Formins are evolutionarily conserved eukaryotic proteins engaged in actin nucleation and other aspects of cytoskeletal organization. Angiosperms have two formin clades with multiple paralogs; typical plant Class I formins are integral membrane proteins that can anchor cytoskeletal structures to membranes. For the main Arabidopsis housekeeping Class I formin, FH1 (At3g25500), plasmalemma localization was documented in heterologous expression and overexpression studies. We previously showed that loss of FH1 function increases cotyledon epidermal pavement cell shape complexity via modification of actin and microtubule organization and dynamics. Here, we employ transgenic Arabidopsis expressing green fluorescent protein-tagged FH1 (FH1-GFP) from its native promoter to investigate in vivo behavior of this formin using advanced microscopy techniques. The fusion protein is functional, since its expression complements the fh1 loss-of-function mutant phenotype. Accidental overexpression of FH1-GFP results in a decrease in trichome branch number, while fh1 mutation has the opposite effect, indicating a general role of this formin in controlling cell shape complexity. Consistent with previous reports, FH1-GFP associates with membranes. However, the protein exhibits surprising actin- and secretory pathway-dependent dynamic localization and relocates between cellular endomembranes and the plasmalemma during cell division and differentiation in root tissues, with transient tonoplast localization at the transition/elongation zones border. FH1-GFP also accumulates in actin-rich regions of cortical cytoplasm and associates with plasmodesmata in both the cotyledon epidermis and root tissues. Together with previous reports from metazoan systems, this suggests that formins might have a shared (ancestral or convergent) role at cell-cell junctions.

• MeSH
• Arabidopsis cytologie   metabolismus   MeSH
• cytoskelet genetika   metabolismus   MeSH
• kořeny rostlin cytologie   metabolismus   MeSH
• plazmodesmy fyziologie   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

FM (Fei-Mao) styryl dyes are commonly used for the fluorescence imaging of plasma membrane (PM) and endocytosis in vivo. Thanks to their amphiphilic character, these dyes are incorporated in the outer leaflet of the PM lipid bilayer and emit fluorescence in its hydrophobic environment. The endocytic pathway of FM dye uptake starts with rapid PM staining and continues in PM invaginations and membrane vesicles during endocytosis, followed by staining of trans-Golgi network (TGN) and ending in tonoplast (vacuolar membrane). FM dyes do not stain endoplasmic reticulum and nuclear membrane. The time-lapse fluorescence microscopy could track endocytic vesicles and characterize the rate of endocytosis in vivo. On the other hand, fixable FM dyes (FX) can be used for the visualization of particular steps in the FM dye uptake in situ. Staining with FM dyes and subsequent microscopic observations could be performed on both tissue and cellular level. Here, we describe simple procedures for the effective FM dye staining and destaining in root tip of Arabidopsis thaliana seedlings and suspension-cultured tobacco cells.

• MeSH
• Arabidopsis cytologie   ultrastruktura   MeSH
• buněčná membrána ultrastruktura   MeSH
• endocytóza * MeSH
• fluorescenční barviva analýza   MeSH
• fluorescenční mikroskopie metody   MeSH
• kořeny rostlin cytologie   ultrastruktura   MeSH
• optické zobrazování metody   MeSH
• tabák cytologie   ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Increasing usage of gold nanoparticles (AuNPs) in different industrial areas inevitably leads to their release into the environment. Thus, living organisms, including plants, may be exposed to a direct contact with nanoparticles (NPs). Despite the growing amount of research on this topic, our knowledge about NPs uptake by plants and their influence on different developmental processes is still insufficient. The first physical barrier for NPs penetration to the plant body is a cell wall which protects cytoplasm from external factors and environmental stresses. The absence of a cell wall may facilitate the internalization of various particles including NPs. Our studies have shown that AuNPs, independently of their surface charge, did not cross the cell wall of Arabidopsis thaliana (L.) roots. However, the research carried out with using light and transmission electron microscope revealed that AuNPs with different surface charge caused diverse changes in the root's histology and ultrastructure. Therefore, we verified whether this is only the wall which protects cells against particles penetration and for this purpose we used protoplasts culture. It has been shown that plasma membrane (PM) is not a barrier for positively charged (+) AuNPs and negatively charged (-) AuNPs, which passage to the cell.

• MeSH
• Arabidopsis cytologie   růst a vývoj   ultrastruktura   MeSH
• buněčná stěna metabolismus   MeSH
• kořeny rostlin cytologie   růst a vývoj   ultrastruktura   MeSH
• kovové nanočástice chemie   ultrastruktura   MeSH
• povrchové vlastnosti MeSH
• protoplasty cytologie   metabolismus   ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH

Uptake of water and nutrients by roots affects the ontogenesis of the whole plant. Nanoparticles, e.g. gold nanoparticles, have a broad range of applications in many fields which leads to the transfer of these materials into the environment. Thus, the understanding of their impact on the growth and development of the root system is an emerging issue. During our studies on the effect of positively charged gold nanoparticles on the barley roots, a hairless phenotype was found. We investigated whether this phenotype correlates with changes in symplasmic communication, which is an important factor that regulates, among others, differentiation of the rhizodermis into hair and non-hair cells. The results showed no restriction in symplasmic communication in the treated roots, in contrast to the control roots, in which the trichoblasts and atrichoblasts were symplasmically isolated during their differentiation. Moreover, differences concerning the root morphology, histology, ultrastructure and the cell wall composition were detected between the control and the treated roots. These findings suggest that the harmful effect of nanoparticles on plant growth may, among others, consist in disrupting the symplasmic communication/isolation, which leads to the development of a hairless root phenotype, thus limiting the functioning of the roots.

