Morphological,genetic and molecular characteristics of barley root hair mutants

Root hairs are tubular outgrowths of specialized epidermal cells called trichoblasts. They affect anchoring plants in soil, the uptake of water and nutrients and are the sites of the interaction between plants and microorganisms. Nineteen root hair mutants of barley representing different stages of root hair development were subjected to detailed morphological and genetic analyses. Each mutant was monogenic and recessive. An allelism test revealed that nine loci were responsible for the mutated root hair phenotypes in the collection and 1–4 mutated allelic forms were identified at each locus. Genetic relationships between the genes responsible for different stages of root hair formation were established. The linkage groups of four loci rhl1, rhp1, rhi1 and rhs1, which had previously been mapped on chromosomes 7H, 1H, 6H and 5H, respectively, were enriched with new markers that flank the genes at a distance of 0.16 cM to 4.6 cM. The chromosomal position of three new genes – two that are responsible for the development of short root hairs (rhs2 and rhs3) and the gene that controls an irregular root hair pattern (rhi2) – were mapped on chromosomes 6H, 2H and 1H, respectively. A comparative analysis of the agrobotanical parameters between some mutants and their respective parental lines showed that mutations in genes responsible for root hair development had no effect on the agrobotanical performance of plants that were grown under controlled conditions. The presented mutant collection is a valuable tool for further identification of genes controlling root hair development in barley.     

SABRE is required for stabilization of root hair patterning in Arabidopsis thaliana

Patterned differentiation of distinct cell types is essential for the development of multicellular organisms. The root epidermis of Arabidopsis thaliana is composed of alternating files of root hair and non‐hair cells and represents a model system for studying the control of cell‐fate acquisition. Epidermal cell fate is regulated by a network of genes that translate positional information from the underlying cortical cell layer into a specific pattern of differentiated cells. While much is known about the genes of this network, new players continue to be discovered. Here we show that the SABRE (SAB) gene, known to mediate microtubule organization, anisotropic cell growth and planar polarity, has an effect on root epidermal hair cell patterning. Loss of SAB function results in ectopic root hair formation and destabilizes the expression of cell fate and differentiation markers in the root epidermis, including expression of the WEREWOLF (WER) and GLABRA2 (GL2) genes. Double mutant analysis reveal that wer and caprice (cpc) mutants, defective in core components of the epidermal patterning pathway, genetically interact with sab. This suggests that SAB may act on epidermal patterning upstream of WER and CPC. Hence, we provide evidence for a role of SAB in root epidermal patterning by affecting cell‐fate stabilization. Our work opens the door for future studies addressing SAB‐dependent functions of the cytoskeleton during root epidermal patterning.     

Multiple cyclic nucleotide‐gated channels coordinate calcium oscillations and polar growth of root hairs

Calcium gradients underlie polarization in eukaryotic cells. In plants, a tip‐focused Ca²⁺‐gradient is fundamental for rapid and unidirectional cell expansion during epidermal root hair development. Here we report that three members of the cyclic nucleotide‐gated channel family are required to maintain cytosolic Ca²⁺ oscillations and the normal growth of root hairs. CNGC6, CNGC9 and CNGC14 were expressed in root hairs, with CNGC9 displaying the highest root hair specificity. In individual channel mutants, morphological defects including root hair swelling and branching, as well as bursting, were observed. The developmental phenotypes were amplified in the three cngc double mutant combinations. Finally, cngc6/9/14 triple mutants only developed bulging trichoblasts and could not form normal root hair protrusions because they burst after the transition to the rapid growth phase. Prior to developmental defects, single and double mutants showed increasingly disturbed patterns of Ca²⁺ oscillations. We conclude that CNGC6, CNGC9 and CNGC14 fulfill partially but not fully redundant functions in generating and maintaining tip‐focused Ca²⁺ oscillations, which are fundamental for proper root hair growth and polarity. Furthermore, the results suggest that these calmodulin‐binding and Ca²⁺‐permeable channels organize a robust tip‐focused oscillatory calcium gradient, which is not essential for root hair initiation but is required to control the integrity of the root hair after the transition to the rapid growth phase. Our findings also show that root hairs possess a large ability to compensate calcium‐signaling defects, and add new players to the regulatory network, which coordinates cell wall properties and cell expansion during polar root hair growth.     

