Progress in the molecular genetic analysis of trichome initiation and morphogenesis in Arabidopsis

Arabidopsis trichomes are large unicellular structures that develop on the surface of most shoot-derived organs. In leaves, the number, spacing and shape of trichomes is tightly regulated, and this process has been used as an experimental system to study the control of cell fate and pattern formation. The control of trichome initiation is complex: both the potential of a cell to adopt the trichome cell fate and an intricate signaling pathway determine the pattern of trichome initiation events. Several important new results suggest that trichome initiation and morphogenesis are redundantly regulated by both positive and negative factors. A testable model for the control of trichome initiation is presented.     

Organized F-actin is essential for normal trichome morphogenesis in Arabidopsis

Actin microfilaments form a three-dimensional cytoskeletal network throughout the cell and constitute an essential throughway for organelle and vesicle transport. Development of Arabidopsis trichomes, unicellular structures derived from the epidermis, is being used as a genetic system in which to study actin-dependent growth in plant cells. The present study indicates that filamentous actin (F-actin) plays an important role during Arabidopsis trichome morphogenesis. For example, immunolocalization of actin filaments during trichome morphogenesis identified rearrangements of the cytoskeletal structure during the development of the mature cell. Moreover, pharmacological experiments indicate that there are distinct requirements for actin- and microtubule-dependent function during trichome morphogenesis. The F-actin-disrupting drug cytochalasin D does not affect the establishment of polarity during trichome development; however, maintenance and coordination of the normal pattern of cell growth are very sensitive to this drug. In contrast, oryzalin, an agent that depolymerizes microtubules, severely inhibits cell polarization. Furthermore, cytochalasin D treatment phenocopies a known class of mutations that cause distorted trichome morphology. Results of an analysis of cell shape and microfilament structure in wild-type, mutant, and drug-treated trichomes are consistent with a role for actin in the maintenance and coordination of an established growth pattern.     

The actin cytoskeleton is required to elaborate and maintain spatial patterning during trichome cell morphogenesis in Arabidopsis thaliana

Arabidopsis thaliana trichomes provide an attractive model system to dissect molecular processes involved in the generation of shape and form in single cell morphogenesis in plants. We have used transgenic Arabidopsis plants carrying a GFP-talin chimeric gene to analyze the role of the actin cytoskeleton in trichome cell morphogenesis. We found that during trichome cell development the actin microfilaments assumed an increasing degree of complexity from fine filaments to thick, longitudinally stretched cables. Disruption of the F-actin cytoskeleton by actin antagonists produced distorted but branched trichomes which phenocopied trichomes of mutants belonging to the 'distorted' class. Subsequent analysis of the actin cytoskeleton in trichomes of the distorted mutants, alien, crooked, distorted1, gnarled, klunker and wurm uncovered actin organization defects in each case. Treatments of wild-type seedlings with microtubule-interacting drugs elicited a radically different trichome phenotype characterized by isotropic growth and a severe inhibition of branch formation; these trichomes did not show defects in actin cytoskeleton organization. A normal actin cytoskeleton was also observed in trichomes of the zwichel mutant which have reduced branching. ZWICHEL, which was previously shown to encode a kinesin-like protein is thought to be involved in microtubule-linked processes. Based on our results we propose that microtubules establish the spatial patterning of trichome branches whilst actin microfilaments elaborate and maintain the overall trichome pattern during development.     

Developmental canalization with no part of stabilizing selection

Referring the developmental canalization to stabilizing selection may be a bias that results from the ignorance of developmental mechanisms. Considering the morphological evolution of one-cell trichomes in Draba plants makes it clear that the transition from continuous variation in morphological traits to developmental creods occurs in the evolution of remote lineages of the genus irrespective of contribution to the net fitness. Morphological diversification of trichome branching is not under selection control, being a physical consequence of the trichome cell volume growth equilibrated by complication of the cell surface shape. At the start of evolution, the trichome development refers not to an individual trichome, but rather to repetitive trichome modules (branches), whose spatiotemporal order is arbitrary, except that some variants of branching depend on events that occur at earlier developmental stages more than others. Under selection fluctuating at random, or with no selection at all, fixing of these variants leads to the formation of trichome ontogeny, in which earlier developmental stages correspond to later stages of developmental evolution.     

Geometry and mechanics of the morphogenesis of active membranes on the example of plant trichome cells of the genus <Emphasis Type="Italic">Draba</Emphasis> L.

The stages of the early morphogenesis of simple (unbranched) and complex (branched) unicellular trichomes are studied in two species of the genus Draba—D. sibirica (Pall.) Thell. and D. daurica DC. The geometry of morphogenesis is estimated by analyzing intraindividual variation of quantitative morphological characteristics of the developing leaf blade and peduncle trichomes. The surface of all types of trichome cells first acquires a spherical shape, followed by a U-shaped configuration with cylindrical proximal and spherical distal regions. In the development of complex trichomes, the area of the distal zone grows at a higher rate, which leads to separation of its volume into individual spherical regions, the morphogenesis of which repeats the early morphogenetic stages of the overall trichome cell, forming simple (unbranched) or complex (branched) trichome rays. As a rule, the lateral polarity of a trichome cell coincides with the proximodistal polarity of the leaf. Quantitative morphological data make it possible to infer an algorithm of the changes in shape common for all trichome cells, namely, the growth cycle comprising alternation of the phases of increase and decrease in the curvature of the outer cell surface. This surface is an active membrane expanded by the internal pressure and concurrently capable of actively increasing its area by incorporation of new structural elements. A distinctive feature of the proposed model is the geometrical inhomogeneity of the surface movement, changing the radius of curvature and creating internal (active) mechanical stresses in this membrane. A decrease in the ratio of the membrane surface area to the volume deprives the spatially homogeneous shape of its stability; correspondingly, the transition from elastic resistance to internal pressure to active resistance with the help of curvature differentiation becomes more energetically favorable. The source for growth and morphogenesis of the active membrane is alternation of the phases of local curvature leveling, which “charges” the membrane with active mechanical stresses and “discharge” of these stresses, leading to differentiation of the membrane’s local curvatures.     

SIAMESE, a gene controlling the endoreduplication cell cycle in Arabidopsis thaliana trichomes

Cell differentiation is generally tightly coordinated with the cell cycle, typically resulting in a nondividing cell with a unique differentiated morphology. The unicellular trichomes of Arabidopsis are a well-established model for the study of plant cell differentiation. Here, we describe a new genetic locus, SIAMESE (SIM), required for coordinating cell division and cell differentiation during the development of Arabidopsis trichomes (epidermal hairs). A recessive mutation in the sim locus on chromosome 5 results in clusters of adjacent trichomes that appeared to be morphologically identical 'twins'. Upon closer inspection, the sim mutant was found to produce multicellular trichomes in contrast to the unicellular trichomes produced by wild-type (WT) plants. Mutant trichomes consisting of up to 15 cells have been observed. Scanning electron microscopy of developing sim trichomes suggests that the cell divisions occur very early in the development of mutant trichomes. WT trichome nuclei continue to replicate their DNA after mitosis and cytokinesis have ceased, and as a consequence have a DNA content much greater than 2C. This phenomenon is known as endoreduplication. Individual nuclei of sim trichomes have a reduced level of endoreduplication relative to WT trichome nuclei. Endoreduplication is also reduced in dark-grown sim hypocotyls relative to WT, but not in light-grown hypocotyls. Double mutants of sim with either of two other mutants affecting endoreduplication, triptychon (try) and glabra3 (gl3) are consistent with a function for SIM in endoreduplication. SIM may function as a repressor of mitosis in the endoreduplication cell cycle. Additionally, the relatively normal morphology of multicellular sim trichomes indicates that trichome morphogenesis can occur relatively normally even when the trichome precursor cell continues to divide. The sim mutant phenotype also has implications for the evolution of multicellular trichomes.     

Morphogenetic bases and parametric system of trichome evolution in plants of the genus <Emphasis Type="Italic">Draba</Emphasis> L.

Using quantitative morphological analysis of light microscopy data, the normal variation of trichome morphogenesis is studied in six whitlow grass species (Draba L.) and the morphological variation of adult trichomes in 11 species. The evolution consists in the transition from a radial morphogenesis pattern to bilateral and replacement of complex (branched) trichome rays with simple (unbranched) rays. A parametric system is constructed for classification of the ray morphology; this system includes two parameters—the ratio of the numbers of complex to simple rays, characterizing the probability of secondary branching of primary buds, and the number of primary buds, characterizing the probability of primary branching on the surface of the trichome cell. In this parametric space, all of the studied species fit well a third-order curve consisting of two ascending branches displaying a positive correlation between the primary and secondary branchings and a descending branch, located between them, where the primary and secondary branches are negatively correlated. The deduced evolutionary direction is almost independent of the size of the trichome cells and is explained exclusively by the mechanics of morphogenesis: acceleration in the development of the primary bud of the ray decreases the probability of its own branching and creates additional elastic extension of the cell surface, preventing other buds from branching. The morphogenesis itself appears to be a mechanically nonholonomic system, filtering in a selective manner the fluctuations of the same sign, which explains the directed pattern of its evolution. In the evolutionarily initial state, trichome ontogenesis is absent because its modules (primary buds) are formed by a mirror duplication. The ontogenesis commences when mirror symmetry in the formation of modules is lost and replaced with an axial pattern; thus, the change in the morphological type of buds is a direct consequence of the emergence of ontogenesis and its further evolution. Its main material is intraindividual variation, the only source of which is the mechanics of morphogenesis itself. It is found that morphological evolution can take place at an initially zero heritability and zero adaptive value of morphological differences.     

DVL genes play a role in the coordination of socket cell recruitment and differentiation

Specialized plant cells arise from undifferentiated cells through a series of developmental steps. The decision to enter into a certain differentiation pathway depends in many cases on signals from neighbouring cells. The ability of cells to engage in short-range intercellular communication permits the coordination of cell actions necessary in many developmental processes. Overexpression of genes from the DEVIL/ROTUNDIFOLIA (DVL/ROT) family results in severe developmental alterations, but very little is known about their mechanism of action. This work presents evidence that suggests a role for these genes in local signalling, specifically in the coordination of socket cell recruitment and differentiation. Overexpression of different DVL genes results in protuberances at the base of the trichomes surrounded by several rows of elongated epidermal cells, morphologically similar to socket cells. Localized overexpression of DVL4 in trichomes and socket cells during early developmental stages activates expression of socket cell markers in additional cells, farther away from the trichome. The same phenomenon is observed in an activation tagged line of DVL1, which also shows an increase in the number of socket cells in contact with the trichome. The roles of individual DVL genes have been difficult to discover since their overexpression phenotypes are quite similar. In gl1 leaves that lack trichomes and socket cells DVL1 expression shows a 69% reduction, suggesting that this gene could be involved in the coordination of socket cell development in wild-type plants.     

Cell polarity in Arabidopsis trichomes

Arabidopsis leaf trichomes are unicellular hairs that display a highly characteristic cell form that has a fixed orientation with respect to the basal-distal leaf axis. The genetic, molecular and cell biological analysis of trichome morphogenesis reveal that various cellular processes need to be coordinated including regulation of the cell cycle, the cell size and the actin and tubulin cytoskeleton. Here we will focus on what is known about the establishment and maintenance of positional information during trichome formation.     

Epidermal differentiation: trichomes in Arabidopsis as a model system

Arabidopsis trichomes are an excellent model system to study all aspects of cell differentiation including cell fate determination, cell cycle regulation, cell polarity and cell expansion. Genetic analysis had initially identified mutants affecting trichome development at different developmental stages. During recent years, molecular analysis of the corresponding genes has revealed a first glimpse of the underlying molecular mechanisms. This paper summarizes some of the recent insights regarding the mechanisms of trichome development.     

Trichome cell growth in Arabidopsis thaliana can be derepressed by mutations in at least five genes

Leaf trichomes in Arabidopsis are unicellular epidermal hairs with a branched morphology. They undergo successive endoreduplication rounds early during cell morphogenesis. Mutations affecting trichome nuclear DNA content, such as triptychon or glabra3, alter trichome branching. We isolated new mutants with supernumerary trichome branches, which fall into three unlinked complementation groups: KAKTUS and the novel loci, POLYCHOME and RASTAFARI. They map to chromosomes IV, II, and V, respectively. The trichomes of these mutants presented an increased DNA content, although to a variable extent. The spindly-5 mutant, which displays a constitutive gibberellin response, also produces overbranched trichomes containing more nuclear DNA. We analyzed genetic interactions using double mutants and propose that two independent pathways, defined by SPINDLY and TRIPTYCHON, act to limit trichome growth. KAKTUS and POLYCHOME might have redundant actions mediating gibberellin control via SPINDLY. The overall leaf polysomaty was not notably affected by these mutations, suggesting that they affect the control of DNA synthesis in a tissue- or cell type-specific manner. Wild-type tetraploids also produce overbranched trichomes; they displayed a shifted polysomaty in trichomes and in the whole leaf, suggesting a developmental program controlling DNA increases via the counting of endoreduplication rounds.     

High‐resolution computational imaging of leaf hair patterning using polarized light microscopy

The leaf hairs (trichomes) on the aerial surface of many plant species play important roles in phytochemical production and herbivore protection, and have significant applications in the chemical and agricultural industries. Trichome formation in the model plant Arabidopsis thaliana also presents a tractable experimental system to study cell differentiation and pattern formation in plants and animals. Studies of this developmental process suggest that trichome positioning may be the result of a self‐forming pattern, emerging from a lateral inhibition mechanism determined by a network of regulatory factors. Critical to the continued success of these studies is the ability to quantitatively characterize trichome pattern phenotypes in response to mutations in the genes that regulate this process. Advanced protocols for the observation of changes in trichome patterns can be expensive and/or time consuming, and lack user‐friendly analysis tools. In order to address some of these challenges, we describe here a strategy based on polarized light microscopy for the quick and accurate measurement of trichome positions, and provide an online tool designed for the quantitative analyses of trichome number, density and patterning.     

Coupling membranes as energy-transmitting cables. II. Cyanobacterial trichomes

Power transmission along trichomes of filamentous cyanobacteria Phormidium uncinatum has been studied with the use of ethylrhodamine fluorescence as a probe for the transmembrane electric potential difference (delta psi). It is found that agents preventing the light-induced delta psi formation (photosynthetic redox chain inhibitor dibromothymoquinone) or dissipating delta psi (uncoupler tetrachlorotrifluoromethylbenzimidazole) strongly decrease the fluorescence of the ethyl-rhodamine-stained trichomes. K+-H+ antiporter nigericin converting delta pH to delta psi increases the fluorescence. These relationships are in agreement with the assumption that ethylrhodamine electrophoretically accumulates inside the cyanobacterial cells. Illumination of a single cell in the P. uncinatum trichome gives rise to quenching of the fluorescence in this cell and usually in one or two neighbor cells, whereas the rest of trichome remains fluorescing. A small light spot (5% of the trichome length) causes an increase in the ethylrhodamine fluorescence not only in the illuminated but also in the nonilluminated parts of the trichome up to the laser-treated cell or its neighbor(s). It is concluded ethylrhodamine can be used to monitor the power transmission which was previously demonstrated by microelectrode studies of the cyanobacterial trichomes. In certain trichomes, several "dark" cells appear during the storage of the trichomes without energy sources. Illumination for several minutes results in dark cells becoming fluorescing. Thus some cells or cell clusters can be reversibly excluded from the lateral delta psi-transmitting system of the trichome, the rest being still electrically connected. This means that filamentous cyanobacteria possess mechanisms to transmit power along the trichome and to switch off this transmission.     

Trichome Development in Arabidopsis thaliana. II. Isolation and Complementation of the GLABROUS1 Gene

We are using the formation of trichomes in Arabidopsis thaliana as a model system to study gene expression during cellular differentiation. To initiate the molecular characterization of this system, we tagged and isolated a gene that is specifically required for the development of the specialized trichome cell. We confirmed the identity of this gene, GLABROUS1 (GL1), by complementation. These results demonstrate that a crucial gene in a plant developmental pathway can be successfully identified by complementation.     

The evolution of gene regulatory networks controlling Arabidopsis thaliana L. trichome development

Background

The variation in structure and function of gene regulatory networks (GRNs) participating in organisms development is a key for understanding species-specific evolutionary strategies. Even the tiniest modification of developmental GRN might result in a substantial change of a complex morphogenetic pattern. Great variety of trichomes and their accessibility makes them a useful model for studying the molecular processes of cell fate determination, cell cycle control and cellular morphogenesis. Nowadays, a large number of genes regulating the morphogenesis of A. thaliana trichomes are described. Here we aimed at a study the evolution of the GRN defining the trichome formation, and evaluation its importance in other developmental processes.

Results

In study of the evolution of trichomes formation GRN we combined classical phylogenetic analysis with information on the GRN topology and composition in major plants taxa. This approach allowed us to estimate both times of evolutionary emergence of the GRN components which are mainly proteins, and the relative rate of their molecular evolution. Various simplifications of protein structure (based on the position of amino acid residues in protein globula, secondary structure type, and structural disorder) allowed us to demonstrate the evolutionary associations between changes in protein globules and speciations/duplications events. We discussed their potential involvement in protein-protein interactions and GRN function.

Conclusions

We hypothesize that the divergence and/or the specialization of the trichome-forming GRN is linked to the emergence of plant taxa. Information about the structural targets of the protein evolution in the GRN may predict switching points in gene networks functioning in course of evolution. We also propose a list of candidate genes responsible for the development of trichomes in a wide range of plant species.

     

Gene expression profiling of the different stages of Arabidopsis thaliana trichome development on the single cell level.

Leaf hairs (trichomes) of Arabidopsis thaliana are a model system for studying cell development, differentiation and cell cycle regulation. To exploit this model system with ultimate spatial resolution we applied single cell sampling, thus avoiding the averaging effect induced by complex tissue mixtures. In particular, we analysed gene expression profiles of two selected stages of the developing trichome: trichome initial cells and mature trichomes, as well as pavement cells. Ten single cells per sample were collected by glass microcapillaries and used for the generation of radioactive probes for subsequent hybridization to nylon filters representing approximately 8000 genes of A. thaliana. Functional categorization of genes transcribed in trichome initials, mature trichomes and pavement cells demonstrated involvement of these surface cells in the stress response. In silico promoter analysis of genes preferentially expressed in trichome initials revealed enrichment in MYB-binding sites and presence of elements involved in hormonal, metal, sulphur response and cell cycle regulation. Three candidate genes preferentially expressed in trichome initials were selected for further analysis: At3g16980 (putative RNA polymerase II), At5g15230 (GASA4) and At4g27260 (GH3.5, WES1). Promoter:GUS studies confirmed expression of the putative RNA polymerase II and the gibberellin responsive GASA4 in trichome initials and partially in mature trichomes. Functional implication of the three selected candidates in trichome development and hence in cell cycle regulation in A. thaliana is discussed. We suggest that these genes are involved in differentiation and initiation of endocycling during trichome development.