TRH1 encodes a potassium transporter required for tip growth in Arabidopsis root hairs

Root hair initiation involves the formation of a bulge at the basal end of the trichoblast by localized diffuse growth. Tip growth occurs subsequently at this initiation site and is accompanied by the establishment of a polarized cytoplasmic organization. Arabidopsis plants homozygous for a complete loss-of-function tiny root hair 1 (trh1) mutation were generated by means of the T-DNA-tagging method. Trichoblasts of trh1 plants form initiation sites but fail to undergo tip growth. A predicted primary structure of TRH1 indicates that it belongs to the AtKT/AtKUP/HAK K(+) transporter family. The proposed function of TRH1 as a K(+) transporter was confirmed in (86)Rb uptake experiments, which demonstrated that trh1 plants are partially impaired in K(+) transport. In line with these results, TRH1 was able to complement the trk1 potassium transporter mutant of Saccharomyces, which is defective in high-affinity K(+) uptake. Surprisingly, the trh1 phenotype was not restored when mutant seedlings were grown at high external potassium concentrations. These data demonstrate that TRH1 mediates K(+) transport in Arabidopsis roots and is responsible for specific K(+) translocation, which is essential for root hair elongation.     

Potassium carrier TRH1 is required for auxin transport in Arabidopsis roots

Disruption of the TRH1 potassium transporter impairs root hair development in Arabidopsis, and also affects root gravitropic behaviour. Rescue of these morphological defects by exogenous auxin indicates a link between TRH1 activity and auxin transport. In agreement with this hypothesis, the rate of auxin translocation from shoots to roots and efflux of [3H]IAA in isolated root segments were reduced in the trh1 mutant, but efflux of radiolabelled auxin was accelerated in yeast cells transformed with the TRH1 gene. In roots, Pro(TRH1):GUS expression was localized to the root cap cells which are known to be the sites of gravity perception and are central for the redistribution of auxin fluxes. Consistent with these findings, auxin-dependent DR5:GUS promoter-reporter construct was misexpressed in the trh1 mutant indicating that partial block of auxin transport through the root cap is associated with upstream accumulation of the phytohormone in protoxylem cells. When [K+] in the medium was reduced from 20 to 0.1 mm, wild type roots showed mild agravitropic phenotype and DR5:GUS misexpression in stelar cells. This pattern of response to low external [K+] was also affected by trh1 mutation. We conclude that the TRH1 carrier is an important part of auxin transport system in Arabidopsis roots.     

Interactions between root hair length and arbuscular mycorrhizal colonisation in phosphorus deficient barley (Hordeum vulgare)

Aims

Phosphorus (P) limits crop yield and P-fertilisers are frequently applied to agricultural soils. However, supplies of quality rock phosphate are diminishing. Plants have evolved mechanisms to improve P-acquisition and understanding these could improve the long-term sustainability of agriculture. Here we examined interactions between root hairs and arbuscular mycorrhizal (AM) colonisation in barley (Hordeum vulgare L.).

Methods

Barley mutants exhibiting different root hair phenotypes, wild type barley and narrowleaf plantain (Plantago lanceolata L.) were grown in the glasshouse in P-sufficient and P-deficient treatments and allowed to develop AM colonization from the natural soil community. Plants were harvested after 6 weeks growth and root hair length, AM-fungal colonisation, shoot biomass and P-accumulation measured.

Results

Under P-deficient conditions, root hair length and AM colonisation were negatively related suggesting that resources are allocated to root hairs rather than to AM fungi in response to P-deficiency. There was evidence that barley and narrowleaf plantain employed different strategies to increase P-acquisition under identical conditions, but root hairs were more effective.

Conclusions

This research suggests future barley breeding programmes should focus on maintaining or improving root hair phenotypes and that pursuing enhancements to AM associations under the prevalent agricultural conditions tested here would be ineffectual.     

