Alterations of hydrogen peroxide (H2O2) levels have a profound impact on numerous signaling cascades orchestrating plant growth, development, and stress signaling, including programmed cell death. To expand the repertoire of known molecular mechanisms implicated in H2O2 signaling, we performed a forward chemical screen to identify small molecules that could alleviate the photorespiratory-induced cell death phenotype of Arabidopsisthaliana mutants lacking H2O2-scavenging capacity by peroxisomal catalase2. Here, we report the characterization of pakerine, an m-sulfamoyl benzamide from the sulfonamide family. Pakerine alleviates the cell death phenotype of cat2 mutants exposed to photorespiration-promoting conditions and delays dark-induced senescence in wild-type Arabidopsis leaves. By using a combination of transcriptomics, metabolomics, and affinity purification, we identified abnormal inflorescence meristem 1 (AIM1) as a putative protein target of pakerine. AIM1 is a 3-hydroxyacyl-CoA dehydrogenase involved in fatty acid β-oxidation that contributes to jasmonic acid (JA) and salicylic acid (SA) biosynthesis. Whereas intact JA biosynthesis was not required for pakerine bioactivity, our results point toward a role for β-oxidation-dependent SA production in the execution of H2O2-mediated cell death.
- MeSH
- Arabidopsis cytologie účinky léků genetika metabolismus MeSH
- buněčná smrt účinky léků MeSH
- buněčné dýchání účinky léků genetika MeSH
- cyklopentany metabolismus MeSH
- fotosyntéza účinky léků genetika MeSH
- fyziologický stres MeSH
- hydroponie metody MeSH
- kyselina salicylová metabolismus MeSH
- listy rostlin cytologie účinky léků metabolismus MeSH
- meristém cytologie účinky léků metabolismus MeSH
- multienzymové komplexy genetika metabolismus MeSH
- oxylipiny metabolismus MeSH
- peroxid vodíku antagonisté a inhibitory farmakologie MeSH
- proteiny huseníčku genetika metabolismus MeSH
- regulace genové exprese u rostlin * MeSH
- rostlinné buňky účinky léků metabolismus MeSH
- semena rostlinná účinky léků MeSH
- signální transdukce MeSH
- stanovení celkové genové exprese MeSH
- sulfonamidy chemická syntéza farmakologie MeSH
- transkriptom MeSH
- výpočetní biologie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Plant hormone cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Subcellular localization of the receptors proposed the endoplasmic reticulum (ER) membrane as a principal cytokinin perception site, while study of cytokinin transport pointed to the plasma membrane (PM)-mediated cytokinin signalling. Here, by detailed monitoring of subcellular localizations of the fluorescently labelled natural cytokinin probe and the receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin receptors can enter the secretory pathway and reach the PM in cells of the root apical meristem, and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. We provide a revised view on cytokinin signalling and the possibility of multiple sites of perception at PM and ER.
- MeSH
- Arabidopsis cytologie genetika metabolismus MeSH
- brefeldin A farmakologie MeSH
- buněčná membrána metabolismus MeSH
- cytokininy chemie metabolismus MeSH
- endoplazmatické retikulum metabolismus MeSH
- fluorescenční barviva chemie metabolismus MeSH
- geneticky modifikované rostliny MeSH
- meristém cytologie metabolismus MeSH
- proteinkinasy genetika metabolismus MeSH
- proteiny huseníčku genetika metabolismus MeSH
- receptory buněčného povrchu genetika metabolismus MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- signální transdukce účinky léků MeSH
- zelené fluorescenční proteiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mutual interactions of the phytohormones, cytokinins and auxin determine root or shoot identity during postembryonic de novo organogenesis in plants. However, our understanding of the role of hormonal metabolism and perception during early stages of cell fate reprogramming is still elusive. Here we show that auxin activates root formation, whereas cytokinins mediate early loss of the root identity, primordia disorganisation and initiation of shoot development. Exogenous and endogenous cytokinins influence the initiation of newly formed organs, as well as the pace of organ development. The process of de novo shoot apical meristem establishment is accompanied by accumulation of endogenous cytokinins, differential regulation of genes for individual cytokinin receptors, strong activation of AHK4-mediated signalling and induction of the shoot-specific homeodomain regulator WUSCHEL. The last is associated with upregulation of isopentenyladenine-type cytokinins, revealing higher shoot-forming potential when compared with trans-zeatin. Moreover, AHK4-controlled cytokinin signalling negatively regulates the root stem cell organiser WUSCHEL RELATED HOMEOBOX 5 in the root quiescent centre. We propose an important role for endogenous cytokinin biosynthesis and AHK4-mediated cytokinin signalling in the control of de novo-induced organ identity.
