormone delivery that could contribute to unraveling cytokinin functions during different developmental processes with high specificity

Fluorescence-activated multi-organelle mapping of subcellular plant hormone distribution

Auxins and cytokinins are two major families of phytohormones that control most aspects of plant growth, development and plasticity. Their distribution in plants has been described, but the importance of cell- and subcellular-type specific phytohormone homeostasis remains undefined. Herein, we revealed auxin and cytokinin distribution maps showing their different organelle-specific allocations within the Arabidopsis plant cell. To do so, we have developed Fluorescence-Activated multi-Organelle Sorting (FAmOS), an innovative subcellular fractionation technique based on flow cytometric principles. FAmOS allows the simultaneous sorting of four differently labelled organelles based on their individual light scatter and fluorescence parameters while ensuring hormone metabolic stability. Our data showed different subcellular distribution of auxin and cytokinins, revealing the formation of phytohormone gradients that have been suggested by the subcellular localization of auxin and cytokinin transporters, receptors and metabolic enzymes. Both hormones showed enrichment in vacuoles, while cytokinins were also accumulated in the endoplasmic reticulum

Profiling of 1-aminocyclopropane-1-carboxylic acid and selected phytohormones in Arabidopsis using liquid chromatography-tandem mass spectrometry

Background Gaseous phytohormone ethylene levels are directly influenced by the production of its immediate non-volatile precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Owing to the strongly acidic character of the ACC molecule, its quantification has been difficult to perform. Here, we present a simple and straightforward validated method for accurate quantification of not only ACC levels, but also major members of other important phytohormonal classes - auxins, cytokinins, jasmonic acid, abscisic acid and salicylic acid from the same biological sample. Results The presented technique facilitates the analysis of 15 compounds by liquid chromatography coupled with tandem mass spectrometry. It was optimized and validated for 10 mg of fresh weight plant material. The extraction procedure is composed of a minimal amount of necessary steps. Accuracy and precision were the basis for evaluating the method, together with process efficiency, recovery and matrix effects as validation parameters. The examined compounds comprise important groups of phytohormones, their active forms and some of their metabolites, including six cytokinins, four auxins, two jasmonates, abscisic acid, salicylic acid and 1-aminocyclopropane-1-carboxylic acid. The resulting method was used to examine their contents in selected Arabidopsis thaliana mutant lines. Conclusion This profiling method enables a very straightforward approach for indirect ethylene study and explores how it interacts, based on content levels, with other phytohormonal groups in plants