Sodium chloride reduces growth and cytosolic calcium,but does not affect cytosolic pH,in root hairs of Arabidopsis thaliana L

The effects of salinity (NaCl) stress on growth, cytosolic Ca(2+) gradients and cytosolic pH homeostasis of root hairs of Arabidopsis thaliana are assessed here. Neither cytosolic Ca(2+) nor pH at the hair apex were significantly affected by 20 min exposure of up to 90 mM NaCl or of up to 5 mM extracellular Ca(2+). Exposure to increasing NaCl concentrations, up to 90 mM, for 2 d or 6 d reduced hair extension, and this inhibition was relieved by supplemental extracellular Ca(2+). Such extended salinity stress reduced the magnitude of the Ca(2+) gradient in the apical 12 microm of hairs at all NaCl concentrations tested (up to 90 mM), including NaCl concentrations that did not reduce hair extension. The magnitude of the tip-focused gradient was also reduced in root hairs of plants grown with low (0.5 mM) extracellular Ca(2+) when compared to those in 5 mM extracellular Ca(2+), regardless of the presence of NaCl. Up to 90 mM NaCl did not affect cytosolic pH of root hairs in any of the treatments. It is concluded that NaCl inhibition of root hair extension in the long term may operate via alterations in the tip-focused Ca(2+) gradient that regulates root hair growth. However, NaCl-induced alterations in this gradient do not always lead to detectably altered growth kinetics. Short-term signalling events in response to NaCl may operate by a means other than altering Ca(2+) at the root hair apex. Salinity stress in root hairs does not appear to be mediated by effects on cytosolic pH.     

Nitric oxide is essential for vesicle formation and trafficking in Arabidopsis root hair growth

The functions of nitric oxide (NO) in processes associated with root hair growth in Arabidopsis were analysed. NO is located at high concentrations in the root hair cell files at any stage of development. NO is detected inside of the vacuole in immature actively growing root hairs and, later, NO is localized in the cytoplasm when they become mature. Experiments performed by depleting NO in Arabidopsis root hairs indicate that NO is required for endocytosis, vesicle formation, and trafficking and it is not involved in nucleus migration, vacuolar development, and transvacuolar strands. The Arabidopsis G'4,3 mutant (double mutant nia1/nia2) is severely impaired in NO production and generates smaller root hairs than the wild type (WT). Root hairs from the Arabidopsis G'4,3 mutant show altered vesicular trafficking and are reminiscent of NO-depleted root hairs from the Arabidopsis WT. Interestingly, normal vesicle formation and trafficking as well as root hair growth is restored by exogenous NO application in the Arabidopsis G'4,3 mutant. All together, these results firmly support the essential role played by NO in the Arabidopsis root-hair-growing process.     

Both vegetative and reproductive actin isovariants complement the stunted root hair phenotype of the Arabidopsis act2-1 mutation

The ACT2 gene, encoding one of eight actin isovariants in Arabidopsis, is the most strongly expressed actin gene in vegetative tissues. A search was conducted for physical defects in act2-1 mutant plants to account for their reduced fitness compared with wild type in population studies. The act2-1 insertion fully disrupted expression of ACT2 RNA and significantly lowered the level of total actin protein in vegetative organs. The root hairs of the act2-1 mutants were 10% to 70% the length of wild-type root hairs, and they bulged severely at the base. The length of the mutant root hairs and degree of bulging at the base were affected by adjusting the osmolarity and gelling agent of the growth medium. The act2-1 mutant phenotypes were fully rescued by an ACT2 genomic transgene. When the act2-1 mutation was combined with another vegetative actin mutation, act7-1, the resulting double mutant exhibited extensive synergistic phenotypes ranging from developmental lethality to severe dwarfism. Transgenic overexpression of the ACT7 vegetative isovariant and ectopic expression of the ACT1 reproductive actin isovariant also rescued the root hair elongation defects of the act2-1 mutant. These results suggest normal ACT2 gene regulation is essential to proper root hair elongation and that even minor differences may cause root defects. However, differences in the actin protein isovariant are not significant to root hair elongation, in sharp contrast to recent reports on the functional nonequivalency of plant actin isovariants. Impairment of root hair functions such as nutrient mining, water uptake, and physical anchoring are the likely cause of the reduced fitness seen for act2-1 mutants in multigenerational studies.     

