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
Chromosome pairing in meiosis usually starts in the vicinity of the telomere attachment to the nuclear membrane and congregation of telomeres in the leptotene bouquet is believed responsible for bringing homologue pairs together. In a heterozygote for an inversion of a rye (Secale cereale L.) chromosome arm in wheat, a distal segment of the normal homologue is capable of chiasmate pairing with its counterpart in the inverted arm, located near the centromere. Using 3D imaging confocal microscopy, we observed that some telomeres failed to be incorporated into the bouquet and occupied various positions throughout the entire volume of the nucleus, including the centromere pole. Rye telomeres appeared ca. 21 times more likely to fail to be included in the telomere bouquet than wheat telomeres. The frequency of the out-of-bouquet rye telomere position in leptotene was virtually identical to the frequency of telomeres deviating from Rabl's orientation in the nuclei of somatic cells, and was similar to the frequency of synapsis of the normal and inverted chromosome arms, but lower than the MI pairing frequency of segments of these two arms normally positioned across the volume of the nucleus. Out-of-position placement of the rye telomeres may be responsible for reduced MI pairing of rye chromosomes in hybrids with wheat and their disproportionate contribution to aneuploidy, but appears responsible for initiating chiasmate pairing of distantly positioned segments of homology in an inversion heterozygote.
- MeSH
- buněčné jádro genetika ultrastruktura MeSH
- centromera chemie ultrastruktura MeSH
- chiméra genetika MeSH
- chromozomální inverze * MeSH
- chromozomy rostlin chemie ultrastruktura MeSH
- druhová specificita MeSH
- heterozygot MeSH
- hybridizace in situ fluorescenční MeSH
- konfokální mikroskopie MeSH
- párování chromozomů MeSH
- počítačové zpracování obrazu statistika a číselné údaje MeSH
- profáze meiózy I * MeSH
- pšenice genetika ultrastruktura MeSH
- rostlinné buňky metabolismus ultrastruktura MeSH
- telomery chemie ultrastruktura MeSH
- žito genetika ultrastruktura MeSH
- zobrazování trojrozměrné metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
During interphase, the chromosomes of eukaryotes decondense and they occupy distinct regions of the nucleus, called chromosome domains or chromosome territories (CTs). In plants, the Rabl's configuration, with telomeres at one pole of nucleus and centromeres at the other, appears to be common, at least in plants with large genomes. It is unclear whether individual chromosomes of plants adopt defined, genetically determined addresses within the nucleus, as is the case in mammals. In this study, the nuclear disposition of alien rye and barley chromosomes and chromosome arm introgressions into wheat while using 3D-FISH in various somatic tissues was analyzed. All of the introgressed chromosomes showed Rabl's orientation, but their relative positions in the nuclei were less clear. While in most cases pairs of introgressed chromosomes occupied discrete positions, their association (proximity) along their entire lengths was rare, and partial association only marginally more frequent. This arrangement is relatively stable in various tissues and during various stages of the cell cycle. On the other hand, the length of a chromosome arm appears to play a role in its positioning in a nucleus: shorter chromosomes or chromosome arms tend to be located closer to the centre of the nucleus, while longer arms are more often positioned at the nuclear periphery.
- MeSH
- buněčné jádro MeSH
- chromatin genetika MeSH
- chromozomy rostlin * MeSH
- hybridizace in situ fluorescenční * metody MeSH
- interfáze * genetika MeSH
- ječmen (rod) genetika MeSH
- počítačové zpracování obrazu MeSH
- průtoková cytometrie MeSH
- pšenice genetika MeSH
- žito genetika MeSH
- Publikační typ
- časopisecké články MeSH
Alien introgressions introduce beneficial alleles into existing crops and hence, are widely used in plant breeding. Generally, introgressed alien chromosomes show reduced meiotic pairing relative to the host genome, and may be eliminated over generations. Reduced pairing appears to result from a failure of some telomeres of alien chromosomes to incorporate into the leptotene bouquet at the onset of meiosis, thereby preventing chiasmate pairing. In this study, we analysed somatic nuclei of rye introgressions in wheat using 3D-FISH and found that while introgressed rye chromosomes or chromosome arms occupied discrete positions in the Rabl's orientation similar to chromosomes of the wheat host, their telomeres frequently occupied positions away from the nuclear periphery. The frequencies of such abnormal telomere positioning were similar to the frequencies of out-of-bouquet telomere positioning at leptotene, and of pairing failure at metaphase I. This study indicates that improper positioning of alien chromosomes that leads to reduced pairing is not a strictly meiotic event but rather a consequence of a more systemic problem. Improper positioning in the nuclei probably impacts the ability of introgressed chromosomes to migrate into the telomere bouquet at the onset of meiosis, preventing synapsis and chiasma establishment, and leading to their gradual elimination over generations.
Phospholipase Dα1 (PLDα1) belongs to phospholipases, a large phospholipid hydrolyzing protein family. PLDα1 has a substrate preference for phosphatidylcholine leading to enzymatic production of phosphatidic acid, a lipid second messenger with multiple cellular functions. PLDα1 itself is implicated in biotic and abiotic stress responses. Here, we present a shot-gun differential proteomic analysis on roots of two Arabidopsis pldα1 mutants compared to the wild type. Interestingly, PLDα1 deficiency leads to altered abundances of proteins involved in diverse processes related to membrane transport including endocytosis and endoplasmic reticulum-Golgi transport. PLDα1 may be involved in the stability of attachment sites of endoplasmic reticulum to the plasma membrane as suggested by increased abundance of synaptotagmin 1, which was validated by immunoblotting and whole-mount immunolabelling analyses. Moreover, we noticed a robust abundance alterations of proteins involved in mitochondrial import and electron transport chain. Notably, the abundances of numerous proteins implicated in glucosinolate biosynthesis were also affected in pldα1 mutants. Our results suggest a broader biological involvement of PLDα1 than anticipated thus far, especially in the processes such as endomembrane transport, mitochondrial protein import and protein quality control, as well as glucosinolate biosynthesis.
- MeSH
- Arabidopsis metabolismus MeSH
- endocytóza MeSH
- fosfolipasa D genetika metabolismus MeSH
- genová ontologie MeSH
- glukosinoláty biosyntéza MeSH
- kořeny rostlin metabolismus MeSH
- mitochondriální proteiny metabolismus MeSH
- proteiny huseníčku genetika metabolismus MeSH
- proteom metabolismus MeSH
- proteomika * MeSH
- synaptotagmin I metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- transport proteinů MeSH
- uncoupling protein 1 metabolismus MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
Isoprenoid cytokinins play a number of crucial roles in the regulation of plant growth and development. To study cytokinin receptor properties in plants, we designed and prepared fluorescent derivatives of 6-[(3-methylbut-2-en-1-yl)amino]purine (N6-isopentenyladenine, iP) with several fluorescent labels attached to the C2 or N9 atom of the purine moiety via a 2- or 6-carbon linker. The fluorescent labels included dansyl (DS), fluorescein (FC), 7-nitrobenzofurazan (NBD), rhodamine B (RhoB), coumarin (Cou), 7-(diethylamino)coumarin (DEAC) and cyanine 5 dye (Cy5). All prepared compounds were screened for affinity for the Arabidopsis thaliana cytokinin receptor (CRE1/AHK4). Although the attachment of the fluorescent labels to iP via the linkers mostly disrupted binding to the receptor, several fluorescent derivatives interacted well. For this reason, three derivatives, two rhodamine B and one 4-chloro-7-nitrobenzofurazan labeled iP were tested for their interaction with CRE1/AHK4 and Zea mays cytokinin receptors in detail. We further showed that the three derivatives were able to activate transcription of cytokinin response regulator ARR5 in Arabidopsis seedlings. The activity of fluorescently labeled cytokinins was compared with corresponding 6-dimethylaminopurine fluorescently labeled negative controls. Selected rhodamine B C2-labeled compounds 17, 18 and 4-chloro-7-nitrobenzofurazan N9-labeled compound 28 and their respective negative controls (19, 20 and 29, respectively) were used for in planta staining experiments in Arabidopsis thaliana cell suspension culture using live cell confocal microscopy.
- MeSH
- 4-chlor-7-nitrobenzofurazan farmakologie MeSH
- adenin analogy a deriváty chemie MeSH
- Arabidopsis metabolismus MeSH
- barvicí látky chemie MeSH
- cytokininy chemie farmakologie MeSH
- fluorescenční barviva chemie MeSH
- isopentenyladenosin chemická syntéza chemie farmakologie MeSH
- karbocyaniny chemie MeSH
- konfokální mikroskopie MeSH
- kukuřice setá metabolismus MeSH
- molekulární struktura MeSH
- proteiny huseníčku metabolismus MeSH
- puriny chemie MeSH
- receptory cytokinové antagonisté a inhibitory chemie MeSH
- regulace genové exprese u rostlin MeSH
- regulátory růstu rostlin metabolismus MeSH
- rhodaminy chemie MeSH
- semenáček metabolismus MeSH
- terpeny metabolismus MeSH
- vývoj rostlin MeSH
- Publikační typ
- časopisecké články MeSH
Plant mitogen-activated protein kinases (MAPKs) constitute a network of signaling cascades responsible for transducing extracellular stimuli and decoding them to dedicated cellular and developmental responses that shape the plant body. Over the last decade, we have accumulated information about how MAPK modules control the development of reproductive tissues and gametes and the embryogenic and postembryonic development of vegetative organs such as roots, root nodules, shoots, and leaves. Of key importance to understanding how MAPKs participate in developmental and environmental signaling is the characterization of their subcellular localization, their interactions with upstream signal perception mechanisms, and the means by which they target their substrates. In this review, we summarize the roles of MAPK signaling in the regulation of key plant developmental processes, and we survey what is known about the mechanisms guiding the subcellular compartmentalization of MAPK modules.
The carnivorous sundew plant (Drosera capensis) captures prey using sticky tentacles. We investigated the tentacle and trap reactions in response to the electrical and jasmonate signalling evoked by different stimuli to reveal how carnivorous sundews recognize digestible captured prey in their traps. We measured the electrical signals, phytohormone concentration, enzyme activities and Chla fluorescence in response to mechanical stimulation, wounding or insect feeding in local and systemic traps. Seven new proteins in the digestive fluid were identified using mass spectrometry. Mechanical stimuli and live prey induced a fast, localized tentacle-bending reaction and enzyme secretion at the place of application. By contrast, repeated wounding induced a nonlocalized convulsive tentacle movement and enzyme secretion in local but also in distant systemic traps. These differences can be explained in terms of the electrical signal propagation and jasmonate accumulation, which also had a significant impact on the photosynthesis in the traps. The electrical signals generated in response to wounding could partially mimic a mechanical stimulation of struggling prey and might trigger a false alarm, confirming that the botanical carnivory and plant defence mechanisms are related. To trigger the full enzyme activity, the traps must detect chemical stimuli from the captured prey.
- MeSH
- biologické modely MeSH
- cyklopentany metabolismus MeSH
- Drosera enzymologie fyziologie MeSH
- elektrofyziologické jevy * MeSH
- fotochemické procesy MeSH
- fotosystém II - proteinový komplex metabolismus MeSH
- oxylipiny metabolismus MeSH
- regulátory růstu rostlin metabolismus MeSH
- rostlinné proteiny metabolismus MeSH
- signální transdukce * MeSH
- Publikační typ
- časopisecké články MeSH
Microtubule organization and dynamics are critical for key developmental processes such as cell division, elongation, and morphogenesis. Microtubule severing is an essential regulator of microtubules and is exclusively executed by KATANIN 1 in Arabidopsis In this study, we comparatively studied the proteome-wide effects in two KATANIN 1 mutants. Thus, shotgun proteomic analysis of roots and aerial parts of single nucleotide mutant fra2 and T-DNA insertion mutant ktn1-2 was carried out. We have detected 42 proteins differentially abundant in both fra2 and ktn1-2 KATANIN 1 dysfunction altered the abundance of proteins involved in development, metabolism, and stress responses. The differential regulation of tubulins and microtubule-destabilizing protein MDP25 implied a feedback microtubule control in KATANIN 1 mutants. Furthermore, deregulation of profilin 1, actin-depolymerizing factor 3, and actin 7 was observed. These findings were confirmed by immunoblotting analysis of actin and by microscopic observation of actin filaments using fluorescently labeled phalloidin. Results obtained by quantitative RT-PCR analysis revealed that changed protein abundances were not a consequence of altered expression levels of corresponding genes in the mutants. In conclusion, we show that abundances of several cytoskeletal proteins as well as organization of microtubules and the actin cytoskeleton are amended in accordance with defective microtubule severing.
- MeSH
- aktiny metabolismus MeSH
- anotace sekvence MeSH
- Arabidopsis genetika metabolismus MeSH
- biologie buňky * MeSH
- genová ontologie MeSH
- katanin genetika MeSH
- mapy interakcí proteinů MeSH
- mikrotubuly metabolismus MeSH
- mutace genetika MeSH
- proteiny huseníčku genetika metabolismus MeSH
- proteom metabolismus MeSH
- proteomika metody MeSH
- rostlinné geny MeSH
- zpětná vazba fyziologická * MeSH
- Publikační typ
- časopisecké články MeSH
