Root Hair Initiation Research Articles

Auxin modulates the enhanced development of root hairs in Arabidopsis thaliana (L.) Heynh. under elevated CO2

Root hairs may play a critical role in nutrient acquisition of plants grown under elevated CO(2) . This study investigated how elevated CO(2) enhanced the development of root hairs in Arabidopsis thaliana (L.) Heynh. The plants under elevated CO(2) (800 µL L(-1)) had denser and longer root hairs, and more H-positioned cells in root epidermis than those under ambient CO(2) (350 µL L(-1)). The elevated CO(2) increased auxin production in roots. Under elevated CO(2) , application of either 1-naphthoxyacetic acid (1-NOA) or N-1-naphthylphthalamic acid (NPA) blocked the enhanced development of root hairs. The opposite was true when the plants under ambient CO(2) were treated with 1-naphthylacetic acid (NAA), an auxin analogue. Furthermore, the elevated CO(2) did not enhance the development of root hairs in auxin-response mutants, axr1-3, and auxin-transporter mutants, axr4-1, aux1-7 and pin1-1. Both elevated CO(2) and NAA application increased expressions of caprice, triptychon and rho-related protein from plants 2, and decreased expressions of werewolf, GLABRA2, GLABRA3 and the transparent testa glabra 1, genes related to root-hair development, while 1-NOA and NPA application had an opposite effect. Our study suggests that elevated CO(2) enhanced the development of root hairs in Arabidopsis via the well-characterized auxin signalling and transport that modulate the initiation of root hairs and the expression of its specific genes.

Conserved and Unique Features of the Maize (Zea mays L.) Root Hair Proteome

Root hairs are unicellular extensions of specialized epidermis cells. Under limiting conditions, they significantly increase the water and nutrient uptake capacity of plants by enlarging their root surface. Thus far, little is known about the initiation and growth of root hairs in the monocot model species maize. To gain a first insight into the protein composition of these specialized cells, the 2573 most abundant proteins of maize root hairs attached to four-day-old primary roots of the inbred line B73 were identified by combining 1DE with nanoLC-MS/MS in a shotgun proteomic experiment. Among the identified proteins, homologues of 252 proteins have been previously associated with root hair formation and development in other species. Comparison of the root hair reference proteome of the monocot species maize with the previously published root hair proteome of the dicot species soybean revealed conserved, but also unique, protein functions in root hairs of these two major groups of flowering plants.

Differential gene expression in leaves and roots of winter rape in response to phosphorus starvation

Genes induced by phosphorus deficiency in leaves and roots of winter rape (Brassica napus) seedlings were isolated and analyzed. The mRNA differential display technique was used to visualize cDNA fragments derived from the phosphorus-tolerant cv. Zy 821 and the phosphorus-sensitive cv. No. 1182 grown under conditions of phosphorous starvation. Approximately 2000 cDNA bands were visualized by differential display using 78 primer pair combinations. A total of 61 phosphorus-starvation-induced cDNA fragments differentially expressed were isolated. Among these, 40 were derived from roots and only 21 from leaves. Sequence analysis of 16 fragments revealed that they represented distinct cDNAs. A subsample of five cDNAs was analyzed by semi-quantitative RT-PCR, which showed that they were truly different products. Transcripts coding for enzymes involved in photosynthesis (thylakoid membrane phosphoprotein) and root hair initiation (xyloglucan endotransglycosylase), and two regulators (RNA-binding/nucleic acid-binding mRNA and a structural constituent of ribosomal mRNA) were found to be differentially expressed. These results show that the mechanism of cv. Zy 821 adaptation to P starvation is complex. Although its P-tolerance trait is controlled by a major gene, many other genes influencing P acquisition and remobilization, carbon and secondary metabolism, developmental processes, and regulatory pathways are also involved.

