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.

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.

Plant Hormonomics: Multiple Phytohormone Profiling by Targeted Metabolomics.

Phytohormones are physiologically important small molecules that play essential roles in intricate signaling networks that regulate diverse processes in plants. We present a method for the simultaneous targeted profiling of 101 phytohormone-related analytes from minute amounts of fresh plant material (less than 20 mg). Rapid and nonselective extraction, fast one-step sample purification, and extremely sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry enable concurrent quantification of the main phytohormone classes: cytokinins, auxins, brassinosteroids, gibberellins, jasmonates, salicylates, and abscisates. We validated this hormonomic approach in salt-stressed and control Arabidopsis (Arabidopsis thaliana) seedlings, quantifying a total of 43 endogenous compounds in both root and shoot samples. Subsequent multivariate statistical data processing and cross-validation with transcriptomic data highlighted the main hormone metabolites involved in plant adaptation to salt stress.

Cell-Type-Specific Cytokinin Distribution within the Arabidopsis Primary Root Apex.

Cytokinins (CKs) play a crucial role in many physiological and developmental processes at the levels of individual plant components (cells, tissues, and organs) and by coordinating activities across these parts. High-resolution measurements of intracellular CKs in different plant tissues can therefore provide insights into their metabolism and mode of action. Here, we applied fluorescence-activated cell sorting of green fluorescent protein (GFP)-marked cell types, combined with solid-phase microextraction and an ultra-high-sensitivity mass spectrometry (MS) method for analysis of CK biosynthesis and homeostasis at cellular resolution. This method was validated by series of control experiments, establishing that protoplast isolation and cell sorting procedures did not greatly alter endogenous CK levels. The MS-based method facilitated the quantification of all the well known CK isoprenoid metabolites in four different transgenic Arabidopsis thaliana lines expressing GFP in specific cell populations within the primary root apex. Our results revealed the presence of a CK gradient within the Arabidopsis root tip, with a concentration maximum in the lateral root cap, columella, columella initials, and quiescent center cells. This distribution, when compared with previously published auxin gradients, implies that the well known antagonistic interactions between the two hormone groups are cell type specific.

Conditional Auxin Response and Differential Cytokinin Profiles in Shoot Branching Mutants.

Strigolactone (SL), auxin, and cytokinin (CK) are hormones that interact to regulate shoot branching. For example, several ramosus (rms) branching mutants in pea (Pisum sativum) have SL defects, perturbed xylem CK levels, and diminished responses to auxin in shoot decapitation assays. In contrast with the last of these characteristics, we discovered that buds on isolated nodes (explants) of rms plants instead respond normally to auxin. We hypothesized that the presence or absence of attached roots would result in transcriptional and hormonal differences in buds and subtending stem tissues, and might underlie the differential auxin response. However, decapitated plants and explants both showed similar up-regulation of CK biosynthesis genes, increased CK levels, and down-regulation of auxin transport genes. Moreover, auxin application counteracted these trends, regardless of the effectiveness of auxin at inhibiting bud growth. Multivariate analysis revealed that stem transcript and CK changes were largely associated with decapitation and/or root removal and auxin response, whereas bud transcript profiles related more to SL defects. CK clustering profiles were indicative of additional zeatin-type CKs in decapitated stems being supplied by roots and thus promoting bud growth in SL-deficient genotypes even in the presence of added auxin. This difference in CK content may explain why rms buds on explants respond better to auxin than those on decapitated plants. We further conclude that rapid changes in CK status in stems are auxin dependent but largely SL independent, suggesting a model in which auxin and CK are dominant regulators of decapitation-induced branching, whereas SLs are more important in intact plants.

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.

The pea TCP transcription factor PsBRC1 acts downstream of Strigolactones to control shoot branching.

The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated. The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application. The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes. The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine. The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth. However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth. In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant. Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1. This phenotype indicates a localized regulation of branching at these nodes specific to pea.

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.

Alterations in hormonal signals spatially coordinate distinct responses to DNA double-strand breaks in Arabidopsis roots.

Plants have a high ability to cope with changing environments and grow continuously throughout life. However, the mechanisms by which plants strike a balance between stress response and organ growth remain elusive. Here, we found that DNA double-strand breaks enhance the accumulation of cytokinin hormones through the DNA damage signaling pathway in the Arabidopsis root tip. Our data showed that activation of cytokinin signaling suppresses the expression of some of the PIN-FORMED genes that encode efflux carriers of another hormone, auxin, thereby decreasing the auxin signals in the root tip and causing cell cycle arrest at G2 phase and stem cell death. Elevated cytokinin signaling also promotes an early transition from cell division to endoreplication in the basal part of the root apex. We propose that plant hormones spatially coordinate differential DNA damage responses, thereby maintaining genome integrity and minimizing cell death to ensure continuous root growth.

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.

Cell-surface receptors enable perception of extracellular cytokinins.

Cytokinins are mobile multifunctional plant hormones with roles in development and stress resilience. Although their Histidine Kinase receptors are substantially localised to the endoplasmic reticulum, cellular sites of cytokinin perception and importance of spatially heterogeneous cytokinin distribution continue to be debated. Here we show that cytokinin perception by plasma membrane receptors is an effective additional path for cytokinin response. Readout from a Two Component Signalling cytokinin-specific reporter (TCSn::GFP) closely matches intracellular cytokinin content in roots, yet we also find cytokinins in extracellular fluid, potentially enabling action at the cell surface. Cytokinins covalently linked to beads that could not pass the plasma membrane increased expression of both TCSn::GFP and Cytokinin Response Factors. Super-resolution microscopy of GFP-labelled receptors and diminished TCSn::GFP response to immobilised cytokinins in cytokinin receptor mutants, further indicate that receptors can function at the cell surface. We argue that dual intracellular and surface locations may augment flexibility of cytokinin responses.

High-Resolution Cell-Type Specific Analysis of Cytokinins in Sorted Root Cell Populations of Arabidopsis thaliana.

We describe a method combining fluorescence-activated cell sorting (FACS) with one-step miniaturized isolation and accurate quantification of cytokinins (CKs) using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to measure these phytohormones in specific cell types of Arabidopsis thaliana roots. The methodology provides information of unprecedented resolution about spatial distributions of CKs, and thus should facilitate attempts to elucidate regulatory networks involved in root developmental processes.

A Pragmatic Approach to Getting Published: 35 Tips for Early Career Researchers.

It is trite to say "publish or perish," yet many early career researchers are often at a loss on how to best get their work published. With strong competition and many manuscripts submitted, it is difficult to convince editors and reviewers to opt for acceptance. A pragmatic approach to publishing may increase one's odds of success. Here, we - a group of postdocs in the field of plant science - present specific recommendations for early career scientists on advanced levels. We cannot provide a recipe-like set of instructions with success guaranteed, but we come from a broad background in plant science, with experience publishing in a number of journals of varying topics and impact factors. We provide tips, tricks, and tools for collaboration, journal selection, and achieving acceptance.