• MeSH
• buněčná diferenciace účinky léků   MeSH
• buněčná membrána metabolismus   MeSH
• epidermis rostlin cytologie   účinky léků   metabolismus   MeSH
• ječmen (rod) účinky léků   genetika   růst a vývoj   metabolismus   MeSH
• kořeny rostlin cytologie   účinky léků   růst a vývoj   metabolismus   MeSH
• kovové nanočástice toxicita   MeSH
• látky znečišťující půdu toxicita   MeSH
• regulace genové exprese u rostlin účinky léků   MeSH
• semenáček účinky léků   růst a vývoj   MeSH
• voda metabolismus   MeSH
• živiny metabolismus   MeSH
• zlato toxicita   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

As non-photosynthesizing organs, roots are dependent on diffusion of oxygen from the external environment and, in some instances, from the shoot for their aerobic metabolism. Establishment of hypoxic niches in the developing tissues of plants has been postulated as a consequence of insufficient diffusion of oxygen to satisfy the demands throughout development. Here, we report that such niches are established at specific stages of lateral root primordia development in Arabidopsis thaliana grown under aerobic conditions. Using gain- and loss-of-function mutants, we show that ERF-VII transcription factors, which mediate hypoxic responses, control root architecture by acting in cells with a high level of auxin signaling. ERF-VIIs repress the expression of the auxin-induced genes LBD16, LBD18, and PUCHI, which are essential for lateral root development, by binding to their promoters. Our results support a model in which the establishment of hypoxic niches in the developing lateral root primordia contributes to the shutting down of key auxin-induced genes and regulates the production of lateral roots.

• MeSH
• Arabidopsis cytologie   genetika   metabolismus   MeSH
• hypoxie buňky MeSH
• kořeny rostlin cytologie   MeSH
• kyseliny indoloctové metabolismus   MeSH
• proteiny huseníčku metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• signální transdukce * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

During plant growth and defense, cell cycle activity needs to be coordinated with cell wall integrity. Little is known about how this coordination is achieved. Here, we investigated coordination in Arabidopsis thaliana seedlings by studying the impact of cell wall damage (CWD, caused by cellulose biosynthesis inhibition) on cytokinin homeostasis, cell cycle gene expression and cell shape in root tips. CWD inhibited cell cycle gene expression and increased transition zone cell width in an osmosensitive manner. These results were correlated with CWD-induced, osmosensitive changes in cytokinin homeostasis. Expression of CYTOKININ OXIDASE/DEHYDROGENASE 2 and 3 (CKX2, CKX3), which encode cytokinin-degrading enzymes, was induced by CWD and reduced by osmoticum treatment. In nitrate reductase1 nitrate reductase2 (nia1 nia2) seedlings, CKX2 and CKX3 transcript levels were not increased and cell cycle gene expression was not repressed by CWD. Moreover, established CWD-induced responses, such as jasmonic acid, salicylic acid and lignin production, were also absent, implying a central role of NIA1/2-mediated processes in regulation of CWD responses. These results suggest that CWD enhances cytokinin degradation rates through a NIA1/2-mediated process, leading to attenuation of cell cycle gene expression.

• MeSH
• Arabidopsis cytologie   účinky léků   genetika   MeSH
• benzamidy farmakologie   MeSH
• biologické modely MeSH
• buněčná stěna účinky léků   metabolismus   MeSH
• buněčný cyklus účinky léků   genetika   MeSH
• cytokininy farmakologie   MeSH
• fenotyp MeSH
• homeostáza účinky léků   MeSH
• kořeny rostlin cytologie   účinky léků   růst a vývoj   MeSH
• messenger RNA genetika   metabolismus   MeSH
• nitrátreduktasa metabolismus   MeSH
• osmóza MeSH
• proteiny huseníčku metabolismus   MeSH
• regulace genové exprese u rostlin * účinky léků   MeSH
• semenáček účinky léků   genetika   MeSH
• sorbitol farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Legume mutants have shown the requirement for receptor-mediated cytokinin signaling in symbiotic nodule organogenesis. While the receptors are central regulators, cytokinin also is accumulated during early phases of symbiotic interaction, but the pathways involved have not yet been fully resolved. To identify the source, timing, and effect of this accumulation, we followed transcript levels of the cytokinin biosynthetic pathway genes in a sliding developmental zone ofLotus japonicusroots.LjIpt2andLjLog4were identified as the major contributors to the first cytokinin burst. The genetic dependence and Nod factor responsiveness of these genes confirm that cytokinin biosynthesis is a key target of the common symbiosis pathway. The accumulation ofLjIpt2andLjLog4transcripts occurs independent of theLjLhk1receptor during nodulation. Together with the rapid repression of both genes by cytokinin, this indicates thatLjIpt2andLjLog4contribute to, rather than respond to, the initial cytokinin buildup. Analysis of the cytokinin response using the synthetic cytokinin sensor,TCSn, showed that this response occurs in cortical cells before spreading to the epidermis inL. japonicusWhile mutant analysis identified redundancy in several biosynthesis families, we found that mutation ofLjIpt4limits nodule numbers. Overexpression ofLjIpt3orLjLog4alone was insufficient to produce the robust formation of spontaneous nodules. In contrast, overexpressing a complete cytokinin biosynthesis pathway leads to large, often fused spontaneous nodules. These results show the importance of cytokinin biosynthesis in initiating and balancing the requirement for cortical cell activation without uncontrolled cell proliferation.

