Constructing a plant cell. The genetic control of root hair development.
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Gene Expression Regulation, Developmental↗
Biomedical subjects
J W Schiefelbein
Publications and source records attributed to J W Schiefelbein.
The ROOT HAIR DEFECTIVE3 gene encodes an evolutionarily conserved protein with GTP-binding motifs and is required for regulated cell enlargement in Arabidopsis.
In plants, morphogenesis is largely determined by the orientation and extent of cell enlargement. To define the molecular mechanisms regulating plant cell enlargement, we have conducted a molecular genetic analysis of the ROOT HAIR DEFECTIVE3 (RHD3) gene of Arabidopsis thaliana. Mutations affecting the RHD3 gene were found to alter cell size, but not cell number, in tissues throughout the plant. Genetic and physiological analyses suggest that the RHD3 gene is not required for proper cell type specification, and it is likely to act downstream of the hormones auxin and ethylene. The RHD3 gene was cloned by a T-DNA tagging method and confirmed by the molecular complementation of the rhd3 mutant phenotype and by the analyses of six rhd3 mutant alleles. Consistent with the global effects of the rhd3 mutations, the RHD3 gene is expressed in all major plant organs. The deduced RHD3 product is a novel 89-kD polypeptide with putative GTP-binding motifs near the amino terminus. RHD3-like genes were identified from a protozoan (Entamoeba histolytica), a fungus (Saccharomyces cerevisiae), and another plant species (Oryza sativa), with the sequence identity including the putative GTP-binding motifs. These results imply that the RHD3 protein is a member of a new class of GTP-binding proteins that is widespread in eukaryotes and required for regulated cell enlargement.
Alleles↗
Growth and ultrastructure of Arabidopsis root hairs: the rhd3 mutation alters vacuole enlargement and tip growth.
The root hairs of plants are tubular projections of root epidermal cells and are suitable for investigating the control of cellular morphogenesis. In wild-type Arabidopsis thaliana (L.) Heynh, growing root hairs were found to exhibit cellular expansion limited to the apical end of the cell, a polarized distribution of organelles in the cytoplasm, and vesicles of several types located near the growing tip. The rhd3 mutant produces short and wavy root hairs with an average volume less than one-third of the wild-type hairs, indicating abnormal cell expansion. The mutant hairs display a striking reduction in vacuole size and a corresponding increase in the relative proportion of cytoplasm throughout hair development. Beadlabeling experiments and ultrastructural analyses indicate that the wavy-hair phenotype of the mutant is caused by asymmetric tip growth, possibly due to abnormally distributed vesicles in cortical areas flanking the hair tips. It is suggested that a major effect of the rhd3 mutation is to inhibit vacuole enlargement which normally accompanies root hair cell expansion.
Arabidopsis↗
Building a root: the control of patterning and morphogenesis during root development.
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Arabidopsis↗
Hormones act downstream of TTG and GL2 to promote root hair outgrowth during epidermis development in the Arabidopsis root.
The Arabidopsis root produces a position-dependent pattern of hair-bearing and hairless cell types during epidermis development. Five loci (TRANSPARENT TESTA GLABRA [TTG], GLABRA2 [GL2], ROOT HAIR DEFECTIVE6 [RHD6], CONSTITUTIVE TRIPLE RESPONSE1 [CTR1], and AUXIN RESISTANT2 [AXR2]) and the plant hormones ethylene and auxin have been reported to affect the production of root hair and hairless cells in the Arabidopsis root. In this study, genetic, molecular, and physiological tests were employed to define the roles of these loci and hormones. Epistasis tests and reporter gene studies indicated that the hairless cell-promoting genes TTG and GL2 are likely to act early to negatively regulate the ethylene and auxin pathways. Studies of the developmental timing of the hormone effects indicated that ethylene and auxin pathways promote root hair outgrowth after cell-type differentiation has been initiated. The genetic analysis of ethylene-and auxin-related mutations showed that root hair formation is influenced by a network of hormone pathways, including a partially redundant ethylene signaling pathway. A model is proposed in which the patterning of root epidermal cells in Arabidopsis is regulated by the cell position-dependent action of the TTG/GL2 pathway, and the ethylene and auxin hormone pathways act to promote root hair outgrowth at a relatively late stage of differentiation.
