To identify novel genes engaged in plant epidermal development, we characterized the phenotypic variability of rosette leaf epidermis of 310 sequenced Arabidopsis thaliana accessions, focusing on trichome shape and distribution, compositional characteristics of the trichome cell wall, and histologically detectable metal ion distribution. Some of these traits correlated with cLimate parameters of our accession's locations of origin, suggesting environmental selection. A novel metal deposition pattern in stomatal guard cells was observed in some accessions. Subsequent GWAS analysis identified 1546 loci with protein sequence-altering SNPs associated with one or more traits, including 5 genes with previously reported relevant mutant phenotypes and 80 additional genes with known or predicted roles in relevant developmental and cellular processes. Some candidates, including GFS9/TT9, exhibited environmentally correlated allele distribution. Several large gene famiLies, namely DUF674, DUF784, DUF1262, DUF1985, DUF3741, cytochrome P450, receptor-Like kinases, Cys/His-rich C1 domain proteins and formins were overrepresented among the candidates for various traits, suggesting epidermal development-related functions. A possible participation of formins in guard cell metal deposition was supported by observations in available loss of function mutants. Screening of candidate gene lists against the STRING interactome database uncovered several predominantly nuclear protein interaction networks with possible novel roles in epidermal development.

• Klíčová slova
• Arabidopsis thaliana, BioClim, GWAS, guard cell, metal accumulation, phenotypic variability, trichome,
• MeSH
• Arabidopsis * genetika   metabolismus   růst a vývoj   MeSH
• celogenomová asociační studie * MeSH
• epidermis rostlin * metabolismus   genetika   růst a vývoj   MeSH
• fenotyp MeSH
• jednonukleotidový polymorfismus genetika   MeSH
• kovy * metabolismus   MeSH
• listy rostlin * genetika   metabolismus   růst a vývoj   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné geny * MeSH
• trichomy * růst a vývoj   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kovy * MeSH
• proteiny huseníčku MeSH

Formins are a large, evolutionarily old family of cytoskeletal regulators whose roles include actin capping and nucleation, as well as modulation of microtubule dynamics. The plant class I formin clade is characterized by a unique domain organization, as most of its members are transmembrane proteins with possible cell wall-binding motifs exposed to the extracytoplasmic space-a structure that appears to be a synapomorphy of the plant kingdom. While such transmembrane formins are traditionally considered mainly as plasmalemma-localized proteins contributing to the organization of the cell cortex, we review, from a cell biology perspective, the growing evidence that they can also, at least temporarily, reside (and in some cases also function) in endomembranes including secretory and endocytotic pathway compartments, the endoplasmic reticulum, the nuclear envelope, and the tonoplast. Based on this evidence, we propose that class I formins may thus serve as 'active cargoes' of membrane trafficking-membrane-embedded proteins that modulate the fate of endo- or exocytotic compartments while being transported by them.

• Klíčová slova
• Actin, biotic interactions, cell growth, cytokinesis, endocytosis, exocytosis, formin, microtubules, plasmalemma, tonoplast,
• MeSH
• buněčná membrána * metabolismus   MeSH
• forminy * metabolismus   MeSH
• membránové proteiny metabolismus   genetika   MeSH
• rostlinné proteiny metabolismus   genetika   MeSH
• transport proteinů * MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• forminy * MeSH
• membránové proteiny MeSH
• rostlinné proteiny MeSH

The phragmoplast separates daughter cells during cytokinesis by constructing the cell plate, which depends on interaction between cytoskeleton and membrane compartments. Proteins responsible for these interactions remain unknown, but formins can link cytoskeleton with membranes and several members of formin protein family localize to the cell plate. Progress in functional characterization of formins in cytokinesis is hindered by functional redundancies within the large formin gene family. We addressed this limitation by employing Small Molecular Inhibitor of Formin Homology 2 (SMIFH2), a small-molecule inhibitor of formins. Treatment of tobacco (Nicotiana tabacum) tissue culture cells with SMIFH2 perturbed localization of actin at the cell plate; slowed down both microtubule polymerization and phragmoplast expansion; diminished association of dynamin-related proteins with the cell plate independently of actin and microtubules; and caused cell plate swelling. Another impact of SMIFH2 was shortening of the END BINDING1b (EB1b) and EB1c comets on the growing microtubule plus ends in N. tabacum tissue culture cells and Arabidopsis thaliana cotyledon epidermis cells. The shape of the EB1 comets in the SMIFH2-treated cells resembled that of the knockdown mutant of plant Xenopus Microtubule-Associated protein of 215 kDa (XMAP215) homolog MICROTUBULE ORGANIZATION 1/GEMINI 1 (MOR1/GEM1). This outcome suggests that formins promote elongation of tubulin flares on the growing plus ends. Formins AtFH1 (A. thaliana Formin Homology 1) and AtFH8 can also interact with EB1. Besides cytokinesis, formins function in the mitotic spindle assembly and metaphase to anaphase transition. Our data suggest that during cytokinesis formins function in: (1) promoting microtubule polymerization; (2) nucleating F-actin at the cell plate; (3) retaining dynamin-related proteins at the cell plate; and (4) remodeling of the cell plate membrane.

• MeSH
• aktiny metabolismus   MeSH
• Arabidopsis účinky léků   genetika   fyziologie   MeSH
• cytokineze účinky léků   genetika   MeSH
• cytoskelet účinky léků   metabolismus   MeSH
• forminy genetika   metabolismus   MeSH
• mikrotubuly účinky léků   metabolismus   MeSH
• tabák účinky léků   genetika   fyziologie   MeSH
• thioketony farmakologie   MeSH
• tubulin metabolismus   MeSH
• uracil analogy a deriváty   farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• aktiny MeSH
• forminy MeSH
• SMIFH2 compound MeSH Prohlížeč
• thioketony MeSH
• tubulin MeSH
• uracil MeSH

The ARP2/3 complex and formins are the only known plant actin nucleators. Besides their actin-related functions, both systems also modulate microtubule organization and dynamics. Loss of the main housekeeping Arabidopsis thaliana Class I membrane-targeted formin FH1 (At3g25500) is known to increase cotyledon pavement cell lobing, while mutations affecting ARP2/3 subunits exhibit an opposite effect. Here we examine the role of FH1 and the ARP2/3 complex subunit ARPC5 (At4g01710) in epidermal cell morphogenesis with focus on pavement cells and trichomes using a model system of single fh1 and arpc5, as well as double fh1 arpc5 mutants. While cotyledon pavement cell shape in double mutants mostly resembled single arpc5 mutants, analysis of true leaf epidermal morphology, as well as actin and microtubule organization and dynamics, revealed a more complex relationship between the two systems and similar, rather than antagonistic, effects on some parameters. Both fh1 and arpc5 mutations increased actin network density and increased cell shape complexity in pavement cells and trichomes of first true leaves, in contrast to cotyledons. Thus, while the two actin nucleation systems have complementary roles in some aspects of cell morphogenesis in cotyledon pavement cells, they may act in parallel in other cell types and developmental stages.

• Klíčová slova
• ARP2/3, At3g25500, At4g01710, actin nucleation, cytoskeleton, formin, pavement cell, trichome,
• Publikační typ
• časopisecké články MeSH

Formins are evolutionarily conserved multi-domain proteins participating in the control of both actin and microtubule dynamics. Angiosperm formins form two evolutionarily distinct families, Class I and Class II, with class-specific domain layouts. The model plant Arabidopsis thaliana has 21 formin-encoding loci, including 10 Class II members. In this study, we analyze the subcellular localization of two A. thaliana Class II formins exhibiting typical domain organization, the so far uncharacterized formin AtFH13 (At5g58160) and its distant homolog AtFH14 (At1g31810), previously reported to bind microtubules. Fluorescent protein-tagged full length formins and their individual domains were transiently expressed in Nicotiana benthamiana leaves under the control of a constitutive promoter and their subcellular localization (including co-localization with cytoskeletal structures and the endoplasmic reticulum) was examined using confocal microscopy. While the two formins exhibit distinct and only partially overlapping localization patterns, they both associate with microtubules via the conserved formin homology 2 (FH2) domain and with the periphery of the endoplasmic reticulum, at least in part via the N-terminal PTEN (Phosphatase and Tensin)-like domain. Surprisingly, FH2 domains of AtFH13 and AtFH14 can form heterodimers in the yeast two-hybrid assay-a first case of potentially biologically relevant formin heterodimerization mediated solely by the FH2 domain.

• Klíčová slova
• At1g31810, At5g58160, AtFH13, AtFH14, FH2 domain, PTEN-like domain, class II formin, confocal laser scanning microscopy,
• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• dimerizace MeSH
• endoplazmatické retikulum metabolismus   MeSH
• exprese genu MeSH
• forminy genetika   metabolismus   MeSH
• mikrotubuly metabolismus   MeSH
• proteinové domény MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• tabák metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• forminy MeSH
• proteiny huseníčku MeSH
• rekombinantní proteiny MeSH

SH3P2 (At4g34660), an Arabidopsis thaliana SH3 and Bin/amphiphysin/Rvs (BAR) domain-containing protein, was reported to have a specific role in cell plate assembly, unlike its paralogs SH3P1 (At1g31440) and SH3P3 (At4g18060). SH3P family members were also predicted to interact with formins-evolutionarily conserved actin nucleators that participate in microtubule organization and in membrane-cytoskeleton interactions. To trace the origin of functional specialization of plant SH3Ps, we performed phylogenetic analysis of SH3P sequences from selected plant lineages. SH3Ps are present in charophytes, liverworts, mosses, lycophytes, gymnosperms, and angiosperms, but not in volvocal algae, suggesting association of these proteins with phragmoplast-, but not phycoplast-based cell division. Separation of three SH3P clades, represented by SH3P1, SH3P2, and SH3P3 of A. thaliana, appears to be a seed plant synapomorphy. In the yeast two hybrid system, Arabidopsis SH3P3, but not SH3P2, binds the FH1 and FH2 domains of the formin FH5 (At5g54650), known to participate in cytokinesis, while an opposite binding specificity was found for the dynamin homolog DRP1A (At5g42080), confirming earlier findings. This suggests that the cytokinetic role of SH3P2 is not due to its interaction with FH5. Possible determinants of interaction specificity of SH3P2 and SH3P3 were identified bioinformatically.

• Klíčová slova
• cell plate, cytokinesis, evolution, formin, interaction specificity, phylogeny,
• MeSH
• Arabidopsis MeSH
• cytokineze * MeSH
• dynaminy metabolismus   MeSH
• fylogeneze MeSH
• molekulární evoluce * MeSH
• proteiny huseníčku klasifikace   genetika   metabolismus   MeSH
• transportní proteiny klasifikace   genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dynaminy MeSH
• proteiny huseníčku MeSH
• SH3P2 protein, Arabidopsis MeSH Prohlížeč
• transportní proteiny MeSH

Cell division and expansion are two fundamental biological processes supporting indeterminate root growth and development of plants. Quantitative evaluations of cell divisions related to root growth analyses have been performed in several model crop and non-crop plant species, but not in important legume plant Medicago sativa. Light-sheet fluorescence microscopy (LSFM) is an advanced imaging technique widely used in animal developmental biology, providing efficient fast optical sectioning under physiological conditions with considerably reduced phototoxicity and photobleaching. Long-term 4D imaging of living plants offers advantages for developmental cell biology not available in other microscopy approaches. Recently, LSFM was implemented in plant developmental biology studies, however, it is largely restricted to the model plant Arabidopsis thaliana. Cellular and subcellular events in crop species and robust plant samples have not been studied by this method yet. Therefore we performed LSFM long-term live imaging of growing root tips of transgenic alfalfa plants expressing the fluorescent molecular marker for the microtubule-binding domain (GFP-MBD), in order to study dynamic patterns of microtubule arrays during mitotic cell division. Quantitative evaluations of cell division progress in the two root tissues (epidermis and cortex) clearly indicate that root growth rate is correlated with duration of cell division in alfalfa roots. Our results favor non-invasive environmental LSFM as one of the most suitable methods for qualitative and quantitative cellular and developmental imaging of living transgenic legume crops.

• Klíčová slova
• Medicago sativa, cell division, developmental imaging, light-sheet microscopy, microtubules, root growth, transgenic crops,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Cytoskeleton can be observed in live plant cells in situ with high spatial and temporal resolution using a combination of specific fluorescent protein tag expression and advanced microscopy methods such as spinning disc confocal microscopy (SDCM) or variable angle epifluorescence microscopy (VAEM). Existing methods for quantifying cytoskeletal dynamics are often either based on laborious manual structure tracking, or depend on costly commercial software. Current automated methods also do not readily allow separate measurements of structure lifetime, lateral mobility, and spatial anisotropy of these parameters. RESULTS: We developed a new freeware-based, operational system-independent semi-manual technique for analyzing VAEM or SDCM data, QuACK (Quantitative Analysis of Cytoskeletal Kymograms), and validated it on data from Arabidopsis thaliana fh1 formin mutants, previously shown by conventional methods to exhibit altered actin and microtubule dynamics compared to the wild type. Besides of confirming the published mutant phenotype, QuACK was used to characterize surprising differential effects of various fluorescent protein tags fused to the Lifeact actin probe on actin dynamics in A. thaliana cotyledon epidermis. In particular, Lifeact-YFP slowed down actin dynamics compared to Lifeact-GFP at marker expression levels causing no macroscopically noticeable phenotypic alterations, although the two fluorophores are nearly identical. We could also demonstrate the expected, but previously undocumented, anisotropy of cytoskeletal dynamics in elongated epidermal cells of A. thaliana petioles and hypocotyls. CONCLUSIONS: Our new method for evaluating plant cytoskeletal dynamics has several advantages over existing techniques. It is intuitive, rapid compared to fully manual approaches, based on the free ImageJ software (including macros we provide here for download), and allows measurement of multiple parameters. Our approach was already used to document unexpected differences in actin mobility in transgenic A. thaliana expressing Lifeact fusion proteins with different fluorophores, highlighting the need for cautious interpretation of experimental results, as well as to reveal hitherto uncharacterized anisotropy of cytoskeletal mobility in elongated plant cells.

• Klíčová slova
• Actin, Anisotropy, FH1 (At3g25500), Kymogram, Lateral mobility, Lifeact, Microtubules, Spinning disc confocal microscopy, Structure stability, Variable angle fluorescence microscopy,
• Publikační typ
• časopisecké články MeSH

Development of the plant aerial organs epidermis involves a complex interplay of cytoskeletal rearrangements, membrane trafficking-dependent cell surface expansion, and intra- and intercellular signaling, resulting in a pattern of perfectly interlocking pavement cells. While recent detailed in vivo observations convincingly identify microtubules rather than actin as key players at the early stages of development of pavement cell lobes in Arabidopsis, mutations affecting the actin-nucleating ARP2/3 complex are long known to reduce pavement cell lobing, suggesting a central role for actin. We have now shown that functional impairment of the Arabidopsis formin FH1 enhances both microtubule dynamics and pavement cell lobing. While formins are best known for their ability to nucleate actin, many members of this old gene family now emerge as direct or indirect regulators of the microtubule cytoskeleton, and our findings suggest that they might co-ordinate action of the two cytoskeletal systems during pavement cell morphogenesis.

• Klíčová slova
• Actin, FH2 proteins, cell growth, epidermal pavement cells, formins, microtubules,
• MeSH
• Arabidopsis růst a vývoj   metabolismus   ultrastruktura   MeSH
• biologické modely MeSH
• cytoskelet metabolismus   fyziologie   ultrastruktura   MeSH
• forminy MeSH
• membránové proteiny genetika   metabolismus   fyziologie   MeSH
• mikrotubuly metabolismus   fyziologie   ultrastruktura   MeSH
• multigenová rodina MeSH
• proteiny huseníčku genetika   metabolismus   fyziologie   MeSH
• rostlinné buňky metabolismus   ultrastruktura   MeSH
• signální transdukce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AFH1 protein, Arabidopsis MeSH Prohlížeč
• forminy MeSH
• membránové proteiny MeSH
• proteiny huseníčku MeSH

The cytoskeleton plays a central part in spatial organization of the plant cytoplasm, including the endomebrane system. However, the mechanisms involved are so far only partially understood. Formins (FH2 proteins), a family of evolutionarily conserved proteins sharing the FH2 domain whose dimer can nucleate actin, mediate the co-ordination between actin and microtubule cytoskeletons in multiple eukaryotic lineages including plants. Moreover, some plant formins contain transmembrane domains and participate in anchoring cytoskeletal structures to the plasmalemma, and possibly to other membranes. Direct or indirect membrane association is well documented even for some fungal and metazoan formins lacking membrane insertion motifs, and FH2 proteins have been shown to associate with endomembranes and modulate their dynamics in both fungi and metazoans. Here we summarize the available evidence suggesting that formins participate in membrane trafficking and endomembrane, especially ER, organization also in plants. We propose that, despite some methodological pitfalls inherent to in vivo studies based on (over)expression of truncated and/or tagged proteins, formins are beginning to emerge as candidates for the so far somewhat elusive link between the plant cytoskeleton and the endomembrane system.

• MeSH
• intracelulární membrány metabolismus   MeSH
• mikrofilamenta metabolismus   MeSH
• proteiny asociované s mikrotubuly chemie   genetika   metabolismus   MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• proteiny huseníčku chemie   genetika   metabolismus   MeSH
• rostlinné buňky metabolismus   MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• FH16 protein, Arabidopsis MeSH Prohlížeč
• FH5 protein, Arabidopsis MeSH Prohlížeč
• proteiny asociované s mikrotubuly MeSH
• proteiny buněčného cyklu MeSH
• proteiny huseníčku MeSH