- MeSH
- Arabidopsis cytologie embryologie genetika MeSH
- cytokininy genetika metabolismus MeSH
- homeodoménové proteiny genetika metabolismus MeSH
- meristém cytologie embryologie MeSH
- organogeneze rostlin fyziologie MeSH
- proteinkinasy genetika metabolismus MeSH
- proteiny huseníčku genetika metabolismus MeSH
- receptory buněčného povrchu genetika metabolismus MeSH
- signální transdukce fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cytokinins are required for symbiotic nodule development in legumes, and cytokinin signaling responses occur locally in nodule primordia and in developing nodules. Here, we show that the Lotus japonicus Ckx3 cytokinin oxidase/dehydrogenase gene is induced by Nod factor during the early phase of nodule initiation. At the cellular level, pCkx3::YFP reporter-gene studies revealed that the Ckx3 promoter is active during the first cortical cell divisions of the nodule primordium and in growing nodules. Cytokinin measurements in ckx3 mutants confirmed that CKX3 activity negatively regulates root cytokinin levels. Particularly, tZ and DHZ type cytokinins in both inoculated and uninoculated roots were elevated in ckx3 mutants, suggesting that these are targets for degradation by the CKX3 cytokinin oxidase/dehydrogenase. The effect of CKX3 on the positive and negative roles of cytokinin in nodule development, infection and regulation was further clarified using ckx3 insertion mutants. Phenotypic analysis indicated that ckx3 mutants have reduced nodulation, infection thread formation and root growth. We also identify a role for cytokinin in regulating nodulation and nitrogen fixation in response to nitrate as ckx3 phenotypes are exaggerated at increased nitrate levels. Together, these findings show that cytokinin accumulation is tightly regulated during nodulation in order to balance the requirement for cell divisions with negative regulatory effects of cytokinin on infection events and root development.
- MeSH
- alely MeSH
- buněčná diferenciace MeSH
- cytokininy metabolismus MeSH
- dusičnany metabolismus MeSH
- fenotyp MeSH
- fixace dusíku genetika MeSH
- fylogeneze MeSH
- homeostáza * MeSH
- kořenové hlízky rostlin genetika růst a vývoj MeSH
- Lotus enzymologie genetika růst a vývoj MeSH
- meristém cytologie růst a vývoj MeSH
- mutace genetika MeSH
- oxidoreduktasy genetika metabolismus MeSH
- promotorové oblasti (genetika) MeSH
- rostlinné geny MeSH
- rostlinné proteiny genetika metabolismus MeSH
- tvorba kořenových hlízek genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Analysis and sorting of plant chromosomes (plant flow cytogenetics) is a special application of flow cytometry in plant genomics and its success depends critically on sample quality. This unit describes the methodology in a stepwise manner, starting with the induction of cell cycle synchrony and accumulation of dividing cells in mitotic metaphase, and continues with the preparation of suspensions of intact mitotic chromosomes, flow analysis and sorting of chromosomes, and finally processing of the sorted chromosomes. Each step of the protocol is described in detail as some procedures have not been used widely. Supporting histograms are presented as well as hints on dealing with plant material; the utility of sorted chromosomes for plant genomics is also discussed. © 2016 by John Wiley & Sons, Inc.