Amino Acids↗
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.
Arabidopsis↗
Responses to iron deficiency in Arabidopsis thaliana: the Turbo iron reductase does not depend on the formation of root hairs and transfer cells.
Arabidopsis thaliana (L.) Heynh. Columbia wild type and a root hair-less mutant RM57 were grown on iron-containing and iron-deficient nutrient solutions. In both genotypes, ferric chelate reductase (FCR) of intact roots was induced upon iron deficiency and followed a Michaelis-Menten kinetic with a Km of 45 and 54 microM FeIII-EDTA and a Vmax of 42 and 33 nmol Fe2+.(g FW)-1.min-1 for the wild type and the mutant, respectively. The pH optimum for the reaction was around pH 5.5. The approximately four fold stimulation of FCR activity was independent of formation of root hairs and/or transfer cells induced by iron deficiency. Iron-deficiency-induced chlorosis and the development of a rigid root habit disappeared when ferric chelate was applied to the leaves, while FCR activity remained unchanged. The time course of the responses to iron deficiency showed that morphological and physiological responses were controlled separately.
Arabidopsis↗
The TTG gene is required to specify epidermal cell fate and cell patterning in the Arabidopsis root.
The control of cell fate was investigated in the root epidermis of Arabidopsis thaliana. Two distinct types of differentiated epidermal cells are normally present: root-hair-bearing cells and hairless cells. In wild-type Arabidopsis roots, epidermal cell fate was found to be correlated with cell position, with root-hair cells located over radial walls between cortical cells, and with hairless cells located directly over cortical cells. This normal positional relationship was absent in ttg (transparent testa glabrous) mutants (lacking trichomes, anthocyanins, and seed coat mucilage); epidermal cells in all positions differentiate into root-hair cells. The opposite condition was generated in roots of transgenic Arabidopsis expressing the maize R (R-Lc) gene product (a putative TTG homologue) under the control of a strong promoter (CaMV35S), which produced hairless epidermal cells in all positions. In both the ttg and R-expressing roots, epidermal cell differentiation was affected at an early stage, prior to the onset of cell elongation or root-hair formation. The ttg mutations were also associated with abnormalities in the morphology and organization of cells within and surrounding the root apical meristem. The results indicate that alterations in TTG activity cause developing epidermal cells to misinterpret their position and differentiate into inappropriate cell types. This suggests that, in wild-type roots, TTG provides, or responds to, positional signals to cause differentiating epidermal cells that lie over cortical cells to adopt a hairless cell fate.
Arabidopsis↗
Getting to the root of plant development: the genetics of Arabidopsis root formation.
Root development in Arabidopsis thaliana is amenable to molecular genetic analyses because of its simplicity and accessibility. Genetic screens have identified a rich collection of mutants that can be used to address a variety of fundamental questions in plant developmental biology. These mutants have defects in genes that govern organ formation, meristem activity, cell differentiation and response to environmental conditions.
Arabidopsis↗
Cell fate and cell morphogenesis in higher plants.
The differentiation of plant cells depends on the regulation of cell fate and cell morphogenesis. Recent studies have led to the identification of mutants and the cloning of genes that influence these processes. In several instances, the genes encode products with homeodomains or Myb or Myc DNA-binding domains.
Arabidopsis↗
Root development in Arabidopsis: four mutants with dramatically altered root morphogenesis.
A genetic analysis of root development in Arabidopsis thaliana has identified mutants that have abnormal morphogenesis. Four of these root morphogenesis mutants show dramatic alterations in post-embryonic root development. The short-root mutation results in a change from indeterminate to determinate root growth and the loss of internal root cell layers. The cobra and lion's tail mutations cause abnormal root cell expansion which is conditional upon the rate of root growth. Expansion is greatest in the epidermal cells in cobra and in the stele cells in lion's tail. The sabre mutation causes abnormal cell expansion that is greatest in the root cortex cell layer and is independent of the root growth rate. The tissue-specific effects of these mutations were characterized with monoclonal antibodies and a transgenic marker line. Genetic combinations of the four mutants have provided insight into the regulation of growth and cell shape during Arabidopsis root development.
