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.
- MeSH
- Arabidopsis metabolismus MeSH
- biologický transport MeSH
- buněčná membrána metabolismus MeSH
- cytokininy metabolismus MeSH
- homeostáza MeSH
- hydrofobní a hydrofilní interakce MeSH
- kinetika MeSH
- kořeny rostlin metabolismus MeSH
- membránové transportní proteiny metabolismus MeSH
- proteiny huseníčku genetika MeSH
- regulace genové exprese u rostlin MeSH
- regulátory růstu rostlin metabolismus MeSH
- signální transdukce fyziologie MeSH
- termodynamika MeSH
- výhonky rostlin metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Auxin concentration gradients are informative for the transduction of many developmental cues, triggering downstream gene expression and other responses. The generation of auxin gradients depends significantly on cell-to-cell auxin transport, which is supported by the activities of auxin efflux and influx carriers. However, at the level of individual plant cell, the co-ordination of auxin efflux and influx largely remains uncharacterized. We addressed this issue by analyzing the contribution of canonical PIN-FORMED (PIN) proteins to the carrier-mediated auxin efflux in Nicotiana tabacum L., cv. Bright Yellow (BY-2) tobacco cells. We show here that a majority of canonical NtPINs are transcribed in cultured cells and in planta. Cloning of NtPIN genes and their inducible overexpression in tobacco cells uncovered high auxin efflux activity of NtPIN11, accompanied by auxin starvation symptoms. Auxin transport parameters after NtPIN11 overexpression were further assessed using radiolabelled auxin accumulation and mathematical modelling. Unexpectedly, these experiments showed notable stimulation of auxin influx, which was accompanied by enhanced transcript levels of genes for a specific auxin influx carrier and by decreased transcript levels of other genes for auxin efflux carriers. A similar transcriptional response was observed upon removal of auxin from the culture medium, which resulted in decreased auxin efflux. Overall, our results revealed an auxin transport-based homeostatic mechanism for the maintenance of endogenous auxin levels. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at http://osf.io/ka97b/.
- MeSH
- biologický transport MeSH
- buněčné linie MeSH
- fylogeneze MeSH
- homeostáza MeSH
- kyseliny indoloctové metabolismus MeSH
- membránové transportní proteiny genetika metabolismus MeSH
- proteiny huseníčku genetika metabolismus MeSH
- regulátory růstu rostlin metabolismus MeSH
- rostlinné proteiny genetika metabolismus MeSH
- tabák genetika fyziologie MeSH
- teoretické modely MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The international symposium "Auxins and Cytokinins in Plant Development" (ACPD), which is held every 4⁻5 years in Prague, Czech Republic, is a meeting of scientists interested in the elucidation of the action of two important plant hormones-auxins and cytokinins. It is organized by a group of researchers from the Laboratory of Hormonal Regulations in Plants at the Institute of Experimental Botany, the Czech Academy of Sciences. The symposia already have a long tradition, having started in 1972. Thanks to the central role of auxins and cytokinins in plant development, the ACPD 2018 symposium was again attended by numerous experts who presented their results in the opening, two plenary lectures, and six regular sessions, including two poster sessions. Due to the open character of the research community, which is traditionally very well displayed during the meeting, a lot of unpublished data were presented and discussed. In this report, we summarize the contributions in individual sessions that attracted our attention.
Developmental responses to auxin are regulated by facilitated uptake and efflux, but detailed molecular understanding of the carrier proteins is incomplete. We have used pharmacological tools to explore the chemical space that defines substrate preferences for the auxin uptake carrier AUX1. Total and partial loss-of-function aux1 mutants were assessed against wild-type for dose-dependent resistance to a range of auxins and analogues. We then developed an auxin accumulation assay with associated mathematical modelling to enumerate accurate IC50 values for a small library of auxin analogues. The structure activity relationship data were analysed using molecular field analyses to create a pharmacophoric atlas of AUX1 substrates. The uptake carrier exhibits a very high level of selectivity towards small substrates including the natural indole-3-acetic acid, and the synthetic auxin 2,4-dichlorophenoxyacetic acid. No AUX1 activity was observed for herbicides based on benzoic acid (dicamba), pyridinyloxyacetic acid (triclopyr) or the 6-arylpicolinates (halauxifen), and very low affinity was found for picolinic acid-based auxins (picloram) and quinolinecarboxylic acids (quinclorac). The atlas demonstrates why some widely used auxin herbicides are not, or are very poor substrates. We list molecular descriptors for AUX1 substrates and discuss our findings in terms of herbicide resistance management.
- MeSH
- Arabidopsis metabolismus MeSH
- biologické modely MeSH
- biotest MeSH
- herbicidy metabolismus MeSH
- indoly metabolismus MeSH
- inhibiční koncentrace 50 MeSH
- kořeny rostlin růst a vývoj MeSH
- kyselina 2,4-dichlorfenoxyoctová metabolismus MeSH
- kyseliny indoloctové metabolismus MeSH
- mutace genetika MeSH
- proteiny huseníčku metabolismus MeSH
- semenáček růst a vývoj MeSH
- substrátová specifita MeSH
- tabák cytologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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.
- MeSH
- aminokyseliny cyklické metabolismus MeSH
- Arabidopsis genetika metabolismus MeSH
- chinolony metabolismus MeSH
- ethyleny metabolismus MeSH
- exprese genu MeSH
- herbicidy chemie MeSH
- homeostáza MeSH
- kyseliny indoloctové metabolismus MeSH
- proteiny huseníčku metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- semenáček metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Parallel determination of auxin and cytokinin levels within plant organs and tissues represents an invaluable tool for studies of their physiological effects and mutual interactions. Thanks to their different chemical structures, auxins, cytokinins and their metabolites are often determined separately, using specialized procedures of sample purification, extraction, and quantification. However, recent progress in the sensitivity of analytical methods of liquid chromatography coupled to mass spectrometry (LC-MS) allows parallel analysis of multiple compounds. Here we describe a method that is based on single step purification protocol followed by LC-MS separation and detection for parallel analysis of auxins, cytokinins and their metabolites in various plant tissues and cell cultures.
- MeSH
- chromatografie kapalinová MeSH
- cytokininy analýza chemie izolace a purifikace MeSH
- kyseliny indoloctové analýza chemie izolace a purifikace MeSH
- metabolomika metody MeSH
- regulátory růstu rostlin analýza MeSH
- rostlinné extrakty analýza chemie izolace a purifikace MeSH
- tandemová hmotnostní spektrometrie MeSH
- Publikační typ
- časopisecké články MeSH
Auxin and cytokinin are key endogenous regulators of plant development. Although cytokinin-mediated modulation of auxin distribution is a developmentally crucial hormonal interaction, its molecular basis is largely unknown. Here we show a direct regulatory link between cytokinin signalling and the auxin transport machinery uncovering a mechanistic framework for cytokinin-auxin cross-talk. We show that the CYTOKININ RESPONSE FACTORS (CRFs), transcription factors downstream of cytokinin perception, transcriptionally control genes encoding PIN-FORMED (PIN) auxin transporters at a specific PIN CYTOKININ RESPONSE ELEMENT (PCRE) domain. Removal of this cis-regulatory element effectively uncouples PIN transcription from the CRF-mediated cytokinin regulation and attenuates plant cytokinin sensitivity. We propose that CRFs represent a missing cross-talk component that fine-tunes auxin transport capacity downstream of cytokinin signalling to control plant development.
- MeSH
- Arabidopsis MeSH
- chromatinová imunoprecipitace MeSH
- cytokininy metabolismus MeSH
- geneticky modifikované rostliny MeSH
- konfokální mikroskopie MeSH
- kořeny rostlin metabolismus MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- kyseliny indoloctové metabolismus MeSH
- membránové transportní proteiny genetika metabolismus MeSH
- proteiny huseníčku genetika metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- responzivní elementy MeSH
- signální transdukce MeSH
- transkripční faktory genetika metabolismus MeSH
- zelené fluorescenční proteiny MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Cytokinins (CKs) are involved in response to various environmental cues, including salinity. It has been previously reported that enhancing CK contents improved salt stress tolerance in tomato. However, the underlying mechanisms of CK metabolism and signaling under salt stress conditions remain to be deciphered. RESULTS: Two tomato isopentenyltransferases, SlIPT3 and SlIPT4, were characterized in tomato and Arabidopsis. Both proteins displayed isopentenyltransferase (IPT) activity in vitro, while their encoding genes exhibited different spatio-temporal expression patterns during tomato plant development. SlIPT3 and SlIPT4 were affected by the endogenous CK status, tightly connected with CKs feedback regulation, as revealed by hormonal treatements. In response to salt stress, SlIPT3 and SlIPT4 were strongly repressed in tomato roots, and differently affected in young and old leaves. SlIPT3 overexpression in tomato resulted in high accumulation of different CK metabolites, following modifications of CK biosynthesis-, signaling- and degradation-gene expression. In addition, 35S::SlIPT3 tomato plants displayed improved tolerance to salinity consecutive to photosynthetic pigments and K(+)/Na(+) ratio retention. Involvement of SlIPT3 and SlIPT4 in salt stress response was also observed in Arabidopsis ipt3 knock-out complemented plants, through maintenance of CK homeostasis. CONCLUSIONS: SlIPT3 and SlIPT4 are functional IPTs encoded by differently expressed genes, distinctively taking part in the salinity response. The substantial participation of SlIPT3 in CK metabolism during salt stress has been determined in 35S::SlIPT3 tomato transformants, where enhancement of CKs accumulation significantly improved plant tolerance to salinity, underlining the importance of this phytohormone in stress response.