- MeSH
- chromozomy rostlin metabolismus MeSH
- DNA rostlinná genetika MeSH
- hybridizace in situ fluorescenční MeSH
- karyotypizace MeSH
- meristém cytologie účinky léků MeSH
- metafáze účinky léků MeSH
- molekulová hmotnost MeSH
- oxid dusný farmakologie MeSH
- proteomika MeSH
- průtoková cytometrie metody MeSH
- rostliny genetika MeSH
- semena rostlinná účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
Stress-activated plant mitogen-activated protein (MAP) kinase pathways play roles in growth adaptation to the environment by modulating cell division through cytoskeletal regulation, but the mechanisms are poorly understood. We performed protein interaction and phosphorylation experiments with cytoskeletal proteins, mass spectrometric identification of MPK6 complexes and immunofluorescence analyses of the microtubular cytoskeleton of mitotic cells using wild-type, mpk6-2 mutant and plants overexpressing the MAP kinase-inactivating phosphatase, AP2C3. We showed that MPK6 interacted with γ-tubulin and co-sedimented with plant microtubules polymerized in vitro. It was the active form of MAP kinase that was enriched with microtubules and followed similar dynamics to γ-tubulin, moving from poles to midzone during the anaphase-to-telophase transition. We found a novel substrate for MPK6, the microtubule plus end protein, EB1c. The mpk6-2 mutant was sensitive to 3-nitro-l-tyrosine (NO2 -Tyr) treatment with respect to mitotic abnormalities, and root cells overexpressing AP2C3 showed defects in chromosome segregation and spindle orientation. Our data suggest that the active form of MAP kinase interacts with γ-tubulin on specific subsets of mitotic microtubules during late mitosis. MPK6 phosphorylates EB1c, but not EB1a, and has a role in maintaining regular planes of cell division under stress conditions.
- MeSH
- anafáze účinky léků MeSH
- aparát dělícího vřeténka účinky léků metabolismus MeSH
- Arabidopsis cytologie účinky léků enzymologie MeSH
- butadieny farmakologie MeSH
- cytokineze účinky léků MeSH
- extracelulárním signálem regulované MAP kinasy metabolismus MeSH
- fosforylace účinky léků MeSH
- fyziologický stres * účinky léků MeSH
- kinetochory účinky léků metabolismus MeSH
- meristém cytologie účinky léků metabolismus MeSH
- mikrotubuly účinky léků metabolismus MeSH
- mitogenem aktivované proteinkinasy metabolismus MeSH
- nitrily farmakologie MeSH
- nitrosace účinky léků MeSH
- proliferace buněk účinky léků MeSH
- proteiny asociované s mikrotubuly metabolismus MeSH
- proteiny huseníčku metabolismus MeSH
- rostlinné buňky účinky léků metabolismus MeSH
- segregace chromozomů účinky léků MeSH
- telofáze účinky léků MeSH
- tubulin metabolismus MeSH
- tyrosin analogy a deriváty farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The role of YODA MITOGEN ACTIVATED PROTEIN KINASE KINASE KINASE 4 (MAPKKK4) upstream of MITOGEN ACTIVATED PROTEIN KINASE 6 (MPK6) was studied during post-embryonic root development of Arabidopsis thaliana. Loss- and gain-of-function mutants of YODA (yda1 and ΔNyda1) were characterized in terms of root patterning, endogenous auxin content and global proteomes. We surveyed morphological and cellular phenotypes of yda1 and ΔNyda1 mutants suggesting possible involvement of auxin. Endogenous indole-3-acetic acid (IAA) levels were up-regulated in both mutants. Proteomic analysis revealed up-regulation of auxin biosynthetic enzymes tryptophan synthase and nitrilases in these mutants. The expression, abundance and phosphorylation of MPK3, MPK6 and MICROTUBULE ASSOCIATED PROTEIN 65-1 (MAP65-1) were characterized by quantitative polymerase chain reaction (PCR) and western blot analyses and interactions between MAP65-1, microtubules and MPK6 were resolved by quantitative co-localization studies and co-immunoprecipitations. yda1 and ΔNyda1 mutants showed disoriented cell divisions in primary and lateral roots, abortive cytokinesis, and differential subcellular localization of MPK6 and MAP65-1. They also showed deregulated expression of TANGLED1 (TAN1), PHRAGMOPLAST ORIENTING KINESIN 1 (POK1), and GAMMA TUBULIN COMPLEX PROTEIN 4 (GCP4). The findings that MPK6 localized to preprophase bands (PPBs) and phragmoplasts while the mpk6-4 mutant transformed with MPK6AEF (alanine (A)-glutamic acid (E)-phenylanine (F)) showed a root phenotype similar to that of yda1 demonstrated that MPK6 is an important player downstream of YODA. These data indicate that YODA and MPK6 are involved in post-embryonic root development through an auxin-dependent mechanism regulating cell division and mitotic microtubule (PPB and phragmoplast) organization.
