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.

• Keywords
• Arabidopsis thaliana, BioClim, GWAS, guard cell, metal accumulation, phenotypic variability, trichome,
• MeSH
• Arabidopsis * genetics   metabolism   growth & development   MeSH
• Genome-Wide Association Study * MeSH
• Plant Epidermis * metabolism   genetics   growth & development   MeSH
• Phenotype MeSH
• Polymorphism, Single Nucleotide genetics   MeSH
• Metals * metabolism   MeSH
• Plant Leaves * genetics   metabolism   growth & development   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Genes, Plant * MeSH
• Trichomes * growth & development   genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Metals * MeSH
• Arabidopsis Proteins 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.

• Keywords
• Actin, biotic interactions, cell growth, cytokinesis, endocytosis, exocytosis, formin, microtubules, plasmalemma, tonoplast,
• MeSH
• Cell Membrane * metabolism   MeSH
• Formins * metabolism   MeSH
• Membrane Proteins metabolism   genetics   MeSH
• Plant Proteins metabolism   genetics   MeSH
• Protein Transport * MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Formins * MeSH
• Membrane Proteins MeSH
• Plant Proteins MeSH

Primary root growth is required by the plant to anchor in the soil and reach out for nutrients and water, while dealing with obstacles. Efficient root elongation and bending depends upon the coordinated action of environmental sensing, signal transduction, and growth responses. The actin cytoskeleton is a highly plastic network that constitutes a point of integration for environmental stimuli and hormonal pathways. In this review, we present a detailed compilation highlighting the importance of the actin cytoskeleton during primary root growth and we describe how actin-binding proteins, plant hormones, and actin-disrupting drugs affect root growth and root actin. We also discuss the feedback loop between actin and root responses to light and gravity. Actin affects cell division and elongation through the control of its own organization. We remark upon the importance of longitudinally oriented actin bundles as a hallmark of cell elongation as well as the role of the actin cytoskeleton in protein trafficking and vacuolar reshaping during this process. The actin network is shaped by a plethora of actin-binding proteins; however, there is still a large gap in connecting the molecular function of these proteins with their developmental effects. Here, we summarize their function and known effects on primary root growth with a focus on their high level of specialization. Light and gravity are key factors that help us understand root growth directionality. The response of the root to gravity relies on hormonal, particularly auxin, homeostasis, and the actin cytoskeleton. Actin is necessary for the perception of the gravity stimulus via the repositioning of sedimenting statoliths, but it is also involved in mediating the growth response via the trafficking of auxin transporters and cell elongation. Furthermore, auxin and auxin analogs can affect the composition of the actin network, indicating a potential feedback loop. Light, in its turn, affects actin organization and hence, root growth, although its precise role remains largely unknown. Recently, fundamental studies with the latest techniques have given us more in-depth knowledge of the role and organization of actin in the coordination of root growth; however, there remains a lot to discover, especially in how actin organization helps cell shaping, and therefore root growth.

• Keywords
• actin, actin-binding protein, auxin, cell elongation, gravitropism, light, root growth,
• Publication type
• Journal Article MeSH
• Review 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
• Actins metabolism   MeSH
• Arabidopsis drug effects   genetics   physiology   MeSH
• Cytokinesis drug effects   genetics   MeSH
• Cytoskeleton drug effects   metabolism   MeSH
• Formins genetics   metabolism   MeSH
• Microtubules drug effects   metabolism   MeSH
• Nicotiana drug effects   genetics   physiology   MeSH
• Thiones pharmacology   MeSH
• Tubulin metabolism   MeSH
• Uracil analogs & derivatives   pharmacology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Actins MeSH
• Formins MeSH
• SMIFH2 compound MeSH Browser
• Thiones MeSH
• Tubulin MeSH
• Uracil MeSH

Formins are a large, evolutionarily conserved family of actin-nucleating proteins with additional roles in regulating microfilament, microtubule, and membrane dynamics. Angiosperm formins, expressed in both sporophytic and gametophytic tissues, can be divided into two subfamilies, Class I and Class II, each often exhibiting characteristic domain organization. Gametophytically expressed Class I formins have been documented to mediate plasma membrane-based actin assembly in pollen grains and pollen tubes, contributing to proper pollen germination and pollen tube tip growth, and a rice Class II formin, FH5/RMD, has been proposed to act as a positive regulator of pollen tube growth based on mutant phenotype and overexpression data. Here we report functional characterization of the Arabidopsis thaliana pollen-expressed typical Class II formin FH13 (At5g58160). Consistent with published transcriptome data, live-cell imaging in transgenic plants expressing fluorescent protein-tagged FH13 under the control of the FH13 promoter revealed expression in pollen and pollen tubes with non-homogeneous signal distribution in pollen tube cytoplasm, suggesting that this formin functions in the male gametophyte. Surprisingly, fh13 loss of function mutations do not affect plant fertility but result in stimulation of in vitro pollen tube growth, while tagged FH13 overexpression inhibits pollen tube elongation. Pollen tubes of mutants expressing a fluorescent actin marker exhibited possible minor alterations of actin organization. Our results thus indicate that FH13 controls or limits pollen tube growth, or, more generally, that typical Class II formins should be understood as modulators of pollen tube elongation rather than merely components of the molecular apparatus executing tip growth.

• Keywords
• Arabidopsis thaliana, At5g58160, Class II formin, pollen tube, tip growth,
• Publication type
• Journal Article 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.

• Keywords
• ARP2/3, At3g25500, At4g01710, actin nucleation, cytoskeleton, formin, pavement cell, trichome,
• Publication type
• Journal Article 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.

• Keywords
• At1g31810, At5g58160, AtFH13, AtFH14, FH2 domain, PTEN-like domain, class II formin, confocal laser scanning microscopy,
• MeSH
• Arabidopsis genetics   metabolism   MeSH
• Dimerization MeSH
• Endoplasmic Reticulum metabolism   MeSH
• Gene Expression MeSH
• Formins genetics   metabolism   MeSH
• Microtubules metabolism   MeSH
• Protein Domains MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Recombinant Proteins genetics   metabolism   MeSH
• Nicotiana metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Formins MeSH
• Arabidopsis Proteins MeSH
• Recombinant Proteins 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.

• Keywords
• cell plate, cytokinesis, evolution, formin, interaction specificity, phylogeny,
• MeSH
• Arabidopsis MeSH
• Cytokinesis * MeSH
• Dynamins metabolism   MeSH
• Phylogeny MeSH
• Evolution, Molecular * MeSH
• Arabidopsis Proteins classification   genetics   metabolism   MeSH
• Carrier Proteins classification   genetics   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Dynamins MeSH
• Arabidopsis Proteins MeSH
• SH3P2 protein, Arabidopsis MeSH Browser
• Carrier Proteins MeSH