We extended the applicability of the BY-2 cell line as a model by introducing two new selection systems. Our protocol provides guidelines for optimising Basta selection in other recalcitrant models. Tobacco BY-2 cell line is the most commonly used cytological model in plant research. It is uniform, can be simply treated by chemicals, synchronised and easily transformed. However, only a few selection systems are available that complicate advanced studies using multiple stacked transgenes and extensive gene editing. In our work, we adopted for BY-2 cell line two other selection systems: sulfadiazine and phosphinothricin (PPT, an active ingredient of Basta herbicide). We show that sulfadiazine can be used in a wide range of concentrations. It is suitable for co-transformation and subsequent double selection with kanamycin or hygromycin, which are standardly used for BY-2 transformation. We also have domesticated the sulfadiazine resistance for the user-friendly GoldenBraid cloning system. Compared to sulfadiazine, establishing selection on phosphinothricin was considerably more challenging. It did not work in any concentration of PPT with standardly cultured cells. Since the selection is based on blocking glutamine synthetase and consequent ammonium toxicity and deficiency of assimilated nitrogen, we tried to manipulate nitrogen availability. We found that the PPT selection reliably works only with nitrogen-starved cells with reduced nitrate reserves that are selected on a medium without ammonium nitrate. Both these adjustments prevent the release of large amounts of ammonium, which can toxify the entire culture in the case of standardly cultured cells. Since high nitrogen reserves can be a common feature of in vitro cultures grown on MS media, nitrogen starvation could be a key step in establishing phosphinothricin resistance in other plant models.

• Keywords
• Bialaphos, Glufosinate ammonium, Selectable markers, Suspension cell lines, Tobacco (Nicotiana tabacum),
• MeSH
• Ammonium Compounds * MeSH
• Nitrogen MeSH
• Plants, Genetically Modified genetics   MeSH
• Sulfadiazine MeSH
• Nicotiana * genetics   MeSH
• Transformation, Genetic MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Ammonium Compounds * MeSH
• Nitrogen MeSH
• phosphinothricin MeSH Browser
• Sulfadiazine MeSH

Together with auxin transport, auxin metabolism is a key determinant of auxin signaling output by plant cells. Enzymatic machinery involved in auxin metabolism is subject to regulation based on numerous inputs, including the concentration of auxin itself. Therefore, experiments characterizing altered auxin availability and subsequent changes in auxin metabolism could elucidate the function and regulatory role of individual elements in the auxin metabolic machinery. Here, we studied auxin metabolism in auxin-dependent tobacco BY-2 cells. We revealed that the concentration of N-(2-oxindole-3-acetyl)-l-aspartic acid (oxIAA-Asp), the most abundant auxin metabolite produced in the control culture, dramatically decreased in auxin-starved BY-2 cells. Analysis of the transcriptome and proteome in auxin-starved cells uncovered significant downregulation of all tobacco (Nicotiana tabacum) homologs of Arabidopsis (Arabidopsis thaliana) DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1), at both transcript and protein levels. Auxin metabolism profiling in BY-2 mutants carrying either siRNA-silenced or CRISPR-Cas9-mutated NtDAO1, as well as in dao1-1 Arabidopsis plants, showed not only the expected lower levels of oxIAA, but also significantly lower abundance of oxIAA-Asp. Finally, ability of DAO1 to oxidize IAA-Asp was confirmed by an enzyme assay in AtDAO1-producing bacterial culture. Our results thus represent direct evidence of DAO1 activity on IAA amino acid conjugates.

• MeSH
• Amino Acids metabolism   MeSH
• Dioxygenases metabolism   MeSH
• Oxidation-Reduction MeSH
• Plant Proteins metabolism   MeSH
• Nicotiana enzymology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Amino Acids MeSH
• Dioxygenases MeSH
• Plant Proteins MeSH

Cytokinins are a class of phytohormones, signalling molecules specific to plants. They act as regulators of diverse physiological processes in complex signalling pathways. It is necessary for plants to continuously regulate cytokinin distribution among different organs, tissues, cells, and compartments. Such regulatory mechanisms include cytokinin biosynthesis, metabolic conversions and degradation, as well as cytokinin membrane transport. In our review, we aim to provide a thorough picture of the latter. We begin by summarizing cytokinin structures and physicochemical properties. Then, we revise the elementary thermodynamic and kinetic aspects of cytokinin membrane transport. Next, we review which membrane-bound carrier proteins and protein families recognize cytokinins as their substrates. Namely, we discuss the families of "equilibrative nucleoside transporters" and "purine permeases", which translocate diverse purine-related compounds, and proteins AtPUP14, AtABCG14, AtAZG1, and AtAZG2, which are specific to cytokinins. We also address long-distance cytokinin transport. Putting all these pieces together, we finally discuss cytokinin distribution as a net result of these processes, diverse in their physicochemical nature but acting together to promote plant fitness.