• MeSH
• biologické modely MeSH
• cytokininy biosyntéza   MeSH
• kořenové hlízky rostlin cytologie   genetika   růst a vývoj   fyziologie   MeSH
• kořeny rostlin cytologie   genetika   růst a vývoj   fyziologie   MeSH
• Lotus cytologie   genetika   růst a vývoj   fyziologie   MeSH
• regulace genové exprese u rostlin MeSH
• regulátory růstu rostlin biosyntéza   MeSH
• Rhizobiaceae fyziologie   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• signální transdukce * MeSH
• symbióza MeSH
• tvorba kořenových hlízek MeSH
• vývojová regulace genové exprese genetika   MeSH
• Publikační typ
• časopisecké články MeSH

Nitrogen (N) is an essential macronutrient that limits agricultural productivity, and both low and high N supply have been suggested to alter plant growth. The overall aim of this work is to study the impact of nitrate (NO3(-)) in maize yield and the possible causes that induce this alteration. High NO3(-) doses did not increase the yield of maize grown neither in the field nor under controlled conditions. In fact, plants grown under controlled conditions for 45 days with NO3(-) concentrations over 5mM showed a decrease in biomass production. This reduction was perceptible in shoots prior to roots, where phytomer expansion was reduced. Cell size and number were also reduced in the leaves of plants with high NO3(-). This alteration was correlated with the increase of 1-aminocyclopropane-1-carboxylic acid in leaves, which was probably translocated from the roots in order to synthesize ethylene. Cytokinins (CKs) also showed a relevant role in this inhibitory effect, increasing in high NO3(-) plants with a reduction in root and shoot growth, inhibition of apical dominance and a strong decrease of leaf expansion, symptoms described previously as "CK syndrome". We propose that high NO3(-) inhibits maize growth by causing hormonal alterations that modify plant growth from cell to whole plant.

• MeSH
• aminokyseliny cyklické metabolismus   MeSH
• biomasa MeSH
• cytokininy metabolismus   MeSH
• dusičnany farmakologie   MeSH
• dusík metabolismus   MeSH
• epidermis rostlin cytologie   účinky léků   růst a vývoj   MeSH
• kořeny rostlin cytologie   účinky léků   růst a vývoj   MeSH
• kukuřice setá cytologie   účinky léků   růst a vývoj   MeSH
• listy rostlin cytologie   účinky léků   růst a vývoj   MeSH
• výhonky rostlin cytologie   účinky léků   růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH

Cytokinins (CKs) play a crucial role in many physiological and developmental processes at the levels of individual plant components (cells, tissues, and organs) and by coordinating activities across these parts. High-resolution measurements of intracellular CKs in different plant tissues can therefore provide insights into their metabolism and mode of action. Here, we applied fluorescence-activated cell sorting of green fluorescent protein (GFP)-marked cell types, combined with solid-phase microextraction and an ultra-high-sensitivity mass spectrometry (MS) method for analysis of CK biosynthesis and homeostasis at cellular resolution. This method was validated by series of control experiments, establishing that protoplast isolation and cell sorting procedures did not greatly alter endogenous CK levels. The MS-based method facilitated the quantification of all the well known CK isoprenoid metabolites in four different transgenic Arabidopsis thaliana lines expressing GFP in specific cell populations within the primary root apex. Our results revealed the presence of a CK gradient within the Arabidopsis root tip, with a concentration maximum in the lateral root cap, columella, columella initials, and quiescent center cells. This distribution, when compared with previously published auxin gradients, implies that the well known antagonistic interactions between the two hormone groups are cell type specific.

• MeSH
• Arabidopsis cytologie   metabolismus   MeSH
• biologický transport MeSH
• cytokininy metabolismus   MeSH
• extrakce na pevné fázi MeSH
• kořeny rostlin cytologie   metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• meristém metabolismus   MeSH
• metabolom MeSH
• miniaturizace MeSH
• orgánová specificita MeSH
• protoplasty metabolismus   MeSH
• průtoková cytometrie MeSH
• separace buněk MeSH
• zelené fluorescenční proteiny metabolismus   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