Ammonium-induced loss of root gravitropism is related to auxin distribution and TRH1 function, and is uncoupled from the inhibition of root elongation in Arabidopsis

Root gravitropism is affected by many environmental stresses, including salinity, drought, and nutrient deficiency. One significant environmental stress, excess ammonium (NH(4)(+)), is well documented to inhibit root elongation and lateral root formation, yet little is known about its effects on the direction of root growth. We show here that inhibition of root elongation upon elevation of external NH(4)(+) is accompanied by a loss in root gravitropism (agravitropism) in Arabidopsis. Addition of potassium (K(+)) to the treatment medium partially rescued the inhibition of root elongation by high NH(4)(+) but did not improve gravitropic root curvature. Expression analysis of the auxin-responsive reporter gene DR5::GUS revealed that NH(4)(+) treatment delayed the development of gravity-induced auxin gradients across the root cap but extended their duration once initiated. Moreover, the β-glucuronidase (GUS) signal intensity in root tip cells was significantly reduced under high NH(4)(+) treatment over time. The potassium carrier mutant trh1 displayed different patterns of root gravitropism and DR5::GUS signal intensity in root apex cells compared with the wild type in response to NH(4)(+). Together, the results demonstrate that the effects of NH(4)(+) on root gravitropism are related to delayed lateral auxin redistribution and the TRH1 pathway, and are largely independent of inhibitory effects on root elongation.     

Nitrogen source interacts with ROP signalling in root hair tip‐growth

Root hairs elongate in a highly polarized manner known as tip growth. Overexpression of constitutively active Rho of Plant (ROP)/RAC GTPases mutants induces swelling of root hairs. Here, we demonstrate that Atrop11^CA‐induced swelling of root hairs depends on the composition of the growth medium. Depletion of ammonium allowed normal root hair elongation in Atrop11^CA plants, induced the development of longer root hairs in wild‐type plants and suppressed the effect of Atrop11^CA expression on actin organization and reactive oxygen species distribution, whereas membrane localization of the protein was not affected. Ammonium at concentrations higher than 1 mM and the presence of nitrate were required for induction of swelling. Oscillations in wall and cytoplasmic pH are known to accompany tip growth in root hairs, and buffering of the growth medium decreased Atrop11^CA‐induced swelling. Fluorescence ratio imaging experiments revealed that in wild‐type root hairs, the addition of NH₄NO₃ to the growth medium induced an increase in the amplitude of extracellular and intracellular pH oscillations and an overall decrease in cytoplasmic pH at the cell apex. Based on these results, we suggest a model in which ROP GTPases and nitrogen‐dependent pH oscillations function in parallel pathways, creating a positive feedback loop during root hair growth.     

OsSNDP1, a Sec14-nodulin domain-containing protein, plays a critical role in root hair elongation in rice

Rice is cultivated in water-logged paddy lands. Thus, rice root hairs on the epidermal layers are exposed to a different redox status of nitrogen species, organic acids, and metal ions than root hairs growing in drained soil. To identify genes that play an important role in root hair growth, a forward genetics approach was used to screen for short-root-hair mutants. A short-root-hair mutant was identified and isolated by using map-based cloning and sequencing. The mutation arose from a single amino acid substitution of OsSNDP1 (Oryza sativa Sec14-nodulin domain protein), which shows high sequence homology with Arabidopsis COW1/AtSFH1 and encodes a phosphatidylinositol transfer protein (PITP). By performing complementation assays with Atsfh1 mutants, we demonstrated that OsSNDP1 is involved in growth of root hairs. Cryo-scanning electron microscopy was utilized to further characterize the effect of the Ossndp1 mutation on root hair morphology. Aberrant morphogenesis was detected in root hair elongation and maturation zones. Many root hairs were branched and showed irregular shapes due to bulged nodes. Many epidermal cells also produced dome-shaped root hairs, which indicated that root hair elongation ceased at an early stage. These studies showed that PITP-mediated phospholipid signaling and metabolism is critical for root hair elongation in rice.     