• Keywords
• ABCG14, AZG1, AZG2, PUP14, cytokinin distribution, cytokinin hydrophobicity, cytokinin transport, membrane transport,
• MeSH
• Arabidopsis metabolism   MeSH
• Biological Transport MeSH
• Cell Membrane metabolism   MeSH
• Cytokinins metabolism   MeSH
• Homeostasis MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Kinetics MeSH
• Plant Roots metabolism   MeSH
• Membrane Transport Proteins metabolism   MeSH
• Arabidopsis Proteins genetics   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Growth Regulators metabolism   MeSH
• Signal Transduction physiology   MeSH
• Thermodynamics MeSH
• Plant Shoots metabolism   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Cytokinins MeSH
• Membrane Transport Proteins MeSH
• Arabidopsis Proteins MeSH
• Plant Growth Regulators MeSH

In over 40 years of research on the cellular uptake of auxin it is somewhat chastening that we have elaborated so little on the original kinetic descriptions of auxin uptake by plant cells made by Rubery and Sheldrake in 1974. Every aspect of that seminal work has been investigated in detail, and the uptake activity they measured is now known to be attributed to the AUX1/LAX family of permeases. Recent pharmacological studies have defined the substrate specificity of AUX1, biochemical studies have evaluated its permeability to auxin in plant cell membranes, and rigourous kinetic studies have confirmed the affinity of AUX1 for IAA and synthetic auxins. Advances in genome sequencing have provided a rich resource for informatic analysis of the ancestry of AUX1 and the LAX proteins and, along with models of topology, suggest mechanistic links to families of eukaryotic proton co-transporters for which crystal structures have been presented. The insights gained from all the accumulated research reflect the brilliance of Rubery and Sheldrake's early work, but recent biochemical analyses are starting to advance further our understanding of this vitally important family of auxin transport proteins.

• Keywords
• auxin transport, development, hormone, kinetics, permeability, structure,
• MeSH
• Biological Transport, Active physiology   MeSH
• Cell Membrane * genetics   metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Membrane Transport Proteins * genetics   metabolism   MeSH
• Plants * genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Indoleacetic Acids MeSH
• Membrane Transport Proteins * MeSH

Plant hormones are master regulators of plant growth and development. Better knowledge of their spatial signaling and homeostasis (transport and metabolism) on the lowest structural levels (cellular and subcellular) is therefore crucial to a better understanding of developmental processes in plants. Recent progress in phytohormone analysis at the cellular and subcellular levels has greatly improved the effectiveness of isolation protocols and the sensitivity of analytical methods. This review is mainly focused on homeostasis of two plant hormone groups, auxins and cytokinins. It will summarize and discuss their tissue- and cell-type specific distributions at the cellular and subcellular levels.

• Keywords
• auxin, cellular level, cytokinin, phytohormone metabolism, phytohormone transport, subcellular level,
• MeSH
• Biological Transport MeSH
• Cytokinins metabolism   MeSH
• Plant Physiological Phenomena * MeSH
• Homeostasis * MeSH
• Intracellular Space metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Metabolic Networks and Pathways MeSH
• Organelles metabolism   MeSH
• Plant Growth Regulators metabolism   MeSH
• Plant Cells metabolism   MeSH
• Plant Development * MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Cytokinins MeSH
• Indoleacetic Acids MeSH
• Plant Growth Regulators MeSH

Auxins mediate various processes that are involved in plant growth and development in response to specific environmental conditions. Its proper spatio-temporal distribution that is driven by polar auxin transport machinery plays a crucial role in the wide range of auxins physiological effects. Numbers of approaches have been developed to either directly or indirectly monitor auxin distribution in vivo in order to elucidate the basis of its precise regulation. Herein, we provide an updated list of valuable techniques used for monitoring auxins in plants, with their utilities and limitations. Because the spatial and temporal resolutions of the presented approaches are different, their combination may provide a comprehensive outcome of auxin distribution in diverse developmental processes.

• Keywords
• auxin, auxin distribution, auxin signalling, auxin transport, direct visualization, indirect visualization, receptor, sensor,
• MeSH
• Arabidopsis metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Development physiology   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Indoleacetic Acids MeSH

The volatile two-carbon hormone ethylene acts in concert with an array of signals to affect etiolated seedling development. From a chemical screen, we isolated a quinoline carboxamide designated ACCERBATIN (AEX) that exacerbates the 1-aminocyclopropane-1-carboxylic acid-induced triple response, typical for ethylene-treated seedlings in darkness. Phenotypic analyses revealed distinct AEX effects including inhibition of root hair development and shortening of the root meristem. Mutant analysis and reporter studies further suggested that AEX most probably acts in parallel to ethylene signaling. We demonstrated that AEX functions at the intersection of auxin metabolism and reactive oxygen species (ROS) homeostasis. AEX inhibited auxin efflux in BY-2 cells and promoted indole-3-acetic acid (IAA) oxidation in the shoot apical meristem and cotyledons of etiolated seedlings. Gene expression studies and superoxide/hydrogen peroxide staining further revealed that the disrupted auxin homeostasis was accompanied by oxidative stress. Interestingly, in light conditions, AEX exhibited properties reminiscent of the quinoline carboxylate-type auxin-like herbicides. We propose that AEX interferes with auxin transport from its major biosynthesis sites, either as a direct consequence of poor basipetal transport from the shoot meristematic region, or indirectly, through excessive IAA oxidation and ROS accumulation. Further investigation of AEX can provide new insights into the mechanisms connecting auxin and ROS homeostasis in plant development and provide useful tools to study auxin-type herbicides.

• Keywords
• Arabidopsis, auxin homeostasis, chemical genetics, ethylene signaling, herbicide, quinoline carboxamide, reactive oxygen species, triple response,
• MeSH
• Amino Acids, Cyclic metabolism   MeSH
• Arabidopsis genetics   metabolism   MeSH
• Quinolones metabolism   MeSH
• Ethylenes metabolism   MeSH
• Gene Expression MeSH
• Herbicides chemistry   MeSH
• Homeostasis MeSH
• Indoleacetic Acids metabolism   MeSH
• Arabidopsis Proteins metabolism   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Seedlings metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1-aminocyclopropane-1-carboxylic acid MeSH Browser
• Amino Acids, Cyclic MeSH
• Quinolones MeSH
• ethylene MeSH Browser
• Ethylenes MeSH
• Herbicides MeSH
• Indoleacetic Acids MeSH
• Arabidopsis Proteins MeSH
• Reactive Oxygen Species MeSH

The phenylpropanoid 3,4-(methylenedioxy)cinnamic acid (MDCA) is a plant-derived compound first extracted from roots of Asparagus officinalis and further characterized as an allelochemical. Later on, MDCA was identified as an efficient inhibitor of 4-COUMARATE-CoA LIGASE (4CL), a key enzyme of the general phenylpropanoid pathway. By blocking 4CL, MDCA affects the biosynthesis of many important metabolites, which might explain its phytotoxicity. To decipher the molecular basis of the allelochemical activity of MDCA, we evaluated the effect of this compound on Arabidopsis thaliana seedlings. Metabolic profiling revealed that MDCA is converted in planta into piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H), the enzyme directly upstream of 4CL. The inhibition of C4H was also reflected in the phenolic profile of MDCA-treated plants. Treatment of in vitro grown plants resulted in an inhibition of primary root growth and a proliferation of lateral and adventitious roots. These observed growth defects were not the consequence of lignin perturbation, but rather the result of disturbing auxin homeostasis. Based on DII-VENUS quantification and direct measurement of cellular auxin transport, we concluded that MDCA disturbs auxin gradients by interfering with auxin efflux. In addition, mass spectrometry was used to show that MDCA triggers auxin biosynthesis, conjugation, and catabolism. A similar shift in auxin homeostasis was found in the c4h mutant ref3-2, indicating that MDCA triggers a cross talk between the phenylpropanoid and auxin biosynthetic pathways independent from the observed auxin efflux inhibition. Altogether, our data provide, to our knowledge, a novel molecular explanation for the phytotoxic properties of MDCA.

• MeSH
• Trans-Cinnamate 4-Monooxygenase antagonists & inhibitors   metabolism   MeSH
• Arabidopsis drug effects   genetics   metabolism   MeSH
• Benzoates metabolism   pharmacology   MeSH
• Biosynthetic Pathways drug effects   MeSH
• Cinnamates chemistry   metabolism   pharmacology   MeSH
• Phenylpropionates chemistry   metabolism   pharmacology   MeSH
• Plants, Genetically Modified MeSH
• Mass Spectrometry MeSH
• Homeostasis drug effects   MeSH
• Coenzyme A Ligases antagonists & inhibitors   metabolism   MeSH
• Microscopy, Confocal MeSH
• Plant Roots drug effects   genetics   metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Lignin biosynthesis   MeSH
• Seedlings drug effects   genetics   growth & development   metabolism   MeSH
• Dose-Response Relationship, Drug MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 4-coumarate-CoA ligase MeSH Browser
• Trans-Cinnamate 4-Monooxygenase MeSH
• Benzoates MeSH
• Cinnamates MeSH
• cinnamic acid MeSH Browser
• Phenylpropionates MeSH
• Coenzyme A Ligases MeSH
• Indoleacetic Acids MeSH
• Lignin MeSH
• phenylpropanoid 3,4-(methylenedioxy)cinnamic acid MeSH Browser
• piperonylic acid MeSH Browser