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.     

Randomization of cortical microtubules in root epidermal cells induces root hair initiation in lettuce (Lactuca sativa L.) seedlings

Root hair formation is induced when lettuce seedlings are transferred from liquid medium at pH 6.0 to fresh medium at pH 4.0. If seedlings are transferred to pH 6.0, no root hairs are formed. We investigated the role of microtubules in this low pH-induced root hair initiation in lettuce. At the hair-forming zone in root epidermal cells, microtubules were perpendicular to the longitudinal axis of the cell just after pre-culture. This arrangement became disordered as early as 5 min after transfer to pH 4.0, and became random by 30 min later. At pH 4.0, the randomization extended to the entire hair-forming zone of seedlings; at pH 6.0, however, randomization did not occur and transverse microtubules were maintained. When seedlings at pH 6.0 were treated with microtubule-depolymerizing drugs, root hairs were formed. In contrast, when a microtubule-stabilizing drug, taxol, was added to the medium, no root hairs formed, even at pH 4.0. These results suggest that the transverse cortical microtubules inhibit root hair formation, and that their destruction is necessary for initiation. Furthermore, the microfilament-disrupting drugs cytochalasin B and latrunculin B inhibited root hair initiation, suggesting that actin filaments are necessary for root hair initiation.     

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.     

Ammonium-Stimulated Root Hair Branching is Enhanced by Methyl Jasmonate and Suppressed by Ethylene in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Root hair development is orchestrated by nutritional factors and plant hormones. We investigated the action of ammonium (NH₄⁺) and its interactions with methyl jasmonate (MeJA) and ethylene in Arabidopsis root hair growth. The formation of root hair branches was dramatically stimulated in media containing 1.25 to 20 mM NH₄⁺ at pH values of 4.0 to 6.5. The NH₄⁺-treated root hairs showed a very short tip growth stage and swells on the sides that indicated the emergence of branches. MeJA (0.08 to 10 μM) worked in synergism with NH₄⁺ to enhance hair branching. In contrast, ethylene had an antagonistic effect; the stimulation of hair branching by NH₄⁺ was suppressed by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and was diminished in ethylene-overproducing mutant eto1-1 seedlings. Moreover, the application of Ag⁺, an ethylene inhibitor, reduced the ACC-induced inhibition of NH₄⁺-stimulated hair branching and restored NH₄⁺-stimulated hair branching in eto1-1 seedlings. Thus, the actions of jasmonate and ethylene appear to be dependent on nutritional conditions such as available nitrogen.     

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.     

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.     

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.     

Interactions between jasmonates and ethylene in the regulation of root hair development in Arabidopsis

Root hair formation is an important model with which to study cell patterning and differentiation in higher plants. Ethylene and auxin are critical regulators of root hair development. The role of jasmonates (JAs) was examined in Arabidopsis root hair development as well as their interactions with ethylene in this process. The results have shown that both methyl jasmonate (MeJA) and jasmonic acid (JA) have a pronounced effect on promoting root hair formation. However, the effect of MeJA and JA on root hair formation was blocked by ethylene inhibitors Ag+ or aminoethoxyvinylglycine (AVG). The stimulatory effects of MeJA and JA were also diminished in ethylene-insensitive mutants etr1-1 and etr1-3. Furthermore, the JA biosynthesis inhibitors ibuprofen and salicylhydroxamic acid (SHAM) suppressed 1-aminocyclopropane-1-carboxylic acid (ACC)-induced root hair formation, and decreased the root hairs in seedlings of the ethylene over-producing mutant eto1-1. These results suggested that JAs promote root hair formation, through an interaction with ethylene.     

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.     

Ethylene is a positive regulator of root hair development in Arabidopsis thaliana

Evidence is provided that ethylene is a positive regulator of hair cell development in the root epidermis of Arabidopsis thaliana. Treatment of seedlings with increasing concentrations of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC) results in progressively more root hair cells developing in positions normally occupied by non-hair cells. Consistent with these findings are observations that treatments that block either ethylene synthesis or its perception reduce the number of root hairs. A model is proposed in which either ethylene or ACC is a signal involved in specifying the pattern of cell differentiation in the Arabidopsis root epidermis.     

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     

Ethylene Controls Adventitious Root Initiation Sites in <Emphasis Type="Italic">Arabidopsis</Emphasis> Hypocotyls Independently of Strigolactones

Adventitious root formation is essential for cutting propagation of diverse species; however, until recently little was known about its regulation. Strigolactones and ethylene have both been shown to inhibit adventitious roots and it has been suggested that ethylene interacts with strigolactones in root hair elongation. We have investigated the interaction between strigolactones and ethylene in regulating adventitious root formation in intact seedlings of Arabidopsis thaliana. We used strigolactone mutants together with 1-aminocyclopropane-1-carboxylic acid (ACC) (ethylene precursor) treatments and ethylene mutants together with GR24 (strigolactone agonist) treatments. Importantly, we conducted a detailed mapping of adventitious root initiation along the hypocotyl and measured ethylene production in strigolactone mutants. ACC treatments resulted in a slight increase in adventitious root formation at low doses and a decrease at higher doses, in both wild-type and strigolactone mutants. Furthermore, the distribution of adventitious roots dramatically changed to the top third of the hypocotyl in a dose-dependent manner with ACC treatments in both wild-type and strigolactone mutants. The ethylene mutants all responded to treatments with GR24. Wild type and max4 (strigolactone-deficient mutant) produced the same amount of ethylene, while emanation from max2 (strigolactone-insensitive mutant) was lower. We conclude that strigolactones and ethylene act largely independently in regulating adventitious root formation with ethylene controlling the distribution of root initiation sites. This role for ethylene may have implications for flood response because both ethylene and adventitious root development are crucial for flood tolerance.     

Response of spring rape (Brassica napus var. oleifera L.) to inoculation with plant growth promoting rhizobacteria containing 1-aminocyclopropane-1-carboxylate deaminase depends on nutrient status of the plant

Responses of rape (Brassica napus var. oleifera L.) to inoculation with plant growth promoting rhizobacteria, Pseudomonas putida Am2, Pseudomonas putida Bm3, Alcaligenes xylosoxidans Cm4, and Pseudomonas sp. Dp2, containing 1-aminocyclopropane-l-carboxylate (ACC) deaminase were studied using growth pouch and soil cultures. In growth pouch culture, the bacteria significantly increased root elongation of phosphorus-sufficient seedlings, whereas root elongation of phosphorus-deficient seedlings was not affected or was even inhibited by the bacteria. Bacterial stimulation of root elongation of phosphorus-sufficient seedlings was eliminated in the presence of a high ammonia concentration (1 mM) in the nutrient solution. Bacterial effects on root elongation of potassium-deficient and potassium-sufficient seedlings were similar. The bacteria also decreased inorganic phosphate content in shoots of potassium- and phosphorus-sufficient seedlings, reduced ethylene production by phosphorus-sufficient seedlings, and inhibited development of root hairs. The effects of treatment with Ag+, a chemical inhibitor of plant ethylene production, on root elongation, ethylene evolution, and root hair formation were similar to bacterial treatments. The number of bacteria on the roots of phosphorus-deficient seedlings was not limited by phosphorus deficiency. In pot experiments with soil culture, inoculation of seeds with bacteria and treatment with aminoethoxyvinylglycine, an inhibitor of ethylene biosynthesis in plants, increased root and (or) shoot biomass of rape plants. Stimulation of plant growth caused by the bacteria was often associated with a decrease in the content of nutrients, such as P, K, S, Mo, and Ba, in shoots, depending on the strain used. The results obtained show that the growth-promoting effects of ACC-utilizing rhizobacteria depend significantly on the nutrient status of the plant.     

Studies on the Physiology of Nodule Formation: IX. The Influence of Combined Nitrogen, Glucose, Light Intensity and Day Length on Root-hair Infection in Clover

Small amounts of nitrate or nitrite salts (10 µg N/plant)in the root medium of Trifolium glomeratum or T. repens delayednodulation, prolonged the initial rapid phase of root infectionand slightly stimulated lateral root formation, whereas equivalentquantities of ammonium sulphate or urea did not. Growth of rootsand root hairs was unaffected by any of these substances at10 µg N/plant. Altering the carbohydrate status of the clover seedlings byadding glucose to the root medium, or by changing day lengthor light intensity, influenced neither the stimulation of root-hairinfection nor the delay in nodulation induced by nitrate at10 fig N/plant, except that plants grown in total darkness hadfewer hairs infected when the root medium contained small amountsof nitrate. The nitrogenous compounds at 100 µg to 1,000 µg N/plant generally delayed and decreased nodulation,increased lateral root formation, slowed hair infection, andincreased root growth.     

Regulation of root development in <Emphasis Type="BoldItalic">Arabidopsis thaliana</Emphasis> by phytohormone-secreting epiphytic methylobacteria

In numerous experimental studies, seedlings of the model dicot Arabidopsis thaliana have been raised on sterile mineral salt agar. However, under natural conditions, no plant has ever grown in an environment without bacteria. Here, we document that germ-free (gnotobiotic) seedlings, raised on mineral salt agar without sucrose, develop very short root hairs. In the presence of a soil extract that contains naturally occurring microbes, root hair elongation is promoted; this effect can be mimicked by the addition of methylobacteria to germ-free seedlings. Using five different bacterial species (Methylobacterium mesophilicum, Methylobacterium extorquens, Methylobacterium oryzae, Methylobacterium podarium, and Methylobacterium radiotolerans), we show that, over 9 days of seedling development in a light-dark cycle, root development (hair elongation, length of the primary root, branching patterns) is regulated by these epiphytic microbes that occur in the rhizosphere of field-grown plants. In a sterile liquid culture test system, auxin (IAA) inhibited root growth with little effect on hair elongation and significantly stimulated hypocotyl enlargement. Cytokinins (trans-zeatin, kinetin) and ethylene (application of the precursor ACC) likewise exerted an inhibitory effect on root growth but, in contrast to IAA, drastically stimulated root hair elongation. Methylobacteria are phytosymbionts that produce/secrete cytokinins. We conclude that, under real-world conditions (soil), the provision of these phytohormones by methylobacteria (and other epiphytic microbes) regulates root development during seedling establishment.     

Ethylene-induced putrescine accumulation modulates K+ partitioning between roots and shoots in barley seedlings

We investigated the cause and effect relationships among ethylene, polyamines, and K⁺ in barley ( Hordeum vulgare L. cv. Amagi) seedlings. Application of 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene, to the growth medium caused a decrease in K⁺ concentration in roots and an increase in shoots. Addition of ACC induced putrescine accumulation in roots, while spermidine and spermine levels remained unchanged. Exogenous supply of putrescine led to putrescine accumulation and reduced K⁺ concentration. Application of Co²⁺, an inhibitor of ethylene biosynthesis, together with ACC, inhibited putrescine accumulation with a decrease in K⁺ concentration in roots. ACC-treated roots showed K⁺ uptake capacity equivalent to that of control roots, implying that the majority of K⁺ is translocated to shoots. These results suggest that ethylene regulates K⁺ partitioning between roots and shoots through the level of accumulation of putrescine in barley seedlings.