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.

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.

Genetic control of trichome branch number in Arabidopsis: the roles of the FURCA loci.

We are using trichome (hair) morphogenesis as a model to study how plant cell shape is controlled. During a screen for new mutations that affect trichome branch initiation in Arabidopsis, we identified seven new mutants that show a reduction in trichome branch number from three branches to two. These mutations were named furca, after the Latin word for two-pronged fork. These seven recessive mutations were placed into four complementation groups that define four new genes: FURCA1, FURCA2, FURCA3 and FURCA4. The trichome branch number phenotype indicates that the FURCA genes encode positive regulators of trichome branch initiation. Analysis of double mutants suggests that primary and secondary branch initiation events are not genetically distinct, but rely on the levels of partially redundant groups of regulators of trichome branch initiation. Based on the analysis of both epistatic and additive genetic interactions between the FURCA genes and other genes that control trichome branch number, we propose a model that explains how these genes interact to control trichome branch initiation. This model successfully predicts the phenotypes of all the single and double mutants examined and suggests points of control of the trichome branch pathway.

Extragenic suppressors of the arabidopsis zwi-3 mutation identify new genes that function in trichome branch formation and pollen tube growth.

The plant cytoskeleton plays a pivotal role in determining the direction of cell wall expansion, and ultimately the cell's final shape. However, the mechanisms by which localized expansion events are initiated remain obscure. Mutational analysis of the trichome (plant hair) morphogenic pathway in Arabidopsis has identified at least eight genes that determine trichome branch number. One of these genes, ZWICHEL (ZWI), encodes a novel member of the kinesin superfamily of motor proteins. Mutations in the ZWI gene cause a reduction in the number of trichome branches. To identify additional genes involved in trichome branch initiation, we screened for extragenic suppressors of the zwi-3 mutation and isolated three suppressors that rescued the branch number defect of zwi-3. These suppressors define three genes, named suz, for suppressor of zwichel-3. All of the suppressors were shown to be allele specific. One of the suppressors, suz2, also rescued the trichome branch number defect of another branch mutant, furca1-2. Plants homozygous for suz2 have more than the wild-type number of trichome branches. This suggests that SUZ2 is a negative regulator of trichome branching and may interact with ZWI and FURCA1. The suz1 and suz3 mutants display no obvious phenotype in the absence of the zwi-3 mutation. The suz1 zwi-3 double mutants also exhibited a male-sterile phenotype due to a defect in pollen tube germination and growth, whereas both the suz1 and the zwi-3 single mutants are fertile. The synthetic male sterility of the suz1 zwi-3 double mutants suggests a role for SUZ1 and ZWI in pollen germination and pollen tube growth. DNA sequence analysis of the zwi-3 mutation indicated that only the tail domain of the zwi-3 protein would be expressed. Thus, the suz mutations show allele-specific suppression of a kinesin mutant that lacks the motor domain.

Genetics of plant cell shape.

Plant cells have a variety of shapes crucial for their functions, yet the mechanisms that generate these shapes are poorly understood. Genetic dissection of the trichome (plant hair) branching pathway in Arabidopsis, has uncovered mechanisms and identified genes that control plant cell morphogenesis. The recent identification of one of these genes, ZWICHEL (ZWI), as a novel member of the kinesin superfamily of microtubule motors provides a starting point for the analysis of the plant cytoskeleton's role in a specific morphogenetic event.

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.

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.

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.

The beta-tubulin gene family of Arabidopsis thaliana: preferential accumulation of the beta 1 transcript in roots.