Nitrate induction of root hair density is mediated by TGA1/TGA4 and CPC transcription factors in Arabidopsis thaliana

Root hairs are specialized cells that are important for nutrient uptake. It is well established that nutrients such as phosphate have a great influence on root hair development in many plant species. Here we investigated the role of nitrate on root hair development at a physiological and molecular level. We showed that nitrate increases root hair density in Arabidopsis thaliana. We found that two different root hair defective mutants have significantly less nitrate than wild‐type plants, suggesting that in A. thaliana root hairs have an important role in the capacity to acquire nitrate. Nitrate reductase‐null mutants exhibited nitrate‐dependent root hair phenotypes comparable with wild‐type plants, indicating that nitrate is the signal that leads to increased formation of root hairs. We examined the role of two key regulators of root hair cell fate, CPC and WER, in response to nitrate treatments. Phenotypic analyses of these mutants showed that CPC is essential for nitrate‐induced responses of root hair development. Moreover, we showed that NRT1.1 and TGA1/TGA4 are required for pathways that induce root hair development by suppression of longitudinal elongation of trichoblast cells in response to nitrate treatments. Our results prompted a model where nitrate signaling via TGA1/TGA4 directly regulates the CPC root hair cell fate specification gene to increase formation of root hairs in A. thaliana.     

NUCLEOLAR SIZE DIFFERENCES IN THE GRASS ROOT EPIDERMIS

Root tips of the festucoid grass, Festuca arundinacea, and 2 panicoid species, Chloris gayana and Panicum virgatum, were processed using 2 different staining techniques. Measurements of nucleolar size were taken on epidermal and cortical cells. Trichoblasts and hair cells of Festuca were found to contain much larger nucleoli than those in hairless initials or hairless cells. Significant nucleolar size differences between hair and hairless cells were also found in the 2 panicoid species. In contrast to Festuca, this difference between the 2 cell types was not as pronounced, and overlapping in nucleolar size occurred between adjacent hair and hairless cells. The cortex was composed of rows of cells in which nucleolar size simply decreased with cell distance from the apex. The significance of the observed nucleolar differences among cell types of the root tip is discussed briefly in relation to systematics, enzyme activity patterns, and differentiation.     

Water-relation parameters of epidermal and cortical cells in the primary root ofTriticum aestivum L.

Water-relation parameters of root hair cells, hairless epidermal cells, and cortical cells in the primary root of wheat have been measured using the pressure-probe technique. Under well-watered conditions the mean cell turgor of cortical cells was 6.8±1.9 (30) bar (mean±SD; the number of observations in brackets). In hairless epidermal and root hair cells the mean cell turgor was 5.5±1.9 (22) and 4.4±1.5 (15) bar, respectively. Despite the large variability, turgor pressure was significantly lower (confidence interval=0.95) in epidermal cells relative to cortical cells. This may be a consequence of the ultrafiltration of ions by the external cell wall and-or plasmalemma of epidermal cells. The volumetric elastic modulus of the cells ranged from 10 to 150 bar. This parameter was dependent on cell volume, but within experimental accuracy, was independent of cell type. No pressure dependence of the volumetric elastic modulus was observed in these cells. The half-times for water exchange ranged from 1.8 to 48.8 s. The mean value increased in the order root hair < hairless epidermal < cortical cells and was directly related to volume to surface area ratio. Thus the hydraulic conductivities of the three cell types were similar and averaged 1.2±0.9·10^-6 (170) cm s^-1 bar^-1. No polarity was observed between inwardly and outwardly directed water flow. The similarity of the hydraulic conductivities of root hairs to those of other cells indicates that the membranes of root hairs are not particularly specialized for water transport. The overall hydraulic conductivity for radial water flow across the root was estimated from the pressure-probe data using a simple model and was compared with that measured directly on whole roots using an osmotic backflow technique. It was tentatively concluded that upon sudden osmotic perturbation, the major pathway for water transfer across the root may be through the symplasm and involve net flow from vacuole to vacuole.     

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.     