- MeSH
- alkyltransferasy a aryltransferasy genetika fyziologie MeSH
- Arabidopsis genetika fyziologie MeSH
- cytokininy metabolismus MeSH
- geneticky modifikované rostliny genetika fyziologie MeSH
- regulace genové exprese u rostlin * MeSH
- Solanum lycopersicum embryologie enzymologie genetika fyziologie MeSH
- tolerance k soli * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Polar auxin transport is a crucial process for control and coordination of plant development. Studies of auxin transport through plant tissues and organs showed that auxin is transported by a combination of phloem flow and the active, carrier-mediated cell-to-cell transport. Since plant organs and even tissues are too complex for determination of the kinetics of carrier-mediated auxin uptake and efflux on the cellular level, simplified models of cell suspension cultures are often used, and several tobacco cell lines have been established for auxin transport assays. However, there are very few data available on the specificity and kinetics of auxin transport across the plasma membrane for Arabidopsis thaliana suspension-cultured cells. In this report, the characteristics of carrier-mediated uptake (influx) and efflux for the native auxin indole-3-acetic acid and synthetic auxins, naphthalene-1-acetic and 2,4-dichlorophenoxyacetic acids (NAA and 2,4-D, respectively) in A. thaliana ecotype Landsberg erecta suspension-cultured cells (LE line) are provided. By auxin competition assays and inhibitor treatments, we show that, similarly to tobacco cells, uptake carriers have high affinity towards 2,4-D and that NAA is a good tool for studies of auxin efflux in LE cells. In contrast to tobacco cells, metabolic profiling showed that only a small proportion of NAA is metabolized in LE cells. These results show that the LE cell line is a useful experimental system for measurements of kinetics of auxin carriers on the cellular level that is complementary to tobacco cells.
- MeSH
- Arabidopsis cytologie růst a vývoj metabolismus MeSH
- biologický transport MeSH
- buněčné kultury MeSH
- fenotyp MeSH
- hypokotyl cytologie růst a vývoj metabolismus MeSH
- kotyledon cytologie růst a vývoj metabolismus MeSH
- kyselina 2,4-dichlorfenoxyoctová metabolismus MeSH
- kyseliny indoloctové metabolismus MeSH
- kyseliny naftalenoctové metabolismus MeSH
- listy rostlin cytologie růst a vývoj metabolismus MeSH
- metabolom MeSH
- regulátory růstu rostlin metabolismus MeSH
- semenáček cytologie růst a vývoj metabolismus MeSH
- tabák cytologie růst a vývoj metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Minimally invasive in vivo measurement of pH in microscopic biological samples of μm or μl size, e.g. plant cells, tissues and saps, may help to explain complex biological processes. Consequently, techniques to achieve such measurements are a focus of interest for botanists. This paper describes a technique for the in vivo measurement of pH in the range pH5.0 to pH7.8 in microscopic plant tissue samples of Arabidopsis thaliana based on a ratiometric fluorescence method using low-loss robust tapered fiber probes. For this purpose tapered fiber probes were prepared and coated with a detection layer containing ion-paired fluorescent pH-transducer 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (c-HPTS). A fluorescence ratiometric approach was employed based on excitation at 415 nm and 450 nm and on the comparison of the fluorescence response at 515 nm. The suitability of tapered fiber probes for local detection of pH between 5.0 and 7.8 was demonstrated. A pH sensitivity of 0.15 pH units was achieved within the pH ranges 5.0-5.9 and 7.1-7.8, and this was improved to 0.04 pH units within the pH range 5.9-7.1. Spatial resolution of the probes was better than 20 μm and a time response within 15-20s was achieved. Despite the minute dimensions of the tapered fiber probes the setup developed was relatively robust and compact in construction and performed reliably. It has been successfully employed for the in vivo local determination of pH of mechanically resistant plant tissues of A. thaliana of microscopic scale. The detection of momentary pH gradients across the intact plant seems to be a good tool for the determination of changes in pH in response to experimental treatments affecting for example enzyme activities, availability of mineral nutrients, hormonal control of plant development and plant responses to environmental cues.