Structural Sterols Are Involved in Both the Initiation and Tip Growth of Root Hairs inArabidopsis thaliana

Structural sterols are abundant in the plasma membrane of root apex cells in Arabidopsis thaliana. They specifically accumulate in trichoblasts during the prebulging and bulge stages and show a polar accumulation in the tip during root hair elongation but are distributed evenly in mature root hairs. Thus, structural sterols may serve as a marker for root hair initiation and growth. In addition, they may predict branching events in mutants with branching root hairs. Structural sterols were detected using the sterol complexing fluorochrome filipin. Application of filipin caused a rapid, concentration-dependent decrease in tip growth. Filipin-complexed sterols accumulated in globular structures that fused to larger FM4-64-positive aggregates in the tip, so-called filipin-induced apical compartments, which were closely associated with the plasma membrane. The plasma membrane appeared malformed and the cytoarchitecture of the tip zone was affected. Trans-Golgi network/early endosomal compartments containing molecular markers, such as small Rab GTPase RabA1d and SNARE Wave line 13 (VTI12), locally accumulated in these filipin-induced apical compartments, while late endosomes, endoplasmic reticulum, mitochondria, plastids, and cytosol were excluded from them. These data suggest that the local distribution and apical accumulation of structural sterols may regulate vesicular trafficking and plasma membrane properties during both initiation and tip growth of root hairs in Arabidopsis.

High resolution imaging to assess oilseed species’ root hair responses to soil water stress

An imaging method was developed to evaluate crop species differences in root hair morphology using high resolution scanners, and to determine if the method could also detect root hair responses to soil water availability. High resolution (1890 picture elements (pixels) cm−1) desktop scanners were buried in containers filled with soil to characterize root hair development under two water availability levels (−63 and −188 kPa) for canola (Brassica napus L. cv Clearwater), camelina (Camelina sativa L. Crantz cv Cheyenne), flax (Linum usitatissimum L. cv CDC Bethune), and lentil (Lens culinaris Medik. cv Brewer). There was notable effect of available moisture on root hair geometry (RHG). At −188 kPa, length from the root tip to the root hair initiation zone decreased and root hair length (RHL) became more variable near the root hair initiation zone as compared to −63 kPa. For the response of primary axial RHL, significant main effects were present for both water availability (P \text{flax} = \text{lentil}} \right) \) had lower RHD \( \left( {\text{canola} = \text{camelina} < \text{flax} < \text{lentil}} \right) \) in primary axial roots and a similar trend was found in lateral RHL. Both water and species had a significant effect on primary axial root surface area (RSA) (P < 0.05) but no significant effect was found for lateral RSA. For primary axial RSA the longest and most dense root hair had the greatest RSA. This novel approach to in situ rhizosphere imaging allowed observation of species differences in root hair development in response to water availability and should be useful in future studies of rhizosphere interactions and crop water and nutrient management.

Arabidopsis IAR4 Modulates Auxin Response by Regulating Auxin Homeostasis

In a screen for enhancers of tir1-1 auxin resistance, we identified two novel alleles of the putative mitochondrial pyruvate dehydrogenase E1alpha-subunit, IAA-Alanine Resistant4 (IAR4). In addition to enhancing the auxin response defects of tir1-1, iar4 single mutants exhibit numerous auxin-related phenotypes including auxin-resistant root growth and reduced lateral root development, as well as defects in primary root growth, root hair initiation, and root hair elongation. Remarkably, all of these iar4 mutant phenotypes were rescued when endogenous indole-3-acetic acid (IAA) levels were increased by growth at high temperature or overexpression of the YUCCA1 IAA biosynthetic enzyme, suggesting that iar4 mutations may alter IAA homeostasis rather than auxin response. Consistent with this possibility, iar4 mutants exhibit increased Aux/IAA stability compared to wild type under basal conditions, but not in response to an auxin treatment. Measurements of free IAA levels detected no significant difference between iar4-3 and wild-type controls. However, we consistently observed significantly higher levels of IAA-amino acid conjugates in the iar4-3 mutant. Furthermore, using stable isotope-labeled IAA precursors, we observed a significant increase in the relative utilization of the Trp-independent IAA biosynthetic pathway in iar4-3. We therefore suggest that the auxin phenotypes of iar4 mutants are the result of altered IAA homeostasis.