Salt-induced plasticity of root hair development is caused by ion disequilibrium in Arabidopsis thaliana

Root hair development is controlled by environmental signals. Studies on root hair plasticity in Arabidopsis thaliana have mainly focused on phosphate and iron deficiency. Root hair growth and development and their physiological role in response to salt stress are largely unknown. Here, we show that root epidermal cell types and root hair development are highly regulated by salt stress. Root hair length and density decreased significantly in a dose-dependent manner on both primary roots and junction sites between roots and shoots. The root hair growth and development were sensitive to inhibition by ions but not to osmotic stress. High salinity also alters anatomical structure of roots, leading to a decrease in cell number in N positions and enlargement of the cells. Moreover, analysis of the salt overly sensitive mutants indicated that salt-induced root hair response is caused by ion disequilibrium and appears to be an adaptive mechanism that reduces excessive ion uptake. Finally, we show that genes WER, GL3, EGL3, CPC, and GL2 might be involved in cell specification of root epidermis in stressed plants. Taken together, data suggests that salt-induced root hair plasticity represents a coordinated strategy for early stress avoidance and tolerance as well as a morphological sign of stress adaptation.     

COMPARATIVE ENZYME DIFFERENTIATION IN GRASS ROOTS. I. ACID PHOSPHATASE

Avers , Charlotte J. (U. Miami, Coral Gables, Fla.), and Robert B. Grimm . Comparative enzyme differentiation in grass roots. I. Acid phosphatase. Amer. Jour. Bot. 46(3) : 190-193. Illus. 1959.—There is a correlation between the pattern of acid phosphatase activity and the particular morphogenetic pattern in the root epidermis of festucoid and panicoid grasses. Four festucoid species all showed intensified enzyme activity in trichoblasts and loss of activity in hairless cell initials prior to the maturation of these cells. The 3 panicoid grasses showed no phosphatase-inactive cells during epidermal development. The festucoid epidermis contains alternating long and short cells which differentiate into hairless and hair cells respectively. The panicoid type shows no such cellular pattern and any epidermal cell seems capable of producing a root hair. Treatment of Phleum roots with 10^-4 M coumarin caused a foreshortening of the growth zones and a concurrent apical shift in differential acid phosphatase activity. This response was interpreted as further evidence of a direct correlation between the morphogenetic and enzymatic differentiations in the root epidermis.     

The Arabidopsis root hair mutants der2-der9 are affected at different stages of root hair development

Root hairs are an excellent model system to study cell developmental processes as they are easily accessible, single-celled, long tubular extensions of root epidermal cells. In a genetic approach to identify loci important for root hair development, we have isolated eight der (deformed root hairs) mutants from an ethylmethanesulfonate (EMS)-mutagenized Arabidopsis population. The der lines represent five new loci involved in root hair development and show a variety of abnormalities in root hair morphology, indicating that different root hair developmental stages are affected. A double mutant analysis with the short root hair actin2 mutant der1-2 confirmed that the der mutants are disturbed at different time points of root hair formation. Auxin and ethylene are known to be important for trichoblast cell fate determination and root hair elongation. Here, we show that they are able to suppress the phenotype of two der mutants. As the auxin- and ethylene-responsive der mutants are affected at different stages of root hair formation, our results demonstrate that the function of auxin and ethylene is not limited to cell differentiation and root hair elongation but that the two hormones are effective throughout the whole root hair developmental process.     

Root hairs are the most important root trait for rhizosheath formation of barley (Hordeum vulgare), maize (Zea mays) and Lotus japonicus (Gifu)

Background and AimsRhizosheaths are defined as the soil adhering to the root system after it is extracted from the ground. Root hairs and mucilage (root exudates) are key root traits involved in rhizosheath formation, but to better understand the mechanisms involved their relative contributions should be distinguished.MethodsThe ability of three species [barley (Hordeum vulgare), maize (Zea mays) and Lotus japonicus (Gifu)] to form a rhizosheath in a sandy loam soil was compared with that of their root-hairless mutants [bald root barley (brb), maize root hairless 3 (rth3) and root hairless 1 (Ljrhl1)]. Root hair traits (length and density) of wild-type (WT) barley and maize were compared along with exudate adhesiveness of both barley and maize genotypes. Furthermore, root hair traits and exudate adhesiveness from different root types (axile versus lateral) were compared within the cereal species.Key ResultsPer unit root length, rhizosheath size diminished in the order of barley > L. japonicus > maize in WT plants. Root hairs significantly increased rhizosheath formation of all species (3.9-, 3.2- and 1.8-fold for barley, L. japonicus and maize, respectively) but there was no consistent genotypic effect on exudate adhesiveness in the cereals. While brb exudates were more and rth3 exudates were less adhesive than their respective WTs, maize rth3 bound more soil than barley brb. Although both maize genotypes produced significantly more adhesive exudate than the barley genotypes, root hair development of WT barley was more extensive than that of WT maize. Thus, the greater density of longer root hairs in WT barley bound more soil than WT maize. Root type did not seem to affect rhizosheath formation, unless these types differed in root length.ConclusionsWhen root hairs were present, greater root hair development better facilitated rhizosheath formation than root exudate adhesiveness. However, when root hairs were absent root exudate adhesiveness was a more dominant trait.     

Flavonol rhamnosylation indirectly modifies the cell wall defects of RHAMNOSE BIOSYNTHESIS1 mutants by altering rhamnose flux

Rhamnose is required in Arabidopsis thaliana for synthesizing pectic polysaccharides and glycosylating flavonols. RHAMNOSE BIOSYNTHESIS1 (RHM1) encodes a UDP‐l ‐rhamnose synthase, and rhm1 mutants exhibit many developmental defects, including short root hairs, hyponastic cotyledons, and left‐handed helically twisted petals and roots. It has been proposed that the hyponastic cotyledons observed in rhm1 mutants are a consequence of abnormal flavonol glycosylation, while the root hair defect is flavonol‐independent. We have recently shown that the helical twisting of rhm1 petals results from decreased levels of rhamnose‐containing cell wall polymers. In this study, we found that flavonols indirectly modify the rhm1 helical petal phenotype by altering rhamnose flux to the cell wall. Given this finding, we further investigated the relationship between flavonols and the cell wall in rhm1 cotyledons. We show that decreased flavonol rhamnosylation is not responsible for the cotyledon phenotype of rhm1 mutants. Instead, blocking flavonol synthesis or rhamnosylation can suppress rhm1 defects by diverting UDP‐l ‐rhamnose to the synthesis of cell wall polysaccharides. Therefore, rhamnose is required in the cell wall for normal expansion of cotyledon epidermal cells. Our findings suggest a broad role for rhamnose‐containing cell wall polysaccharides in the morphogenesis of epidermal cells.     

Histology and symplasmic tracer distribution during development of barley androgenic embryos

The present study concerns three aspects of barley androgenesis: (1) the morphology and histology of the embryos during their development, (2) the time course of fluorescent symplasmic tracers’ distribution, and (3) the correlation between symplasmic communication and cell differentiation. The results indicate that barley embryos, which are developing via an androgenic pathway, resemble their zygotic counterparts with respect to their developmental stages, morphology and histology. Analysis of the distribution of the symplasmic tracers, HPTS, and uncaged fluorescein indicates the symplasmic isolation of (1) the protodermis from the underlying cells of the late globular stage onwards, and (2) the embryonic organs at the mature stage of development.     

Cell biology and genetics of root hair formation inArabidopsis thaliana

Summary In this review we integrate the information available on the cell biology of root hair formation with recent findings from the analysis of root hair mutants ofArabidopsis thaliana. The mature Arabidopsis root epidermis consists of root-hair-producing cells and non-root-hair-producing cells. Root hair growth begins with a swelling of the outer epidermal wall. It has been postulated that this is due to a pH-mediated localised cell wall loosening. From the bulge a single root hair emerges which grows by tip growth. The root hair tip consists of a vesicle-rich zone and an organelle-rich subapical zone. The vesicles supply new plasma membrane and cell wall material for elongation. The cytoskeleton and its associated regulatory proteins such as profilin and spectrin are proposed to be involved in the targeting of vesicles. Ca²⁺ influxes and gradients are present in hair tips, but their function is still unclear. Mutants have been isolated with lesions in various parts of the root hair developmental pathway from bulge identity and initiation, to control of tip diameter and extent and polarity of elongation.Abbreviations [Ca²⁺]_c cytosolic calcium concentration - MT microtubule - PM plasma membrane - VRZ vesicle-rich zone - WT wild type Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday