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 TRANSPARENT TESTA GLABRA1 locus, which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis, encodes a WD40 repeat protein.

The TRANSPARENT TESTA GLABRA1 (TTG1) locus regulates several developmental and biochemical pathways in Arabidopsis, including the formation of hairs on leaves, stems, and roots, and the production of seed mucilage and anthocyanin pigments. The TTG1 locus has been isolated by positional cloning, and its identity was confirmed by complementation of a ttg1 mutant. The locus encodes a protein of 341 amino acid residues with four WD40 repeats. The protein is similar to AN11, a regulator of anthocyanin biosynthesis in petunia, and more distantly related to those of the beta subunits of heterotrimeric G proteins, which suggests a role for TTG1 in signal transduction to downstream transcription factors. The 1.5-kb TTG1 transcript is present in all major organs of Arabidopsis. Sequence analysis of six mutant alleles has identified base changes producing truncations or single amino acid changes in the TTG1 protein.

GLABROUS1 overexpression and TRIPTYCHON alter the cell cycle and trichome cell fate in Arabidopsis.

Cellular competence, initiation cues, and inhibition signals control the distribution of trichomes on the Arabidopsis leaf. The GLABROUS1 (GL1) gene has a dual role in that it is required for trichome initiation, but GL1 overexpression reduces trichome number. We have found that a mutation in the TRIPTYCHON (TRY) gene partially suppresses the GL1 overexpression phenotype but not in a way that indicates that TRY directly controls an epidermal inhibition pathway. Surprisingly, cauliflower mosaic virus 35S::GL1 try plants contain a subclass of trichomes derived from the subepidermal layer. Altered cell cycle control was also detected in 35S::GL1 and try plants. A mutation in TRY led to increased epidermal and mesophyll cell number, a reduction in endoreduplication in the epidermis, and an increase in endoreduplication in trichomes. GL1 overexpression also reduced endoreduplication levels in both the epidermis and trichomes; however, in the presence of try, it synergistically enhanced trichome endoreduplication. Interactions with the COTYLEDON TRICHOME1 (COT1) gene indicate that GL1 and TRY control trichome development and may be involved in cell cycle control during leaf development.

cot1: a regulator of Arabidopsis trichome initiation.

In Arabidopsis, the timing and spatial arrangement of trichome initiation is tightly regulated and requires the activity of the GLABROUS1 (GL1) gene. The COTYLEDON TRICHOME 1 (COT1) gene affects trichome initiation during late stages of leaf development and is described in this article. In the wild-type background, cot1 has no observable effect on trichome initiation. GL1 overexpression in wild-type plants leads to a modest number of ectopic trichomes and to a decrease in trichome number on the adaxial leaf surface. The cot1 mutation enhances GL1-overexpression-dependent ectopic trichome formation and also induces increased leaf trichome initiation. The expressivity of the cot1 phenotype is sensitive to cot1 and 35S::GL1 gene dosage, and the most severe phenotypes are observed when cot1 and 35S::GL1 are homozygous. The COT1 locus is located on chromosome 2 15.3 cM north of er. Analysis of the interaction between cot1, try, and 35S::GL1 suggests that COT1 is part of a complex signal transduction pathway that regulates GL1-dependent adoption of the trichome cell fate.

Control of GL2 expression in Arabidopsis leaves and trichomes.

More than twenty genes are required for the correct initiation, spacing, and morphogenesis of trichomes in Arabidopsis. The initial selection of trichome precursors requires the activity of both the GLABROUS1 (GL1) and TRANSPARENT TESTA GLABROUS (TTG) genes. The GLABRA2 (GL2) gene is required for subsequent phases of trichome morphogenesis such as cell expansion, branching, and maturation of the trichome cell wall. Previous studies have shown that GL2 is a member of the homeodomain class of transcription factors. Here we report a detailed analysis of GL2 expression in the shoot using anti-GL2 antibodies and the GUS reporter gene fused to the GL2 promoter. The GL2 expression profile in the shoot is complex, and involves spatial and temporal variation in developing leaves and trichomes. Two separate promoter domains that are expressed in trichomes were identified. GL2, like GL1, is expressed in developing trichomes and in cells surrounding trichomes during early stages of trichome development. Unlike GL1, GL2 expression persists in mature trichomes. It was found that while GL1 and TTG were not required for the initiation of GL2 expression in the non-trichome cells, the presence of a functional GL1 or TTG gene was able to increase GL2 expression in these cells compared to ttg gl1 plants. The hypothesis that GL1 regulates aspects of GL2 expression is consistent with epistatic analysis of gl1 and gl2 and the expression patterns of GL1 and GL2. In support of this hypothesis, it was found that ectopic expression of GL1 in the presence of ectopic expression of the maize R gene, which can bypass the requirement for TTG, can ectopically activate GL2 transcription.

A common position-dependent mechanism controls cell-type patterning and GLABRA2 regulation in the root and hypocotyl epidermis of Arabidopsis.

A position-dependent pattern of epidermal cell types is produced during root development in Arabidopsis thaliana. This pattern is reflected in the expression pattern of GLABRA2 (GL2), a homeobox gene that regulates cell differentiation in the root epidermis. GL2 promoter::GUS fusions were used to show that the TTG gene, a regulator of root epidermis development, is necessary for maximal GL2 activity but is not required for the pattern of GL2 expression. Furthermore, GL2-promoter activity is influenced by expression of the myc-like maize R gene (35S::R) in Arabidopsis but is not affected by gl2 mutations. A position-dependent pattern of cell differentiation and GL2-promoter activity was also discovered in the hypocotyl epidermis that was analogous to the pattern in the root. Non-GL2-expressing cell files in the hypocotyl epidermis located outside anticlinal cortical cell walls exhibit reduced cell length and form stomata. Like the root, the hypocotyl GL2 activity was shown to be influenced by ttg and 35S::R but not by gl2. The parallel pattern of cell differentiation in the root and hypocotyl indicates that TTG and GL2 participate in a common position-dependent mechanism to control cell-type patterning throughout the apical-basal axis of the Arabidopsis seedling.

Essential role of a kinesin-like protein in Arabidopsis trichome morphogenesis.

Little is known about how cell shape is controlled. We are using the morphogenesis of trichomes (plant hairs) on the plant Arabidopsis thaliana as a model to study how cell shape is controlled. Wild-type Arabidopsis trichomes are large, single epidermal cells with a stalk and three or four branches, whereas in zwichel (zwi) mutants the trichomes have a shortened stalk and only two branches. To further understand the role of the ZWI gene in trichome morphogenesis we have cloned the wild-type ZWICHEL (ZWI) gene by T-DNA tagging, and report here that it encodes a member of the kinesin superfamily of microtubule motor proteins. Kinesin proteins transport diverse cellular materials in a directional manner along microtubules. Kinesin-like proteins are characterized by a highly conserved "head" region that comprises the motor domain, and a nonconserved "tail" region that is thought to participate in recognition and binding of the appropriate cargo.

The homeobox gene GLABRA2 is required for position-dependent cell differentiation in the root epidermis of Arabidopsis thaliana.

The role of the Arabidopsis homeobox gene, GLABRA 2 (GL2), in the development of the root epidermis has been investigated. The wild-type epidermis is composed of two cell types, root-hair cells and hairless cells, which are located at distinct positions within the root, implying that positional cues control cell-type differentiation. During the development of the root epidermis, the differentiating root-hair cells (trichoblasts) and the differentiating hairless cells (atrichoblasts) can be distinguished by their cytoplasmic density, vacuole formation, and extent of elongation. We have determined that mutations in the GL2 gene specifically alter the differentiation of the hairless epidermal cells, causing them to produce root hairs, which indicates that GL2 affects epidermal cell identity. Detailed analyses of these differentiating cells showed that, despite forming root hairs, they are similar to atrichoblasts of the wild type in their cytoplasmic characteristics, timing of vacuolation, and extent of cell elongation. The results of in situ nucleic acid hybridization and GUS reporter gene fusion studies show that the GL2 gene is preferentially expressed in the differentiating hairless cells of the wild type, during a period in which epidermal cell identity is believed to be established. These results indicate that the GL2 homeodomain protein normally regulates a subset of the processes that occur during the differentiation of hairless epidermal cells of the Arabidopsis root. Specifically, GL2 appears to act in a cell-position-dependent manner to suppress hair formation in differentiating hairless cells.

The control of trichome spacing and number in Arabidopsis.

Arabidopsis trichomes are single-celled epidermal hairs that serve as a useful model for the study of plant cell differentiation. An examination of the distribution of trichomes early in their development revealed that developing trichomes occur adjacent to another trichome much less frequently than would be expected by chance. Clonal analysis of epidermal cell lineages ruled out a role for cell lineage in generating the observed minimum-distance spacing pattern. Taken together, these results are consistent with a role for lateral inhibition in the control of trichome development. We also report the identification of a new locus, Reduced Trichome Number (RTN), which affects the initiation of trichomes. This locus was initially detected by the reduced number of leaf trichomes on Landsberg erecta plants compared to that on Columbia plants. Quantitative Trait Locus mapping revealed that more than 73% of the variation in trichome number was due to a major locus near erecta on chromosome 2. The reduced number of trichomes conditioned by the Landsberg erecta allele of this locus appeared to be due to an early cessation of trichome initiation. The implications of these observations are discussed with regard to previously published models of trichome development.

Roles of the GLABROUS1 and TRANSPARENT TESTA GLABRA Genes in Arabidopsis Trichome Development.

Arabidopsis trichomes are branched, single-celled epidermal hairs. These specialized cells provide a convenient model for investigating the specification of cell fate in plants. Two key genes regulating the initiation of trichome development are GLABROUS1 (GL1) and TRANSPARENT TESTA GLABRA (TTG). GL1 is a member of the myb gene family. The maize R gene, which can functionally complement the Arabidopsis ttg mutation, encodes a basic helix-loop-helix protein. We used constitutively expressed copies of the GL1 and R genes to test hypotheses about the roles of GL1 and TTG in trichome development. The results support the hypothesis that TTG and GL1 cooperate at the same point in the trichome developmental pathway. Furthermore, the constitutive expression of both GL1 and R in the same plant caused trichomes to develop on all shoot epidermal surfaces. Results were also obtained indicating that TTG plays an additional role in inhibiting neighboring cells from becoming trichomes.

Plant development. The making of a plant hair.

The identification of genes required for leaf-hair formation in the plant Arabidopsis thaliana is leading to the detailed dissection of a cell-differentiation pathway.

The GLABRA2 gene encodes a homeo domain protein required for normal trichome development in Arabidopsis.

The GLABRA2 gene (GL2) is one of several genes known to have a role in trichome development in Arabidopsis. Mutations at this locus result in abnormal trichome expansion. We have identified several gl2 mutants from a T-DNA-mutagenized population of plants. The T-DNA insert in one of the mutant lines cosegregated with the recessive gl2 phenotype and thus served as a molecular tag to isolate genomic DNA at the putative GL2 locus. RFLP analysis of the segregating population and subsequent molecular complementation experiments established that the GL2 gene had been cloned. The predicted polypeptide from one of the ORFs contained on this fragment showed significant identity to the homeo domain sequence. The construction of a full-length cDNA by RT-PCR confirmed the presence of a homeo box in the GL2 gene and showed that it is substantially different from other recently cloned homeo box genes in plants. The expression pattern of GL2, as demonstrated by in situ hybridization, indicated that the gene is expressed in trichome progenitor cells and at stages associated with trichome development. This suggests that GL2 may regulate events required for the directional cell expansion observed during trichome formation.

Characterization of a weak allele of the GL1 gene of Arabidopsis thaliana.

A genomic clone containing the gl1-2 allele has been isolated and sequenced. The predicted amino acid sequence of the gl1-2 protein is identical to that of the GL1-Col allele up to position 201. At this point in the coding region of gl1-2 there is a deletion relative to the wild-type sequence that results in an in-frame stop codon at position 202. This deletion removes 27 amino acid residues, including a highly negatively charged region, from the predicted gl1-2 polypeptide. The loss of this negatively charged carboxy-terminal region from the gl1-2 product is most likely the cause of the partial loss of gene activity which results in a reduction in leaf trichome initiation.

Arabidopsis GLABROUS1 Gene Requires Downstream Sequences for Function.

The Arabidopsis GLABROUS1 (GL1) gene is a myb gene homolog required for the initiation of trichome development. In situ hybridization revealed that the highest levels of GL1 transcripts were present in developing trichomes. In contrast, previous work had shown that putative promoter sequences from the 5[prime] noncoding region of the GL1 gene directed the expression of a [beta]-glucuronidase (GUS) reporter gene only in stipules. Deletion analysis of the 3[prime] noncoding region of GL1 has identified an enhancer that is essential for GL1 function. Sequences from the region containing the enhancer, in conjunction with GL1 upstream sequences, direct the expression of a GUS reporter gene in leaf primordia and developing trichomes in addition to stipules, indicating that the downstream enhancer is required for the normal expression pattern of GL1.

A myb gene required for leaf trichome differentiation in Arabidopsis is expressed in stipules.

The GL1 gene is required for the initiation of differentiation of hair cells (trichomes) on the crucifer, Arabidopsis thaliana. This gene has been localized to a 4.5 kb DNA fragment by molecular complementation of gl1 mutants. DNA sequence analysis has shown that the protein encoded by GL1 contains a Myb DNA-binding motif. Southern analysis and subsequence analysis of isolated lambda clones has established that GL1 is a member of an extensive myb gene family in Arabidopsis. The putative GL1 promoter directs the expression of the GUS reporter gene in non-trichome-bearing structures that appear to be stipules. This pattern of expression suggests that GL1 may control the synthesis of a diffusible signal that activates the developmental pathway for trichome differentiation.

A model for cell-type determination and differentiation in plants.

We are using trichome formation on the plant Arabidopsis thaliana as a model for the study of plant cell determination and differentiation. Several of the genes that are required for trichome formation are defined by mutations. Two mutations, ttg and gl1, prevent the initiation of trichome differentiation. Thus, these mutations define products that are involved in the signalling of trichome determination. Other mutations, gl2, gl3, dis1, and dis2, define genes that are involved in trichome maturation. Our immediate goal has been to isolate the genes defined by these mutations and determine the role that they play in trichome formation. Our general goals are (1) to identify counterparts to these genes that are involved in other cell type determination and differentiation processes; (2) to manipulate cell development by altering the normal expression of these genes; and (3) to determine if this information can be used to improve crop plants. Presently, most of our progress has centered on the GL1 gene, which has been isolated and characterized. We have found that GL1 is a myb-related gene that is uniquely required for trichome initiation. As in other plants, Arabidopsis has a family of myb-related genes. We are currently investigating the possibility that some of these other myb-related genes are also uniquely required for other types of cell determination events.

Trichome Development in Arabidopsis thaliana. I. T-DNA Tagging of the GLABROUS1 Gene.

Progeny from a transformed Arabidopsis plant (produced by the Agrobacterium-mediated seed transformation procedure) were found to be segregating for an altered trichome phenotype. The mutant plants have normal leaf trichomes but completely lack trichomes usually found on the stem. The mutation is tightly linked to a T-DNA insert. Complementation analysis with genetically characterized trichome mutants revealed that the new mutation is an allele of the GL1 locus. The new trichome mutant has been designated gl1-43. DNA gel blot analysis indicated that the insert site contains a complex array of at least four tandemly linked T-DNA units oriented as both direct and inverted repeats. A genomic library, constructed using DNA from gl1-43 plants, was used to clone DNA that flanks the left end of the T-DNA insert. The availability of DNA from the region interrupted by the insert has allowed initial characterization of the wild-type GL1 gene and will permit the eventual cloning and sequencing of this developmentally interesting gene.