Microtubule reorganization as a response to implementation of NO signals in plant cells

The effects of an exogeneous NO donor, sodium nitroprusside, on the orientation and organization of cortical microtubules in Arabidopsis thaliana root cells expressing GFP-MAP4 were studied in vivo. It was found that sodium nitroprusside treatment (10–500 μM, 24 h) caused the acceleration of primary root growth and enhanced initiation of root hairs in the differentiation zone. The influence of sodium nitroprusside revealed in changes in the orientation and organization of cortical microtubules in different types of cells of A. thaliana root. The most sensitive to sodium nitroprusside exposure were microtubules in epidermal cells of the elongation zone, where native transverse orientation of cortical microtubules turned into random, oblique, or longitudinal relative to the primary root axis. We suggest that NO, as one of the intracellular secondary messengers, triggers cell differentiation by reorientation of cortical microtubules, possibly via tubulin nitrotyrosination.

Parasitic angiosperms, semagenesis and general strategies for plant–plant signaling in the rhizosphere

In addition to their roles in eukaryotic defense and development, reactive oxygen species (ROS) have recently been identified as critical for host attachment by the parasitic angiosperms. In a process known as semagenesis, ROS generated at the root tip of Striga asiatica (L.) Kuntze (Scrophulariaceae) function together with host peroxidases to oxidize monolignols at the host root surface. As a result, para-benzoquinone products accumulate as both necessary and sufficient components for inducing development of the host attachment organ, the haustorium. This event constitutes the critical vegetative/pathogenic transition in the parasite. New evidence is presented that semagenesis occurs broadly in plant-plant signaling. Eudicotyledenous seedlings are more sensitive to the xenognostic benzoquinones than monocots, but general root development, including root elongation, root hair initiation and root hair growth, is impacted in both clades. Specific inhibitors of haustorial development in S. asiatica also inhibit benzoquinone-mediated root development in the non-parasites. These results suggest a common mechanism for benzoquinone perception. Semagenesis enriches our understanding of the mechanisms available for small-molecule underground information exchange among plants. Critical differences in this process, as used by parasitic plants, are beginning to emerge and point towards new strategies for managing parasitic angiosperms in agricultural settings.

Dynamics of growth, proliferation and differentiation of wheat root cells exposed to a high nickel concentration

The dynamics of root growth, proliferation of initial cells of the root cap, rhizodermis, and central metaxylem, as well as structural changes in the cells induced by a 72-h exposure to a high (0.1 mM) concentration of NiSO4 were studied in 3-day-old wheat (Triticum aestivum L.) seedlings. In the roots of control plants, we observed a 12-h rhythm of changes in the length of the cells that completed elongating. Upon the treatment with nickel, this effect was negated, and a considerable reduction in the root length increment was observed in 12 h. In 24 h, root growth essentially ceased. Cell elongation was suppressed acropetally, and the cells, whose elongation was over, became shorter. In the meristem and apical part of the elongation zone, slow cell growth continued during the second and even third days. Autoradiography showed that the earliest effect of nickel on the processes of root morphogenesis observed in 6 h was a suppression of cell transition to DNA synthesis. The cells, where DNA synthesis has already started or which were in other stages of the cycle, continued to pass slowly through the cycle and completed it. Sister cells formed as a result of division subsequently left the cycle in the phase G1 and transited to dormancy. It was found that the main mechanism of cell proliferation cessation was the suppression of cell transition to DNA synthesis. In the cells elongating when exposed to nickel, tissue-specific changes in the nucleus structure were observed (chromatolysis in the rhizodermis and cortex, pycnosis in the endodermis, a disturbance of the nucleus structure in the central metaxylem). These disorders were only observed after cessation of elongation. Root incubation in 0.1 mM nickel solution did not affect the onset of cell differentiation in the xylem and metaphloem and shifted its beginning to the root tip. However, in 24 h the initiation and growth of root hairs were suppressed. It was concluded that tissue-specific nickel-induced changes in the nucleus structure in the elongating cells do not cause the cessation of root growth, although point to nickel toxic effect on the cells in the course of elongation.

Phytotoxic effects of leukamenin E (an ent-kaurene diterpenoid) on root growth and root hair development in Lactuca sativa L. seedlings.

Leukamenin E, an ent-kaurene diterpenoid isolated from Isodon racemosa (Hemsl) Hara, showed phytotoxic effects on root growth and root hair development of lettuce seedlings (Lactuca sativa L.). Lower concentrations (10 microM) of leukamenin E did not affect root growth, but at concentrations higher than 50 microM, the rate was inhibited. The influence of leukamenin E on root growth rate was closely correlated with alterations in the mitotic index. A low incidence of aberrant mitosis image was observed when lettuce roots were treated with higher concentrations (100 and 200 microM) of leukamenin E. This suggests that inhibition of root growth may be due to inhibition of cell division. All tested concentrations of the diterpenoid (10 microM or more) inhibited root hair development in a dose-dependent manner. At a concentration of 80 microM, leukamenin E completely blocked root hair initiation. Application of Ag(+)-an ethylene action inhibitor-to lettuce seedlings inhibited root hair elongation similar to the diterpenoid. Enhanced root hair length was stimulated by exogenous ethephon-an ethylene-releasing agent-and could be reversed by addition of leukamenin E. This suggests that leukamenin E may act as a potential ethylene action antagonist in the inhibition of lettuce root hair development. We conclude that leukamenin E may curb root hair development by interfering with ethylene action at concentrations above 10 microM and inhibits root growth via inhibition of cell division at concentrations above 50 microM.

Stage-specific crosstalk between light, auxin, and ethylene during low-pH-induced root hair formation in lettuce (Lactuca sativa L.) seedlings

In the light, transfer of lettuce seedlings precultured on liquid medium at pH 6.0 to fresh medium at pH 4.0 induces root hair formation. However, no root hairs form in the dark. Here, we investigated how light induces root hair formation. Randomization of the transverse cortical microtubule (CMT) arrays which occurs in root epidermal cells in the light prior to root hair initiation was not observed in the dark. However, addition of indole-3-acetic acid (IAA) or 1-aminocyclopropane-1-carboxylic acid (ACC) induced CMT randomization and root hair formation. In these cases, CMT randomization occurred in almost the same time-dependent manner as under light. However, root hair initiation was delayed for several hours in the dark. These results suggest that light promotes CMT randomization and root hair initiation via auxin and ethylene signaling but light additionally influences root hair initiation independently of these signaling mechanisms. Furthermore, addition of a microtubule-depolymerizing drug in the dark disrupted the transverse CMT arrays and initiated root hair formation; however, root hair elongation was still suppressed. Root hairs elongated when IAA or ACC was applied with the drug. These results suggest that light also promotes root hair elongation via auxin and ethylene signaling.

Ultrastructure of Medicago sativa rhizodermis cells over their early development

Ultrastructure was investigated along the files of developing epidermal cells in the root tip of a model plant Medicago sativa, in which all rhizodermal cells are potential hair-forming trichoblasts. Differentiation at subcellular level was observed up to the stage of bulge initiation in the trichoblasts. Root hair initiation indicated by the emergence of bulges from trichoblasts was detected at various distances from the root tip and, it was independent of the trichoblast size. During rhizodermal cell differentiation, starch grains accumulated in the plastids. Nuclei located in the central part of the young, meristematic cells moved towards the inner periclinal wall as the central vacuole enlarged. The bulging region of the trichoblasts located opposite the nucleus and was rich in mitochondria, ER, ribosomes, and Golgi bodies, and contained also vesicles enclosing fibrillar material. This material responded positively to phosphotungstic acid, which was used for detection of cell wall polysaccharides. The cell wall thickness within the bulging domain was significantly lower than in other parts of trichoblasts. We suggest that internalization of cell wall polysaccharides occurs within the bulging area, contributing to local thinning of the cell wall and providing a source of osmotically active compounds for maintaining turgor in the trichoblast. Thus, the internalization process might be necessary for root hair outgrowth.

NaRALF, a peptide signal essential for the regulation of root hair tip apoplastic pH in Nicotiana attenuata, is required for root hair development and plant growth in native soils

Rapid alkalinization factor (RALF) is a 49-amino-acid peptide that rapidly alkalinizes cultivated tobacco cell cultures. In the native tobacco Nicotiana attenuata, NaRALF occurs as a single-copy gene and is highly expressed in roots and petioles. Silencing the NaRALF transcript by transforming N. attenuata with an inverted-repeat construct generated plants (irRALF) with normal wild-type (WT) above-ground parts, but with roots that grew longer and produced trichoblasts that developed into abnormal root hairs. Most trichoblasts produced a localized 'bulge' without commencing root hair tip growth; fewer trichoblasts grew, but were only 10% as long as those of WT plants. The root hair phenotype was associated with slowed apoplastic pH oscillations, increased pH at the tips of trichoblasts and decreased accumulation of reactive oxygen species in the root hair initiation zone. The root hair growth phenotype was partially restored when irRALF lines were grown in a low-pH-buffered medium, and reproduced in WT plants grown in a high-pH-buffered medium. When irRALF plants were grown in pH 5.6, 6.7 and 8.1 soils together with WT plants in glasshouse experiments, they were out-competed by WT plants in basic, but not acidic, soils. When WT and irRALF lines were planted into the basic soils of the native habitat of N. attenuata in the Great Basin Desert, irRALF plants had smaller leaves, shorter stalks, and produced fewer flowers and seed capsules than did WT plants. We conclude that NaRALF is required for regulating root hair extracellular pH, the transition from root hair initiation to tip growth and plant growth in basic soils.

Rapid Kinetic Analysis of Ethylene Growth Responses in Seedlings: New Insights into Ethylene Signal Transduction

Ethylene is a phytohormone that influences diverse processes in plants. Ethylene causes various changes in etiolated seedlings that differ between species and include reduced growth of shoots and roots, increased diameter of shoots, agravitropic growth, initiation of root hairs, and increased curvature of the apical hook. The inhibition of growth in etiolated seedlings has become widely used to screen for and identify mutants. This approach has led to an increased understanding of ethylene signaling. Most studies use end-point analysis after several days of exposure to assess the effects of ethylene. Recently, the use of time-lapse imaging has re-emerged as an experimental method to study the rapid kinetics of ethylene responses. This review outlines the historical use of ethylene growth kinetic studies and summarizes the recent use of this approach coupled with molecular biology to provide new insights into ethylene signaling.

The role of auxin in root-hair initiation

The role of auxin in root-hair initiation

Temporal and positional relationships between Mn uptake and low-pH-induced root hair formation in Lactuca sativa cv. Grand Rapids seedlings.

We investigated whether low-pH-induced manganese (Mn) deficiency causes low-pH-induced root hair formation in lettuce seedlings. Both the number and length of root hairs increased in 0 microM Mn (Mn-free) at pH 6 and decreased in 3 mM Mn (excess Mn) at pH 4 compared with the values in 10 microM Mn (normal Mn). These results indicate an inhibitory effect of Mn on both root hair initiation and elongation. The time dependency of root hair induction caused by Mn deficiency corresponded to that caused by low pH. Within 1 h after the pH of the culture medium was reduced from pH 6 to pH 4, the Mn uptake by roots decreased to 43% of that at pH 6. These results suggest that low-pH-induced Mn deficiency promotes root hair formation. At low pH, the rate of Mn uptake was reduced in areas >2 mm from the root tip. Roots with low-pH-induced root hairs still showed low Mn uptake during 3 h of incubation at pH 6. Therefore, the additional root hairs induced by low pH did not compensate for the low-pH-induced decrease in Mn uptake.

Molecular Cloning and Characterization of β-Expansin Gene Related to Root Hair Formation in Barley

Root hairs are specialized epidermal cells that play a role in the uptake of water and nutrients from the rhizosphere and serve as a site of interaction with soil microorganisms. The process of root hair formation is well characterized in Arabidopsis (Arabidopsis thaliana); however, there is a very little information about the genetic and molecular basis of root hair development in monocots. Here, we report on isolation and cloning of the beta-expansin (EXPB) gene HvEXPB1, tightly related to root hair initiation in barley (Hordeum vulgare). Using root transcriptome differentiation in the wild-type/root-hairless mutant system, a cDNA fragment present in roots of wild-type plants only was identified. After cloning of full-length cDNA and genomic sequences flanking the identified fragment, the subsequent bioinformatics analyses revealed homology of the protein coded by the identified gene to the EXPB family. Reverse transcription-PCR showed that expression of HvEXPB1 cosegregated with the root hair phenotype in F2 progeny of the cross between the hairless mutant rhl1.a and the wild-type Karat parent variety. Expression of the HvEXPB1 gene was root specific; it was expressed in roots of wild-type forms, but not in coleoptiles, leaves, tillers, and spikes. The identified gene was active in roots of two other analyzed root hair mutants: rhp1.a developing root hair primordia only and rhs1.a with very short root hairs. Contrary to this, a complete lack of HvEXPB1 expression was observed in roots of the spontaneous root-hairless mutant bald root barley. All these observations suggest a role of the HvEXPB1 gene in the process of root hair formation in barley.

An Arabidopsis ACT2 dominant-negative mutation, which disturbs F-actin polymerization, reveals its distinctive function in root development.

Eight functional actin genes are present in Arabidopsis: The functional characterization of these genes in loss-of-function mutants is difficult, because highly conserved isovariants are generally expressed in the same tissue. We isolated a novel semi-dominant mutant allele (act2-2D) of an actin gene, ACT2, with a missense mutation which causes an amino acid substitution at the surface of the ACT2 protein. ACT2 promoter::ACT2-2D transgenic plants showed the same phenotype as act2-2D, indicating that act2-2D is a dominant-negative mutant. act2-2D exhibited defects in the initiation and elongation of root hairs, the elongation of root epidermal cells, and growth in aerial portions. Specifically, radial cell expansion was reduced and occasional cell death occurred in trichoblasts but not in atrichoblasts of the root epidermis. In contrast, cell division patterns in the root meristem were not affected. act2-3, a loss-of-function ACT2 mutant, did not develop most of these morphological abnormalities. Actin filament (F-actin) bundles in root epidermal cells of act2-2D were shorter than in the wild type and in the loss-of-function mutant. We conclude that defective F-actin polymerization caused the aberrant cell morphology in a dominant-negative manner, and that ACT2 functions in cell elongation and root hair formation.

AXR3 and SHY2 interact to regulate root hair development.

Signal transduction of the plant hormone auxin centres on the regulation of the abundance of members of the Aux/IAA family of transcriptional regulators, of which there are 29 in Arabidopsis. Auxin can influence Aux/IAA abundance by promoting the transcription of Aux/IAA genes and by reducing the half-life of Aux/IAA proteins. Stabilising mutations, which render Aux/IAA proteins resistant to auxin-mediated degradation, confer a wide range of phenotypes consistent with disruptions in auxin response. Interestingly, similar mutations in different family members can confer opposite phenotypic effects. To understand the molecular basis for this functional specificity in the Aux/IAA family, we have studied a pair of Aux/IAAs, which have contrasting roles in root hair development. We have found that stabilising mutations in AXR3/IAA17 blocks root hair initiation and elongation, whereas similar mutations in SHY2/IAA3 result in early initiation of root hair development and prolonged hair elongation, giving longer root hairs. The phenotypes resulting from double mutant combinations, the transient induction of expression of the proteins, and the pattern of transcription of the cognate genes suggest that root hair initiation is controlled by the relative abundance of SHY2 and AXR3 in a cell. These results suggest a general model for auxin signalling in which the modulation of the relative abundance of different Aux/IAA proteins can determine which down-stream responses are induced.

Ethylene promotes the induction by auxin of the cortical microtubule randomization required for low-pH-induced root hair initiation in lettuce (Lactuca sativa L.) seedlings.

Transverse cortical microtubule (CMT) arrays in lettuce root epidermal cells randomize soon after a shift from pH 6.0 to pH 4.0, and this randomization is essential for root hair initiation. We investigated the hormonal regulation of CMT randomization. At pH 4.0, 1 micro M of the auxin competitive inhibitor 2-(p-chlorophenoxy)-2-methylpropionic acid (PCIB), 0.1 micro M of the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) or 0.1 micro M of the ethylene action inhibitor Ag(+) suppressed CMT randomization and root hair initiation. At pH 6.0, addition of 0.1 micro M indole-3-acetic acid (IAA) or 1 micro M of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) induced CMT randomization and root hair initiation. Culturing with 0.1 micro M IAA plus 0.1 micro M AVG, or 1 micro M ACC plus 1 micro M PCIB also induced these phenomena. ACC (1 micro M) plus 100 micro M PCIB inhibited CMT randomization and root hair initiation, but 1 micro M AVG with 0.1 micro M Ag(+) and 0.1 micro M IAA induced them. These results suggest that auxin is essential for CMT randomization. As a higher concentration of PCIB was required to suppress CMT randomization when ACC was added, the greater amount of ethylene produced at pH 4.0 may promote the induction by auxin of CMT randomization in hair-forming cells.