Victor Kirik

TRIPTYCHON and CAPRICE mediate lateral inhibition during trichome and root hair patterning in Arabidopsis.

Trichome patterning in Arabidopsis is a model for the generation of a spacing pattern from initially equivalent cells. We show that the TRIPTYCHON gene that functions in lateral inhibition encodes a single‐repeat MYB‐related transcription factor that lacks a recognizable activation domain. It has high sequence simi larity to the root hair patterning gene CAPRICE. Both genes are expressed in trichomes and act together during lateral inhibition. We further show that TRIPTYCHON and CAPRICE act redundantly in the position‐dependent cell fate determination in the root epidermis. Thus, the same lateral inhibition mechanism seems to be involved in both de novo patterning and position‐dependent cell determination. We propose a model explaining trichome and root hair patterning by a common mechanism.

Arabidopsis CROOKED encodes for the smallest subunit of the ARP2/3 complex and controls cell shape by region specific fine F-actin formation.

The generation of a specific cell shape requires differential growth, whereby specific regions of the cell expand more relative to others. The Arabidopsis crooked mutant exhibits aberrant cell shapes that develop because of mis-directed expansion, especially during a rapid growth phase. GFP-aided visualization of the F-actin cytoskeleton and the behavior of subcellular organelles in different cell-types in crooked and wild-type Arabidopsis revealed that localized expansion is promoted in cellular regions with fine F-actin arrays but is restricted in areas that maintain dense F-actin. This suggested that a spatiotemporal distinction between fine versus dense F-actin in a growing cell could determine the final shape of the cell. CROOKED was molecularly identified as the plant homolog of ARPC5, the smallest sub-unit of the ARP2/3 complex that in other organisms is renowned for its role in creating dendritic arrays of fine F-actin. Rescue of crooked phenotype by the human ortholog provides the first molecular evidence for the presence and functional conservation of the complex in higher plants. Our cell-biological and molecular characterization of CROOKED suggests a general actin-based mechanism for regulating differential growth and generating cell shape diversity.

ENHANCER of TRY and CPC 2 (ETC2) reveals redundancy in the region-specific control of trichome development of Arabidopsis

Abstract>An evolutionarily conserved set of proteins consisting of MYB and bHLH transcription factors and a WD40 domain protein is known to act in concert to control various developmental processes including trichome and root hair development. Their function is difficult to assess because most of them belong to multigene families and appear to act in a redundant fashion. In this study we identified an enhancer of the two root hair and trichome patterning mutants triptychon (try) and caprice (cpc), enhancer of try and cpc2 (etc2). The ETC2 gene shows high sequence similarity to the single-repeat MYB genes CPC and TRY. Overexpression results in the suppression of trichomes and overproduction of root hairs similarly as observed for TRY and CPC suggesting that ETC2 has similar biochemical properties. The etc2 single mutant shows an increase in trichome number on leaves and petiols. Double and triple mutant analysis indicates that the ETC2 gene acts redundant with TRY and CPC in trichome patterning.

Functional diversification of MYB23 and GL1 genes in trichome morphogenesis and initiation

The functional diversification of duplicated genes is one of the driving forces in evolution. To understand the molecular mechanisms of gene diversification, we studied the functional relationship of the two Arabidopsis paralogous MYB-related genes GL1 and MYB23. We show that MYB23 controls trichome branching and trichome initiation at leaf edges. The latter is controlled redundantly together with GL1. We show that the two proteins are functionally equivalent during trichome initiation but not during trichome branching. RT-PCR and reporter construct analysis revealed spatial, temporal and genetic differences in transcriptional regulation of the GL1 and MYB23 genes. Presented data indicate that the diversification of GL1 and MYB23 gene functions occurred at the level of cis-regulatory sequences with respect to trichome initiation, and that, in parallel, the diversification with respect to regulation of trichome branching also involved changes in respective proteins.

The cell morphogenesis gene ANGUSTIFOLIA encodes a CtBP/BARS‐like protein and is involved in the control of the microtubule cytoskeleton

The ANGUSTIFOLIA (AN) gene is required for leaf hair (trichome) branching and is also involved in polarized expansion underlying organ shape. Here we show that the AN gene encodes a C‐terminal binding proteins/brefeldin A ADP‐ribosylated substrates (CtBP/BARS) related protein. AN is expressed at low levels in all organs and the AN protein is localized in the cytoplasm. In an mutant trichomes, the organization of the actin cytoskeleton is normal but the distribution of microtubules is aberrant. A role of AN in the control of the microtubule cytoskeleton is further supported by the finding that AN genetically and physically interacts with ZWICHEL, a kinesin motor molecule involved in trichome branching. Our data suggest that CtBP/BARS‐like protein function in plants is directly associated with the microtubule cytoskeleton.

