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.     

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.     

Effects of glucose and ethylene on root hair initiation and elongation in lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L.) seedlings

Root hair formation occurs in lettuce seedlings after transfer to an acidic medium (pH 4.0). This process requires cortical microtubule (CMT) randomization in root epidermal cells and the plant hormone ethylene. We investigated the interaction between ethylene and glucose, a new signaling molecule in plants, in lettuce root development, with an emphasis on root hair formation. Dark-grown seedlings were used to exclude the effect of photosynthetically produced glucose. In the dark, neither root hair formation nor the CMT randomization preceding it occurred, even after transfer to the acidic medium (pH 4.0). Adding 1-aminocyclopropane-1-carboxylic-acid (ACC) to the medium rescued the induction, while adding glucose did not. Although CMT randomization occurred when glucose was applied together with ACC, it was somewhat suppressed compared to that in ACC-treated seedlings. This was not due to a decrease in the speed of randomization, but due to lowering of the maximum degree of randomization. Despite the negative effect of glucose on ACC-induced CMT randomization, the density and length of ACC-induced root hairs increased when glucose was also added. The hair-cell length of the ACC-treated seedlings was comparable to that in the combined-treatment seedlings, indicating that the increase in hair density caused by glucose results from an increase in the root hair number. Furthermore, quantitative RT-PCR revealed that glucose suppressed ethylene signaling. These results suggest that glucose has a negative and positive effect on the earlier and later stages of root hair formation, respectively, and that the promotion of the initiation and elongation of root hairs by glucose may be mediated in an ethylene-independent manner.     

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 μM Mn (Mn-free) at pH 6 and decreased in 3 mM Mn (excess Mn) at pH 4 compared with the values in 10 μM 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     

Differential expression of ACC oxidase genes during low-pH-induced root hair formation in lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L.) seedlings

Root hair formation is induced in lettuce seedlings when the seedlings are transferred from a liquid medium at pH 6.0 to one at pH 4.0. Auxin, ethylene, and light are also required for the induction of root hair formation. To investigate the mechanism by which ethylene production is regulated during root hair formation, we isolated three 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase genes (Ls-ACO1, 2, and 3) from lettuce, each of which exists as a single copy in the genome. Analysis of the deduced amino acid sequences of the three ACO proteins as well as a phylogenetic analysis revealed that Ls-ACO3 was the most divergent among the ACO family. Northern hybridization analyses revealed that the mRNA levels of Ls-ACO2, but not Ls-ACO1 and Ls-ACO3, increased in the primary root after the transfer to a pH 4.0 medium. Addition of ACC or indole-3-acetic acid (IAA) to the pH 6.0 medium induced root hair formation, and a concomitant accumulation of Ls-ACO2 mRNA was observed. In contrast, the mRNA levels of Ls-ACO1 and Ls-ACO3 were unaffected by either ACC or IAA treatment. Furthermore, white light irradiation of dark-grown seedlings following the transfer to pH 4.0 medium induced the accumulation of all three ACO mRNAs. However, accumulation of Ls-ACO2 mRNA was also observed in non-irradiated seedlings, suggesting that the expression of Ls-ACO2 was induced not by light but by low pH. These results suggest that among the differentially regulated ACO genes, Ls-ACO2 plays a key role in ethylene production during low-pH-induced root hair formation in lettuce.     

Light-induced root hair formation in lettuce (Lactuca sativa L. cv. Grand Rapids) roots at low pH is brought by chlorogenic acid synthesis and sugar

Previously, we reported that chlorogenic acid (CGA) facilitated root hair formation at pH 4.0 in lettuce (Lactuca sativa L. cv. Grand Rapids). Light was essential for this process. In the present study, we determined relationships between CGA, light, and sugar during root hair formation in lettuce seedlings. The amount of CGA increased with white light in intact seedlings. Exogenously applied CGA restored root hair formation in dark-grown intact seedlings at pH 4.0. However, no root hair formation was induced in decapitated seedlings regardless of light exposure and CGA application. Application of sucrose or glucose induced both root hair formation and CGA synthesis in light-grown decapitated seedlings at pH 4.0. Blue light was the most effective for both root hair formation and CGA synthesis when supplied with sucrose to decapitated seedlings. Addition of sucrose and CGA together induced root hair formation at pH 4.0 in dark-grown decapitated seedlings. Results suggest that light induced CGA synthesis from sugar in the roots. Sugar was also required for root hair formation other than starting material of CGA synthesis. In addition, an unknown low pH-induced factor was essential for lettuce root hair formation.     

Role of manganese in low-pH-induced root hair formation in<Emphasis Type="Italic"> Lactuca sativa</Emphasis> cv. Grand Rapids seedlings

Root hair formation is induced by low pH in lettuce (Lactuca sativa L. cv. Grand Rapids) seedlings cultured in mineral medium. The role of mineral concentrations in this phenomenon was investigated, especially for manganese. When lettuce seedlings were cultured in media that were deficient in calcium (Ca), manganese (Mn), boron (B) or molybdenum (Mo), morphological changes were induced in roots. Deficiency of other nutrients had little effect on root hair formation. Ca or B deficiency inhibited the growth of the main root and the formation of root hairs, regardless of pH. Mn or Mo deficiency increased root hair formation at pH 6 and suppressed main root growth slightly. In contrast, increasing the Mn concentration suppressed low-pH-induced root hair formation. The Mn content of roots grown at pH 4 was only about 15% of that at pH 6. In contrast, the Mo content of roots grown at low pH was about six times that of roots grown at neutral pH. These results suggest that root hair formation induced by low pH is at least partly mediated by decreased Mn uptake in root cells.     

The COW1 locus of arabidopsis acts after RHD2, and in parallel with RHD3 and TIP1, to determine the shape, rate of elongation, and number of root hairs produced from each site of hair formation.

Two recessive mutant alleles at CAN OF WORMS1 (COW1), a new locus involved in root hair morphogenesis, have been identified in Arabidopsis thaliana L. Heynh. Root hairs on Cow1- mutants are short and wide and occasionally formed as pairs at a single site of hair formation. The COW1 locus maps to chromosome 4. Root hairs on Cow1- plants form in the usual positions, suggesting that the phenotype is not the result of abnormal positional signals. Root hairs on Cow1- roots begin hair formation normally, forming a small bulge, or root hair initiation site, of normal size and shape and in the usual position on the hair-forming cell. However, when Cow1- root hairs start to elongate by tip growth, abnormalities in the shape and elongation rate of the hairs become apparent. Genetic evidence from double-mutant analysis of cow1-1 and other loci involved in root hair development supports our conclusion that COW1 is required during root hair elongation.     

Auxin and ethylene promote root hair elongation in Arabidopsis

Genetic and physiological studies implicate the phytohormones auxin and ethylene in root hair development. To learn more about the role of these compounds, we have examined the root hair phenotype of a number of auxin- and ethylene-related mutants. In a previous study, Masucci and Schiefelbein (1996) showed that neither the auxin response mutations aux1 and axr1 nor the ethylene response mutations etr1 and ein2 have a significant effect on root hair initiation. In this study, we found that mutants deficient in either auxin or ethylene response have a pronounced effect on root hair length. Treatment of wild-type, axr1 and etr1 seedlings with the synthetic auxin, 2,4-D, or the ethylene precursor ACC, led to the development of longer root hairs than untreated seedlings. Furthermore, axr1 seedlings grown in the presence of ACC produce ectopic root hairs and an unusual pattern of long root hairs followed by regions that completely lack root hairs. These studies indicate that both auxin and ethylene are required for normal root hair elongation.     

Position and cell type-dependent microtubule reorientation characterizes the early response of the Arabidopsis root epidermis to ethylene

The involvement of cortical microtubules in the control of plant cell expansion was studied in the Arabidopsis root epidermis. In the zone of fast elongation microtubules were transverse to the root axis in all epidermal cells. However when cells entered the differentiation zone cell type-specific microtubule reorientation took place. In the trichoblasts that were then approximately 130 µm long and formed the root hair bulge, the microtubules switched to a random distribution. In the adjoining atrichoblasts microtubules adopted a slightly oblique orientation. In more proximal parts of the differentiation zone atrichoblast microtubules were found in a more oblique and finally in a longitudinal orientation. Upon exposure to ethylene or 1-aminocyclopropane-1-carboxylic acid (ACC – the precursor of ethylene) at a saturating dose, cell elongation abruptly stopped. From then on trichoblast cells reached only a length of about 35 µm, and developed root hairs. Cortical microtubules changed orientation within 10 min. In trichoblasts they adopted the typical random orientation, in atrichoblasts however, they took up a longitudinal orientation. Microtubule reorientation was complete within 60 min. The possible role of microtubules in the control of cell elongation is discussed.     