Arabidopsis↗
Alternative 3' splice acceptor sites modulate enzymic activity in derivative alleles of the maize bronze1-mutable 13 allele.
The defective Suppressor-mutator (dSpm)-induced allele bronze1-mutable 13 (bz1-m13) and many of its derivative alleles are leaky mutants with measurable levels of flavonol O3-glucosyltransferase activity. This activity results from splicing at acceptor site-1, one of two cryptic 3' splice sites within the dSpm insertion in bz1-m13. In this study, splicing in bz1-m13 change-in-state (CS) alleles CS-3 and CS-64 was shown to be altered from bz1-m13; previous work found altered splicing in CS-9. CS-64 is a null allele and lacks the acceptor site-1-spliced transcript because this site is deleted. CS-3 and CS-9 had increased levels of the acceptor site-1 transcript relative to bz1-m13 and increased enzymic activities. A deletion in CS-9 altered splicing by eliminating acceptor site-2. Both acceptor sites were intact in CS-3, but a deletion removed most of a 275-bp GC-rich sequence in dSpm. This suggests that GC-rich sequences affect splicing and is consistent with models postulating a role for AU content in the splicing of plant introns. Splicing does not necessarily occur, however, at the junction of AU-rich intron sequences and GC-rich exon sequences.
Alleles↗
The development of plant roots: new approaches to underground problems.
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Mutation↗
Tissue-specific effects of maize bronze gene promoter mutations induced by Ds1 insertion and excision.
Bz-wm is an allele of the Bz locus of maize isolated by McClintock (1962) as a derivative of bz-m2. It contains a Ds1 insertion 63 bp upstream of the start of transcription and a 3 bp insertion in the coding region at the site of the Ac element that was present in bz-m2. Bz-wm produces, in the aleurone layer of the endosperm, low amounts (approximately 1% of wild-type) of a Bz-gene encoded UDP-glucose: flavoid 3-0-glucosyltransferase (UFGT) polypeptide with altered thermal stability. Three phenotypically wild-type derivatives, Bz' (wm)-1, Bz' (wm)-2 and Bz' (wm)-3, were isolated in the presence of Ac and shown to have excised the Ds1 element but not fully restored UFGT activity in endosperm assays. In the studies reported here, we have further analyzed these Bz' derivatives of Bz-wm by determining the DNA sequences left behind on Ds1 excision, and by measuring the amount of UFGT activity and/or Bz mRNA conditioned by Bz-wm and the Bz' derivatives in different tissues. The data indicate that tissue-specific differences in expression of the Bz gene have been produced in alleles with mutations caused by transposable elements Ac and Ds. These mutations may affect either the amount of Bz transcription or the stability of the UFGT polypeptide. The sequence or spacing in the -63 region of the Bz promoter appears to be critical for maximum expression in aleurone and husk but not in pollen and pigmented seedling tissue.
Alleles↗
Molecular characterization of suppressor-mutator (Spm)-induced mutations at the bronze-1 locus in maize: the bz-m13 alleles.
The bz-m13 allele of maize contains a defective Suppressor-mutator (dSpm) transposable element and gives rise to a variety of stable and unstable derivatives in the presence of an autonomous Suppressor-mutator (Spm) element. The dSpm-13 element of bz-m13 consists of 2,241 base pairs (bp) and is located within the second exon of the bronze-1 (bz) gene. A number of the stable derivatives, both functional and nonfunctional, derived from bz-m13 were characterized molecularly. Results from genomic DNA blotting experiments indicate that the dSpm-13 element had excised from the locus in each stable derivative analyzed. The unstable derivatives bz-m13CS9 and bz-m13CS6 contain dSpm elements in the same position and orientation as the dSpm-13 element, but they differ in the length of the element. The dSpm-13CS9 element is 902 bp and arose via a deletion between two 5-bp direct repeats within the dSpm-13 element. The dSpm-13CS6 element is 2,239 bp and only differs from dSpm-13 by a 2-bp deletion at the end of one of the 13-bp terminal inverted repeats. The effect of these deletions on the frequency and timing of Spm-induced excision is discussed herein. In the absence of Spm, each of the bz-m13 alleles conditions a nonmutant phenotype despite the presence of the insertions in the second exon. The role of RNA splicing in this phenomenon and the recent finding of an acceptor splice site within the terminal inverted repeat are also discussed.
Alleles↗
Two mutations in a maize bronze-1 allele caused by transposable elements of the Ac-Ds family alter the quantity and quality of the gene product.
The Dissociation (Ds) mutant, Bz-wm, of the maize bronze-1 (bz) locus conditions a leaky phenotype. Plants carrying this mutant allele synthesize a low amount of an altered Bz gene product, which leads to reduced anthocyanin pigmentation in the seed. The molecular analysis reported here shows that the Bz-wm mutant has a 406-bp Ds1 insertion located 63 bp 5' to the start of Bz transcription. Furthermore, the Bz-wm allele contains three additional base pairs within the second exon, relative to the wild-type Bz allele. These additional nucleotides are believed to be derived from the 8-bp target site duplication created by an Activator (Ac) element in a previous allele in the series. The biochemical and molecular analyses of Bz-wm and revertants of Bz-wm indicate that the three additional nucleotides are responsible for the altered enzyme stability, while the Ds1 element affects the steady-state level of Bz-specific protein and RNA. Since the two mutations present in the Bz-wm mutant were each caused by the action of the Ac-Ds transposable element system, these results provide new insights into the ways that transposable elements can modify maize gene expression.
Alleles↗
RNA splicing permits expression of a maize gene with a defective Suppressor-mutator transposable element insertion in an exon.
The bz-m13CS9 allele of the bronze-1 gene in maize contains a 902-base-pair defective Suppressor-mutator (dSpm) transposable element in the second exon. Nevertheless, 40-50% of the enzymatic activity conditioned by a nonmutant allele at the bronze-1 locus is routinely recovered in crude extracts prepared from plants carrying bz-m13CS9 in the absence of an autonomous Suppressor-mutator element. Analyses of RNAs produced by such plants show that transcription proceeds through the dSpm. The dSpm sequence of the messenger RNA precursor is then removed by RNA splicing using the donor site of the single bronze-1 intron and an acceptor site within the inverted terminal repeat of the dSpm. This results in a messenger RNA with the proper reading frame that could produce a functional enzyme. These data demonstrate that this dSpm insertion in an exon of a structural gene has produced a functional allele with a novel intron consisting, in part, of the dSpm. This mechanism appears to allow dSpm elements to reduce the impact of their insertions on gene expression.
Alleles↗
Deletions within a defective suppressor-mutator element in maize affect the frequency and developmental timing of its excision from the bronze locus.
Six independent derivatives of the bz-m13 allele, which contains a 2.2-kilobase-pair defective Suppressor-mutator (dSpm) insertion at the bronze (bz) locus, have been isolated and analyzed. The derivatives were selected for alterations in the frequency and timing of somatic reversion; such derivatives have previously been analyzed genetically and designated "changes in state" by McClintock [McClintock, B. (1955) Carnegie Inst. Washington, Yearb. 54, 245-255]. All of the derivatives analyzed in the present study revert substantially later in development than the original insertion mutation and some show a very low frequency of reversion as well. All of the derivatives contain insertions at the same site as the parent bz-m13 allele. Deletions of 400-1300 base pairs were found in the dSpm elements in four of the six derivatives; the remaining derivatives could not be distinguished structurally from the original mutant allele. The results suggest that changes in the frequency and developmental timing of excision are attributable to alterations in the dSpm element. Furthermore, these data suggest that DNA sequences near the ends of the element are important for responding to the two transacting functions supplied by the transposition-competent Suppressor-mutator (Spm) element.
Base Sequence↗