- MeSH
- Arabidopsis cytologie účinky léků embryologie enzymologie MeSH
- buněčné dělení * účinky léků MeSH
- cytokineze účinky léků MeSH
- epidermis rostlin cytologie MeSH
- fenotyp MeSH
- fluorescenční protilátková technika MeSH
- fosforylace účinky léků MeSH
- interfáze MeSH
- kořeny rostlin anatomie a histologie cytologie embryologie MeSH
- kyseliny indoloctové metabolismus farmakologie MeSH
- MAP kinasy kinas (kinas) metabolismus MeSH
- meristém cytologie účinky léků MeSH
- mikrotubuly účinky léků metabolismus MeSH
- mitogenem aktivované proteinkinasy metabolismus MeSH
- mitóza účinky léků MeSH
- mutace genetika MeSH
- proteiny huseníčku metabolismus MeSH
- proteomika MeSH
- transport proteinů účinky léků MeSH
- upregulace * účinky léků MeSH
- vazba proteinů účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In higher plants, cell cycle activation in the meristems at germination is essential for the initiation of post-embryonic development. We previously identified the signaling pathways of homeobox transcription factor STIMPY and metabolic sugars as two interacting branches of the regulatory network that is responsible for activating meristematic tissue proliferation in Arabidopsis. In this study, we found that CYCP2;1 is both a direct target of STIMPY transcriptional activation and an early responder to sugar signals. Genetic and molecular studies show that CYCP2;1 physically interacts with three of the five mitotic CDKs in Arabidopsis, and is required for the G2 to M transition during meristem activation. Taken together, our results suggest that CYCP2;1 acts as a permissive control of cell cycle progression during seedling establishment by directly linking genetic control and nutritional cues with the activity of the core cell cycle machinery.
- MeSH
- aktivace transkripce MeSH
- Arabidopsis embryologie MeSH
- buněčné dělení genetika MeSH
- cyklin-dependentní kinasy biosyntéza MeSH
- cykliny biosyntéza genetika metabolismus MeSH
- homeodoménové proteiny genetika MeSH
- meristém cytologie embryologie MeSH
- proliferace buněk MeSH
- proteiny huseníčku biosyntéza genetika metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- rostlinné geny MeSH
- sacharosa farmakologie MeSH
- semenáček genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Plant vascular meristems are sets of pluripotent cells that enable radial growth by giving rise to vascular tissues and are therefore crucial to plant development. However, the overall dynamics of cellular determination and patterning in and around vascular meristems is still unexplored. We study this process in the shoot vascular tissue of Arabidopsis thaliana, which is organized in vascular bundles that contain three basic cell types (procambium, xylem and phloem). A set of molecules involved in this process has now been identified and partially characterized, but it is not yet clear how the regulatory interactions among them, in conjunction with cellular communication processes, give rise to the steady patterns that accompany cell-fate determination and arrangement within vascular bundles. We put forward a dynamic model factoring in the interactions between molecules (genes, peptides, mRNA and hormones) that have been reported to be central in this process, as well as the relevant communication mechanisms. When a few proposed interactions (unverified, but based on related data) are postulated, the model reproduces the hormonal and molecular patterns expected for the three regions within vascular bundles. In order to test the model, we simulated mutant and hormone-depleted systems and compared the results with experimentally reported phenotypes. The proposed model provides a formal framework integrating a set of growing experimental data and renders a dynamic account of how the collective action of hormones, genes, and other molecules may result in the specification of the three main cell types within shoot vascular bundles. It also offers a tool to test the necessity and sufficiency of particular interactions and conditions for vascular patterning and yields novel predictions that may be experimentally tested. Finally, this model provides a reference for further studies comparing the overall dynamics of tissue organization and formation by meristems in other plant organs and species.
- MeSH
- algoritmy MeSH
- Arabidopsis cytologie genetika metabolismus MeSH
- biologické modely MeSH
- buněčná diferenciace MeSH
- cévní svazky rostlin cytologie genetika metabolismus MeSH
- cytokininy fyziologie MeSH
- genové regulační sítě MeSH
- meristém cytologie genetika metabolismus MeSH
- počítačová simulace MeSH
- proteinkinasy fyziologie MeSH
- proteiny huseníčku fyziologie MeSH
- regulace genové exprese u rostlin MeSH
- signální transdukce MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND AND AIMS: The maize lrt1 (lateral rootless1) mutant is impaired in its development of lateral roots during early post-embryonic development. The aim of this study was to characterize, in detail, the influences that the mutation exerts on lateral root initiation and the subsequent developments, as well as to describe the behaviour of the entire plant under variable environmental conditions. METHODS: Mutant lrt1 plants were cultivated under different conditions of hydroponics, and in between sheets of moist paper. Cleared whole mounts and anatomical sections were used in combination with both selected staining procedures and histochemical tests to follow root development. Root surface permeability tests and the biochemical quantification of lignin were performed to complement the structural data. KEY RESULTS: The data presented suggest a redefinition of lrt1 function in lateral roots as a promoter of later development; however, neither the complete absence of lateral roots nor the frequency of their initiation is linked to lrt1 function. The developmental effects of lrt1 are under strong environmental influences. Mutant primordia are affected in structure, growth and emergence; and the majority of primordia terminate their growth during this last step, or shortly thereafter. The lateral roots are impaired in the maintenance of the root apical meristem. The primary root shows disturbances in the organization of both epidermal and subepidermal layers. The lrt1-related cell-wall modifications include: lignification in peripheral layers, the deposition of polyphenolic substances and a higher activity of peroxidase. CONCLUSIONS: The present study provides novel insights into the function of the lrt1 gene in root system development. The lrt1 gene participates in the spatial distribution of initiation, but not in its frequency. Later, the development of lateral roots is strongly affected. The effect of the lrt1 mutation is not as obvious in the primary root, with no influences observed on the root apical meristem structure and maintenance; however, development of the epidermis and cortex are impaired.
- MeSH
- buněčná stěna metabolismus MeSH
- epidermis rostlin anatomie a histologie genetika růst a vývoj MeSH
- hydroponie MeSH
- kořeny rostlin cytologie genetika růst a vývoj MeSH
- kukuřice setá cytologie genetika růst a vývoj MeSH
- lignin metabolismus MeSH
- meristém cytologie genetika růst a vývoj MeSH
- mutace MeSH
- polyfenoly metabolismus MeSH
- regulace genové exprese u rostlin * MeSH
- rostlinné proteiny genetika metabolismus MeSH
- semenáček cytologie genetika růst a vývoj MeSH
- výhonky rostlin cytologie genetika růst a vývoj MeSH
- vývojová regulace genové exprese MeSH
- životní prostředí MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH