David G. Oppenheimer

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.

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.

SIAMESE, a Plant-Specific Cell Cycle Regulator, Controls Endoreplication Onset in Arabidopsis thaliana

Recessive mutations in the SIAMESE (SIM) gene of Arabidopsis thaliana result in multicellular trichomes harboring individual nuclei with a low ploidy level, a phenotype strikingly different from that of wild-type trichomes, which are single cells with a nuclear DNA content of ∼16C to 32C. These observations suggested that SIM is required to suppress mitosis as part of the switch to endoreplication in trichomes. Here, we demonstrate that SIM encodes a nuclear-localized 14-kD protein containing a cyclin binding motif and a motif found in ICK/KRP (for Interactors of Cdc2 kinase/Kip-related protein) cell cycle inhibitor proteins. Accordingly, SIM was found to associate with D-type cyclins and CDKA;1. Homologs of SIM were detected in other dicots and in monocots but not in mammals or fungi. SIM proteins are expressed throughout the shoot apical meristem, in leaf primordia, and in the elongation zone of the root and are localized to the nucleus. Plants overexpressing SIM are slow-growing and have narrow leaves and enlarged epidermal cells with an increased DNA content resulting from additional endocycles. We hypothesize that SIM encodes a plant-specific CDK inhibitor with a key function in the mitosis-to-endoreplication transition.

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.

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.

Floral gene resources from basal angiosperms for comparative genomics research

BackgroundThe Floral Genome Project was initiated to bridge the genomic gap between the most broadly studied plant model systems. Arabidopsis and rice, although now completely sequenced and under intensive comparative genomic investigation, are separated by at least 125 million years of evolutionary time, and cannot in isolation provide a comprehensive perspective on structural and functional aspects of flowering plant genome dynamics. Here we discuss new genomic resources available to the scientific community, comprising cDNA libraries and Expressed Sequence Tag (EST) sequences for a suite of phylogenetically basal angiosperms specifically selected to bridge the evolutionary gaps between model plants and provide insights into gene content and genome structure in the earliest flowering plants.ResultsRandom sequencing of cDNAs from representatives of phylogenetically important eudicot, non-grass monocot, and gymnosperm lineages has so far (as of 12/1/04) generated 70,514 ESTs and 48,170 assembled unigenes. Efficient sorting of EST sequences into putative gene families based on whole Arabidopsis/rice proteome comparison has permitted ready identification of cDNA clones for finished sequencing. Preliminarily, (i) proportions of functional categories among sequenced floral genes seem representative of the entire Arabidopsis transcriptome, (ii) many known floral gene homologues have been captured, and (iii) phylogenetic analyses of ESTs are providing new insights into the process of gene family evolution in relation to the origin and diversification of the angiosperms.ConclusionInitial comparisons illustrate the utility of the EST data sets toward discovery of the basic floral transcriptome. These first findings also afford the opportunity to address a number of conspicuous evolutionary genomic questions, including reproductive organ transcriptome overlap between angiosperms and gymnosperms, genome-wide duplication history, lineage-specific gene duplication and functional divergence, and analyses of adaptive molecular evolution. Since not all genes in the floral transcriptome will be associated with flowering, these EST resources will also be of interest to plant scientists working on other functions, such as photosynthesis, signal transduction, and metabolic pathways.

IRREGULAR TRICHOME BRANCH1 in Arabidopsis Encodes a Plant Homolog of the Actin-Related Protein2/3 Complex Activator Scar/WAVE That Regulates Actin and Microtubule Organization

The dynamic actin cytoskeleton is important for a myriad of cellular functions, including intracellular transport, cell division, and cell shape. An important regulator of actin polymerization is the actin-related protein2/3 (Arp2/3) complex, which nucleates the polymerization of new actin filaments. In animals, Scar/WAVE family members activate Arp2/3 complex-dependent actin nucleation through interactions with Abi1, Nap1, PIR121, and HSCP300. Mutations in the Arabidopsis thaliana genes encoding homologs of Arp2/3 complex subunits PIR121 and NAP1 all show distorted trichomes as well as additional epidermal cell expansion defects, suggesting that a Scar/WAVE homolog functions in association with PIR121 and NAP1 to activate the Arp2/3 complex in Arabidopsis. In a screen for trichome branching defects, we isolated a mutant that showed irregularities in trichome branch positioning and expansion. We named this gene IRREGULAR TRICHOME BRANCH1 (ITB1). Positional cloning of the ITB1 gene showed that it encodes SCAR2, an Arabidopsis protein related to Scar/WAVE. Here, we show that itb1 mutants display cell expansion defects similar to those reported for the distorted class of trichome mutants, including disruption of actin and microtubule organization. In addition, we show that the scar homology domain (SHD) of ITB1/SCAR2 is necessary and sufficient for in vitro binding to Arabidopsis BRK1, the plant homolog of HSPC300. Overexpression of the SHD in transgenic plants causes a dominant negative phenotype. Our results extend the evidence that the Scar/WAVE pathway of Arp2/3 complex regulation exists in plants and plays an important role in regulating cell expansion.

The PRETTY FEW SEEDS2 gene encodes an Arabidopsis homeodomain protein that regulates ovule development

The PRETTY FEW SEEDS2 gene encodes a homeodomain protein that regulates ovule development. In peptide alignments spanning the homeodomain and the WOX domain, PFS2 shared 95% amino acid identity with the PRESSED FLOWER and WUSCHEL proteins. In the pfs2-1 allele, the integuments display morphological abnormalities and 95% of the embryo sacs fail to develop properly, which results in reduced fecundity. PFS2 transcripts were most abundant in developing ovules, which accounts for the ovule phenotype in pfs2 mutants. In addition, PFS2 transcripts were present in developing primordia and differentiating organs, but, interestingly, they were absent during cell maturation. Ectopic PFS2 expression interfered with differentiation of primordia from meristems. For most plants, this resulted in fasciated stems, altered phyllotaxy, a cessation of primordia differentiation, or a combination of these. In the plants that made ovules, ectopic PFS2 expression blocked megaspore mother cell differentiation and often impeded polarized growth of the outer integument. PFS2 activity altered AGAMOUS expression, which accounts for some of the gain- and loss-of-function phenotypes. Based on analyses presented here, PFS2 affects either ovule patterning or differentiation.

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.

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 cytoskeletons role in a specific morphogenetic event.