Ultrastructure,differentiation and cell wall texture of trichoblasts and root hairs of ceratopteris thalictroides (L.) brongn. (Parkeriaceae)

Trichoblasts and root hairs of Ceratopteris thalictroides (L.) Brongn. were studied by different techniques to survey their morphological features. Trichoblasts could be identified at an early stage by an intra-vacuolar precipitate appearing during fixation. Special attention was paid to root-hair initiation. No structures or changes were observed that play a role in the initiation of papilla formation. When the papilla is formed, vesicles and periplasmic membranes can be observed which may play a role in the weakening of the cell wall during the papilla outgrowth.During root-hair growth, the nucleus of the trichoblast moves from the trichoblast to a subapical position in the root hair. The nuclei of all root cells contain 2 types of nuclear inclusions, one of which is proteinaceous.The cell wall of the Ceratopteris root hair has a helicoidal texture and because the cortical microtubules run longitudinally in the root hair, no correlation can be made between the directions of microtubules and microfibrils in these root hairs.     

Role of a positive regulator of root hair development,CAPRICE, in Arabidopsis root epidermal cell differentiation

In Arabidopsis, root hairs are formed only from a set of epidermal cells named trichoblasts or hair-forming cells. Previous studies showed CAPRICE (CPC) promotes differentiation of hair-forming cells by controlling a negative regulator, GLABRA2 (GL2), which is preferentially expressed in hairless cells. Here, we show that CPC is also predominantly expressed in the hairless cells, but not in the neighboring hair-forming cells, and that CPC protein moves to the hair-forming cells and represses the GL2 expression. We also show that the N terminus of bHLH protein interacts with CPC and is responsible for the GL2 expression. We propose a model in which CPC plays a key role in the fate-determination of hair-forming cells.     

Cellular analysis of UV-B-induced barley root subapical swelling

UV-B irradiation of barley (Hordeum vulgare L.) roots (1 W/m², 15 min) or leaves (3 W/m², 3.3 h) and also one-day-long root incubation in the Knop solution supplemented with 1–4 μM ABA, 1 mM salicylic acid, 16 μM ionomycin, or 0.1 mM colchicine induced growth retardation and subapical root swelling. All factors, except for colchicine, initiated growth of root hairs on the surface of swellings and suppressed their initiation and growth in more basal root region. During the first hour after unilateral root UV-B irradiation, their growth sharply retarded and hydraulic conductivity of membranes in the rhizodermis of growth zone rose 1.5-fold. In 2.5 h, root tips bent toward the source of irradiation. In 4.5 h, the ratio of longitudinal to transverse root extensibility in the root growth zone reduced twofold. In 8 h, root diameter in the subapical zone increased and root hairs appeared in this zone and attained 300 μm in length. In a day after irradiation, on unirradiated root side, meristematic cells continued to divide and grow, although at a much lower rate. On the irradiated root side, the cells of the rhizodermis and outer cortex ceased to divide and produced vacuoles. Vacuolation did not occur in the cells of the quiescent center and a distal part of the meristem. The lower part of the elongation zone swelled due to cortical cell expansion (except for the endodermis) in both irradiated and unirradiated root sides. It is supposed that cortical microtubule randomization plays an important role in the changed anisotropy of cell wall extensibility and cytosolic calcium is involved in this process. The role of oxidative stress and hormonal shifts in the development of subapical root swelling and root hair formation caused by UV-B radiation is discussed.     

The Arabidopsis Rop2 GTPase is a positive regulator of both root hair initiation and tip growth

Root hairs provide a model system for the study of cell polarity. We examined the possibility that one or more members of the distinct plant subfamily of RHO monomeric GTPases, termed Rop, may function as molecular switches regulating root hair growth. Specific Rops are known to control polar growth in pollen tubes. Overexpressing Rop2 (Rop2 OX) resulted in a strong root hair phenotype, whereas overexpressing Rop7 appeared to inhibit root hair tip growth. Overexpressing Rops from other phylogenetic subgroups of Rop did not give a root hair phenotype. We confirmed that Rop2 was expressed throughout hair development. Rop2 OX and constitutively active GTP-bound rop2 (CA-rop2) led to additional and misplaced hairs on the cell surface as well as longer hairs. Furthermore, CA-rop2 depolarized root hair tip growth, whereas Rop2 OX resulted in hairs with multiple tips. Dominant negative GDP-bound Rop2 reduced the number of hair-forming sites and led to shorter and wavy hairs. Green fluorescent protein-Rop2 localized to the future site of hair formation well before swelling formation and to the tip throughout hair development. We conclude that the Arabidopsis Rop2 GTPase acts as a positive regulatory switch in the earliest visible stage in hair development, swelling formation, and in tip growth.     

Expression of AtPRP3, a proline-rich structural cell wall protein from Arabidopsis, is regulated by cell-type-specific developmental pathways involved in root hair formation

The tightly regulated expression patterns of structural cell wall proteins in several plant species indicate that they play a crucial role in determining the extracellular matrix structure for specific cell types. We demonstrate that AtPRP3, a proline-rich cell wall protein in Arabidopsis, is expressed in root-hair-bearing epidermal cells at the root/shoot junction and within the root differentiation zone of light-grown seedlings. Several lines of evidence support a direct relationship between AtPRP3 expression and root hair development. AtPRP3/beta-glucuronidase (GUS) expression increased in roots of transgenic seedlings treated with either 1-aminocyclopropane-1-carboxylic acid (ACC) or alpha-naphthaleneacetic acid (alpha-NAA), compounds known to promote root hair formation. In the presence of 1-alpha-(2-aminoethoxyvinyl)glycine (AVG), an inhibitor of ethylene biosynthesis, AtPRP3/GUS expression was strongly reduced, but could be rescued by co-addition of ACC or alpha-NAA to the growth medium. In addition, AtPRP3/GUS activity was enhanced in ttg and gl2 mutant backgrounds that exhibit ectopic root hairs, but was reduced in rhd6 and 35S-R root-hair-less mutant seedlings. These results indicate that AtPRP3 is regulated by developmental pathways involved in root hair formation, and are consistent with AtPRP3's contributing to cell wall structure in Arabidopsis root hairs.     

Microtubule array formation during root hair infection thread initiation and elongation in the Mesorhizobium-Lotus symbiosis

Nuclear migration during infection thread (IT) development in root hairs is essential for legume-Rhizobium symbiosis. However, little is known about the relationships between IT formation, nuclear migration, and microtubule dynamics. To this aim, we used transgenic Lotus japonicus expressing a fusion of the green fluorescent protein and tubulin-α6 from Arabidopsis thaliana to visualize in vivo dynamics of cortical microtubules (CMT) and endoplasmic microtubules (EMTs) in root hairs in the presence or absence of Mesorhizobium loti inoculation. We also examined the effect of microtubule-depolymerizing herbicide, cremart, on IT initiation and growth, since cremart is known to inhibit nuclear migration. In live imaging studies of M. loti-treated L. japonicus root hairs, EMTs were found in deformed, curled, and infected root hairs. The continuous reorganization of the EMT array linked to the nucleus appeared to be essential for the reorientation, curling, and IT initiation and the growth of zone II root hairs which are susceptible to rhizobial infection. During IT initiation, the EMTs appeared to be linked to the root hair surface surrounding the M. loti microcolonies. During IT growth, EMTs dissociated from the curled root hair tip, remained linked to the nucleus, and appeared to surround the IT tip. Lack or disorganized EMT arrays that were no longer linked to the nucleus were observed only in infection-aborted root hairs. Cremart affected IT formation and nodulation in a concentration-dependent manner, suggesting that the microtubule (MT) organization and successive nuclear migration are essential for successful nodulation in L. japonicus by M. loti.     

Early cytological events in the infection of the root hairs ofTrifolium repens byRhizobium leguminosarum biovartrifolii observed by glass slide culture in living seedlings

This report describes the early cytological events in the infection byRhizobium leguminosarum biovartrifolii of the root hairs ofTrifolium repens seedlings kept alive on agar medium in glass slide culture experiment. The infection threads bearing rhizobia were formed as soon as the epidermal cells began to emerge as root hairs. On the top of some of these infected emerging root hairs, there were smoky, cell-debris-like bodies, which appeared to be derived from the cell wall dug by rhizobia. Similar bodies were also observed in longer root hairs. None of the root hair cells along the length of the roots which contained infection threads were curled or distorted. A substantial number of pink-colored nodules were later formed on the roots with non-curled infected root hairs.     

Root Hair Initiation Is Coupled to a Highly Localized Increase of Xyloglucan Endotransglycosylase Action in Arabidopsis Roots

Root hairs are formed by two separate processes: initiation and subsequent tip growth. Root hair initiation is always accompanied by a highly localized increase in xyloglucan endotransglycosylase (XET) action at the site of future bulge formation, where the trichoblast locally loosens its cell wall. This suggests an important role of XET in the first stages of root hair initiation. The tip of growing root hairs is not marked by localized high XET action. Experiments in which root hair initiation was modulated and observations on root hair mutants support this view. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid shifts both root hair initiation and the local increase in XET action toward the root tip. On the other hand, roots treated with the ethylene inhibitor aminoethoxyvinyl-glycine, as well as roots of mutants affected in root hair initiation (rhl1, rhd6-1, and axr2-1) revealed no localized increases of XET action at all and consequently did not initiate root hairs. Disruption of actin and microtubules did not prevent the localized increase in XET action. Also, the temporal and spatial pattern of action as the specific pH dependence suggest that different isoforms of XET act in different processes of root development.