root tip
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Tip growth is one of the most preferable models in the study of plant cell polarity; cell wall deposition is restricted mainly to a certain area of the cell, and cell expansion at this specific area leads to the development of tubular outgrowth. Tip-growing root hairs are well-established systems for such studies, because their lateral position within the root makes them easily accessible for experimental approaches and microscopic observations. Fundamental structural and molecular processes driving tip growth are exocytosis, endocytosis, and all aspects of vesicular and endosomal dynamic trafficking, as related to targeted membrane flow. Study of vesicles and endosomes in living root hairs, however, is rather difficult, due to their small size and due to the resolution limits of conventional light microscopes. Here we present noninvasive approaches for visualizing vesicular and endosomal compartments in the tip of growing root hairs using electronic light microscopy, contrast-enhanced video light microscopy, and confocal laser scanning microscopy (CLSM). These methods allow utilizing the maximum resolution of the light microscope. Together with protocols for appropriate preparation of living plant samples, the described methods should help improve our understanding on how tiny vesicles and endosomes support the process of tip growth in root hairs.
- MeSH
- Arabidopsis růst a vývoj ultrastruktura MeSH
- endozomy metabolismus ultrastruktura MeSH
- exocytóza genetika MeSH
- konfokální mikroskopie MeSH
- kořeny rostlin růst a vývoj ultrastruktura MeSH
- molekulární biologie metody MeSH
- polarita buněk MeSH
- transport proteinů MeSH
- transportní vezikuly metabolismus ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Arabidopsis PIN2 protein directs transport of the phytohormone auxin from the root tip into the root elongation zone. Variation in hormone transport, which depends on a delicate interplay between PIN2 sorting to and from polar plasma membrane domains, determines root growth. By employing a constitutively degraded version of PIN2, we identify brassinolides as antagonists of PIN2 endocytosis. This response does not require de novo protein synthesis, but involves early events in canonical brassinolide signaling. Brassinolide-controlled adjustments in PIN2 sorting and intracellular distribution governs formation of a lateral PIN2 gradient in gravistimulated roots, coinciding with adjustments in auxin signaling and directional root growth. Strikingly, simulations indicate that PIN2 gradient formation is no prerequisite for root bending but rather dampens asymmetric auxin flow and signaling. Crosstalk between brassinolide signaling and endocytic PIN2 sorting, thus, appears essential for determining the rate of gravity-induced root curvature via attenuation of differential cell elongation.
- MeSH
- Arabidopsis účinky léků metabolismus MeSH
- biologický transport účinky léků MeSH
- brassinosteroidy metabolismus farmakologie MeSH
- endocytóza účinky léků MeSH
- gravitropismus účinky léků fyziologie MeSH
- kořeny rostlin účinky léků metabolismus MeSH
- kyseliny indoloctové metabolismus MeSH
- meristém účinky léků metabolismus MeSH
- proteiny huseníčku metabolismus MeSH
- regulátory růstu rostlin metabolismus farmakologie MeSH
- signální transdukce MeSH
- steroidy heterocyklické metabolismus farmakologie 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
Redirection of intercellular auxin fluxes via relocalization of the PIN-FORMED 3 (PIN3) and PIN7 auxin efflux carriers has been suggested to be necessary for the root gravitropic response. Cytokinins have also been proposed to play a role in controlling root gravitropism, but conclusive evidence is lacking. We present a detailed study of the dynamics of root bending early after gravistimulation, which revealed a delayed gravitropic response in transgenic lines with depleted endogenous cytokinins (Pro35S:AtCKX) and cytokinin signaling mutants. Pro35S:AtCKX lines, as well as a cytokinin receptor mutant ahk3, showed aberrations in the auxin response distribution in columella cells consistent with defects in the auxin transport machinery. Using in vivo real-time imaging of PIN3-GFP and PIN7-GFP in AtCKX3 overexpression and ahk3 backgrounds, we observed wild-type-like relocalization of PIN proteins in the columella early after gravistimulation, with gravity-induced relocalization of PIN7 faster than that of PIN3. Nonetheless, the cellular distribution of PIN3 and PIN7 and expression of PIN7 and the auxin influx carrier AUX1 was affected in AtCKX overexpression lines. Based on the retained cytokinin sensitivity in pin3 pin4 pin7 mutant, we propose the AUX1-mediated auxin transport rather than columella-located PIN proteins as a target of endogenous cytokinins in the control of root gravitropism.
- MeSH
- Arabidopsis účinky léků fyziologie MeSH
- biologické modely MeSH
- biologický transport účinky léků MeSH
- cytokininy farmakologie MeSH
- gravitace MeSH
- gravitropismus účinky léků MeSH
- kořeny rostlin účinky léků fyziologie MeSH
- kyseliny indoloctové metabolismus MeSH
- meristém účinky léků fyziologie MeSH
- proteiny huseníčku metabolismus MeSH
- signální transdukce účinky léků MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Plant growth depends on stem cell niches in meristems. In the root apical meristem, the quiescent center (QC) cells form a niche together with the surrounding stem cells. Stem cells produce daughter cells that are displaced into a transit-amplifying (TA) domain of the root meristem. TA cells divide several times to provide cells for growth. SHORTROOT (SHR) and SCARECROW (SCR) are key regulators of the stem cell niche. Cytokinin controls TA cell activities in a dose-dependent manner. Although the regulatory programs in each compartment of the root meristem have been identified, it is still unclear how they coordinate one another. Here, we investigate how PHABULOSA (PHB), under the posttranscriptional control of SHR and SCR, regulates TA cell activities. The root meristem and growth defects in shr or scr mutants were significantly recovered in the shr phb or scr phb double mutant, respectively. This rescue in root growth occurs in the absence of a QC. Conversely, when the modified PHB, which is highly resistant to microRNA, was expressed throughout the stele of the wild-type root meristem, root growth became very similar to that observed in the shr; however, the identity of the QC was unaffected. Interestingly, a moderate increase in PHB resulted in a root meristem phenotype similar to that observed following the application of high levels of cytokinin. Our protoplast assay and transgenic approach using ARR10 suggest that the depletion of TA cells by high PHB in the stele occurs via the repression of B-ARR activities. This regulatory mechanism seems to help to maintain the cytokinin homeostasis in the meristem. Taken together, our study suggests that PHB can dynamically regulate TA cell activities in a QC-independent manner, and that the SHR-PHB pathway enables a robust root growth system by coordinating the stem cell niche and TA domain.
- MeSH
- Arabidopsis genetika růst a vývoj MeSH
- buněčné dělení genetika MeSH
- cytokininy genetika metabolismus MeSH
- DNA vazebné proteiny genetika MeSH
- fenotyp MeSH
- geneticky modifikované rostliny růst a vývoj MeSH
- homeodoménové proteiny biosyntéza genetika MeSH
- homeostáza MeSH
- kořeny rostlin genetika růst a vývoj MeSH
- meristém genetika růst a vývoj MeSH
- nika kmenových buněk genetika MeSH
- proteiny huseníčku biosyntéza genetika metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- transkripční faktory genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Programmed cell death in plants occurs both during stress responses and as an integral part of regular plant development. Despite the undisputed importance of developmentally controlled cell death processes for plant growth and reproduction, we are only beginning to understand the underlying molecular genetic regulation. Exploiting the Arabidopsis thaliana root cap as a cell death model system, we identified two NAC transcription factors, the little-characterized ANAC087 and the leaf-senescence regulator ANAC046, as being sufficient to activate the expression of cell death-associated genes and to induce ectopic programmed cell death. In the root cap, these transcription factors are involved in the regulation of distinct aspects of programmed cell death. ANAC087 orchestrates postmortem chromatin degradation in the lateral root cap via the nuclease BFN1. In addition, both ANAC087 and ANAC046 redundantly control the onset of cell death execution in the columella root cap during and after its shedding from the root tip. Besides identifying two regulators of developmental programmed cell death, our analyses reveal the existence of an actively controlled cell death program in Arabidopsis columella root cap cells.
- MeSH
- Arabidopsis genetika metabolismus MeSH
- kořeny rostlin genetika metabolismus MeSH
- meristém genetika metabolismus MeSH
- proteiny huseníčku genetika metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- transkripční faktory genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Background and Aims: The actin cytoskeleton forms a dynamic network in plant cells. A single-point mutation in the DER1 (deformed root hairs1) locus located in the sequence of ACTIN2, a gene for major actin in vegetative tissues of Arabidopsis thaliana, leads to impaired root hair development (Ringli C, Baumberger N, Diet A, Frey B, Keller B. 2002. ACTIN2 is essential for bulge site selection and tip growth during root hair development of Arabidopsis. Plant Physiology129: 1464-1472). Only root hair phenotypes have been described so far in der1 mutants, but here we demonstrate obvious aberrations in the organization of the actin cytoskeleton and overall plant development. Methods: Organization of the actin cytoskeleton in epidermal cells of cotyledons, hypocotyls and roots was studied qualitatively and quantitatively by live-cell imaging of transgenic lines carrying the GFP-FABD2 fusion protein and in fixed cells after phalloidin labelling. Patterns of root growth were characterized by FM4-64 vital staining, light-sheet microscopy imaging and microtubule immunolabelling. Plant phenotyping included analyses of germination, root growth and plant biomass. Key Results: Speed of germination, plant fresh weight and total leaf area were significantly reduced in the der1-3 mutant in comparison with the C24 wild-type. Actin filaments in root, hypocotyl and cotyledon epidermal cells of the der1-3 mutant were shorter, thinner and arranged in more random orientations, while actin bundles were shorter and had altered orientations. The wavy pattern of root growth in der1-3 mutant was connected with higher frequencies of shifted cell division planes (CDPs) in root cells, which was consistent with the shifted positioning of microtubule-based preprophase bands and phragmoplasts. The organization of cortical microtubules in the root cells of the der1-3 mutant, however, was not altered. Conclusions: Root growth rate of the der1-3 mutant is not reduced, but changes in the actin cytoskeleton organization can induce a wavy root growth pattern through deregulation of CDP orientation. The results suggest that the der1-3 mutation in the ACT2 gene does not influence solely root hair formation process, but also has more general effects on the actin cytoskeleton, plant growth and development.