The genome of Arabidopsis thaliana (L.) Heynh. was shown to contain a beta-tubulin gene family consisting of at least seven distinct genes and/or pseudogenes. Genomic clones of five different beta-tubulin genes and/or pseudogenes have been isolated and partially characterized. The complete nucleotide sequence of one A. thaliana beta-tubulin gene, designated beta 1, has been determined. A comparison of the predicted amino acid sequence of the A. thaliana beta 1-tubulin with the predicted sequences of beta-tubulins of animals and protists indicated that this plant beta-tubulin shows a high degree of homology with other beta-tubulins. However, the beta 1-tubulin contains a novel single amino acid insertion at position 41. The A. thaliana beta 1-tubulin gene is transcribed, as shown by RNA blot hybridization and S1 nuclease analyses. A 3'-noncoding gene-specific probe was used to examine the expression of the beta 1-tubulin gene in leaves, roots, and flowers by blot hybridization analyses of total RNA isolated from these tissues. The results showed that the transcript of the beta 1 gene accumulates predominantly in roots, with low levels of transcript in flowers, and barely detectable levels of transcript in leaves. A second genomic clone was shown to contain two essentially identical beta-tubulin coding sequences in direct tandem orientation and separated by 1 kb.

The α1-tubulin gene of Arabidopsis thaliana: primary structure and preferential expression in flowers.

The primary structure of the α1-tubulin gene of Arabidopsis thaliana was determined and the 5' and 3' ends of its transcript were identified by S1 nuclease mapping experiments. The information obtained was used to (i) predict the amino acid sequence of the α1-tubulin, (ii) deduce the positions of introns within the α1-tubulin gene, and (iii) construct 3' noncoding gene-specific hybridization probes with which to study the pattern of α1-tubulin transcript accumulation in different tissues and at different stages of development. The predicted amino acid sequence of the α1-tubulin has 92% identity with the predicted product of the previously characterized A. thaliana α3-tubulin gene. The coding sequence of the α1-tubulin gene is interrupted by four introns located at positions identical to those of the four introns in the α3 gene. RNA blot hybridization studies carried out with an α1-tubulin gene-specific probe showed that the α1 gene transcript accumulates primarily in flowers, with little transcript present in RNA isolated from roots or leaves. In order to investigate the pattern of α-tubulin gene expression in developing flowers, RNA was isolated from flowers at five different stages of development: flower buds, unopened flowers with pollen, open flowers, flowers with elongating carpels, and green seed pods. RNA blot hybridizations performed with 3' noncoding gene-specific probes showed that the α3 tubulin gene transcript is present in flowers at all stages of development, whereas the α1-tubulin gene transcript could only be detected in RNA from unopened flowers with pollen, open flowers, and flowers with elongating carpels.

Characterization of the alpha-tubulin gene family of Arabidopsis thaliana.

The genome of Arabidopsis thaliana (Linnaeus) Heynhold was shown to contain an alpha-tubulin gene family consisting of at least four genes and/or pseudogenes. The primary structure of a transcribed alpha-tubulin gene was determined. A comparison of the predicted amino acid sequence of the A. thaliana alpha-tubulin with the predicted amino acid sequences of alpha-tubulins of Chlamydomonas reinhardtii, Stylonychia lemnae, and Homo spaiens reveals a high degree of homology; 90%, 87%, and 83% identity, respectively. Thus, a plant alpha-tubulin exhibits a high degree of homology to the alpha-tubulins of protists and animals. The coding sequence of the A. thaliana alpha-tubulin gene is interrupted by four introns, which occur at positions different from those of the less numerous introns of C. reinhardtii and rat alpha-tubulin genes. S1 nuclease mapping data showed that transcription is initiated 99 +/- 1 base pairs upstream from the translation initiation codon. Both 5' and 3' noncoding gene-specific probes were used to examine the expression of the alpha-tubulin gene in leaves, roots, and flowers by hybridization to total RNA isolated from these tissues. The results showed that the alpha-tubulin gene was transcribed in all three tissues.

The bacteriophage T4 regulatory protein gpunf/alc binds to DNA in the absence of RNA polymerase.

DNA-cellulose chromatography and two-dimensional gel electrophoresis have been used to demonstrate the DNA-binding capacity of bacteriophage T4 gpunf/alc. The unf/alc protein does not bind to DNA via an association with RNA polymerase; gpunf/alc was shown to bind to DNA after separation from RNA polymerase and other large proteins by Sephadex chromatography.