CPR5 is involved in cell proliferation and cell death control and encodes a novel transmembrane protein

Plants often respond to pathogens by sacrificing cells at the infection site. This type of programmed cell death is mimicked by the constitutive pathogene response5 (cpr5) mutant in Arabidopsis in the absence of pathogens, suggesting a role for CPR5 in programmed cell death control. The analysis of the cellular phenotypes of two T-DNA-tagged cpr5 alleles revealed an additional role for CPR5 in the regulation of endoreduplication and cell division. In cpr5 mutant trichomes, endoreduplication cycles stop after two rounds instead of four, and trichome cells have fewer branches than normal. Eventually, cpr5 trichomes die, the nucleus disintegrates, and the cell collapses. Similarly, leaf growth stops earlier than in wild-type, and, frequently, regions displaying spontaneous cell death are observed. The cloning of the CPR5 gene revealed a novel putative transmembrane protein with a cytosolic domain containing a nuclear-targeting sequence. The dual role of CPR5 in cell proliferation and cell death control suggests a regulatory link between these two processes.

The MYB23 Gene Provides a Positive Feedback Loop for Cell Fate Specification in the Arabidopsis Root Epidermis

The specification of cell fates during development requires precise regulatory mechanisms to ensure robust cell type patterns. Theoretical models of pattern formation suggest that a combination of negative and positive feedback mechanisms are necessary for efficient specification of distinct fates in a field of differentiating cells. Here, we examine the role of the R2R3-MYB transcription factor gene, AtMYB23 (MYB23), in the establishment of the root epidermal cell type pattern in Arabidopsis thaliana. MYB23 is closely related to, and is positively regulated by, the WEREWOLF (WER) MYB gene during root epidermis development. Furthermore, MYB23 is able to substitute for the function of WER and to induce its own expression when controlled by WER regulatory sequences. We also show that the MYB23 protein binds to its own promoter, suggesting a MYB23 positive feedback loop. The localization of MYB23 transcripts and MYB23-green fluorescent protein (GFP) fusion protein, as well as the effect of a chimeric MYB23-SRDX repressor construct, links MYB23 function to the developing non-hair cell type. Using mutational analyses, we find that MYB23 is necessary for precise establishment of the root epidermal pattern, particularly under conditions that compromise the cell specification process. These results suggest that MYB23 participates in a positive feedback loop to reinforce cell fate decisions and ensure robust establishment of the cell type pattern in the Arabidopsis root epidermis.

Two novel MYB homologues with changed expression in late embryogenesis-defective Arabidopsis mutants.

Two novel MYB genes (ATMYBR1 and ATMYBR2) were isolated from Arabidopsis thaliana. Binding to a conserved MYB recognition sequence is demonstrated for the ATMYBR1 protein. The expression of both genes is affected by the fus3, lec1 and abi3 mutations causing pleiotropic defects during late embryogenesis and seed maturation including the loss of dormancy and desiccation tolerance. The strong increase of the transcript levels of both MYB genes during very late stages of embryogenesis typically found in wild type is missing in the mutants. Furthermore, the expression of both MYB genes is developmentally regulated in vegetative tissues. The highly conserved repeats (R2 and R3) of the DNA binding MYB domain of both proteins represent chimeric structures combining features typical of plant and animal derived proteins. This demonstrates the existence of a distinct subfamily of animal-like MYB factors in plant genomes.

The Arabidopsis TUBULIN-FOLDING COFACTOR A Gene Is Involved in the Control of the α/β-Tubulin Monomer Balance

The control of the stoichiometric balance of α- and β-tubulin is important during microtubule biogenesis. This process involves several tubulin-folding cofactors (TFCs), of which only TFC A is not essential in mammalian in vitro systems or in vivo in yeast. Here, we show that the TFC A gene is important in vivo in plants. The Arabidopsis gene KIESEL (KIS) shows sequence similarity to the TFC A gene. Expression of the mouse TFC A gene under the control of the 35S promoter rescues the kis mutation, indicating that KIS is the Arabidopsis ortholog of TFC A. kis plants exhibit a range of defects similar to the phenotypes associated with impaired microtubule function: plants are reduced in size and show meiotic defects, cell division is impaired, and trichomes are bulged and less branched. Microtubule density was indistinguishable from that of the wild type, but microtubule organization was affected in trichomes and hypocotyl cells of dark-grown kis plants. The kis phenotype was rescued by overexpression of an α-tubulin, indicating that KIS is involved in the control of the correct balance of α- and β-tubulin monomers.

The Arabidopsis STICHEL Gene Is a Regulator of Trichome Branch Number and Encodes a Novel Protein

Here, we analyze the STICHEL(STI) gene, which plays an important role in the regulation of branch number of the unicellular trichomes in Arabidopsis. We have isolated the STI locus by positional cloning and confirmed the identity by sequencing seven independent sti alleles. The STI gene encodes a protein of 1,218 amino acid residues containing a domain with sequence similarity to the ATP-binding eubacterial DNA-polymerase III γ-subunits. Because endoreduplication was found to be normal insti mutants the molecular function of STIin cell morphogenesis is not linked to DNA replication and, therefore, postulated to represent a novel pathway. Northern-blot analysis shows that STI is expressed in all organs suggesting thatSTI function is not trichome specific. The analysis ofsti alleles and transgenic lines overexpressingSTI suggests that STI regulates branching in a dosage-dependent manner.