The supply of nutrient ions by diffusion to plant roots in soil

Summary Observation of soil grown roots of rye-grass shows that an approximately cylindrical volume of soil, the root hair cylinder, is densely occupied by root hairs. Estimates are given of the concentration of labile and solution potassium within the root hair cylinder during experiments measuring potassium uptake from two soils by single roots. Calculations, using a diffusion model, suggest that labile potassium concentrations may be reduced to between 99.3 and 53 per cent of the initial, depending on the diffusion characteristics of the soil and nutrient demand by the root. Of the total potassium absorbed by a root in 4 days, the proportion which is supplied from within the root hair cylinder is small (0.8 to 6.3 per cent) indicating that diffusion to the root from the soil outside the root hair cylinder is of paramount importance. When root demand is high, diffusion appears to limit uptake to between 71 and 59 per cent of that which roots of comparable physiology would be expected to absorb from stirred solution of the same concentration. Nevertheless, the presence of root hairs is calculated to have enhanced uptake by up to 77 per cent compared with roots without hairs because they virtually increase the root diameter. Diffusion does not appear to be a limiting factor when root demand is low and hairs can then add little to the efficiency of the root system in potassium absorption.     

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.     

The Arabidopsis Phosphatidylinositol Phosphate 5-Kinase PIP5K3 is a key regulator of root hair tip growth

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] functions as a site-specific signal on membranes to promote cytoskeletal reorganization and membrane trafficking. Localization of PtdIns(4,5)P2 to apices of growing root hairs and pollen tubes suggests that it plays an important role in tip growth. However, its regulation and mode of action remain unclear. We found that Arabidopsis thaliana PIP5K3 (for Phosphatidylinositol Phosphate 5-Kinase 3) encodes a phosphatidylinositol 4-phosphate 5-kinase, a key enzyme producing PtdIns(4,5)P2, that is preferentially expressed in growing root hairs. T-DNA insertion mutations that substantially reduced the expression of PIP5K3 caused significantly shorter root hairs than in the wild type. By contrast, overexpression caused longer root hairs and multiple protruding sites on a single trichoblast. A yellow fluorescent protein (YFP) fusion of PIP5K3, driven by the PIP5K3 promoter, complemented the short-root-hair phenotype. PIP5K3-YFP localized to the plasma membrane and cytoplasmic space of elongating root hair apices, to growing root hair bulges, and, notably, to sites about to form root hair bulges. The signal was greatest in rapidly growing root hairs and quickly disappeared when elongation ceased. These results provide evidence that PIP5K3 is involved in localizing PtdIns(4,5)P2 to the elongating root hair apex and is a key regulator of the machinery that initiates and promotes root hair tip growth.     

OsCSLD1, a cellulose synthase-like D1 gene, is required for root hair morphogenesis in rice

Root hairs are long tubular outgrowths that form on the surface of specialized epidermal cells. They are required for nutrient and water uptake and interact with the soil microflora. Here we show that the Oryza sativa cellulose synthase-like D1 (OsCSLD1) gene is required for root hair development, as rice (Oryza sativa) mutants that lack OsCSLD1 function develop abnormal root hairs. In these mutants, while hair development is initiated normally, the hairs elongate less than the wild-type hairs and they have kinks and swellings along their length. Because the csld1 mutants develop the same density and number of root hairs along their seminal root as the wild-type plants, we propose that OsCSLD1 function is required for hair elongation but not initiation. Both gene trap expression pattern and in situ hybridization analyses indicate that OsCSLD1 is expressed in only root hair cells. Furthermore, OsCSLD1 is the only member of the four rice CSLD genes that shows root-specific expression. Given that the Arabidopsis (Arabidopsis thaliana) gene KOJAK/AtCSLD3 is required for root hair elongation and is expressed in the root hair, it appears that OsCSLD1 may be the functional ortholog of KOJAK/AtCSLD3 and that these two genes represent the root hair-specific members of this family of proteins. Thus, at least part of the mechanism of root hair morphogenesis in Arabidopsis is conserved in rice.     

The Microtubule Plus-End Tracking Protein ARMADILLO-REPEAT KINESIN1 Promotes Microtubule Catastrophe in Arabidopsis

Microtubule dynamics are critically important for plant cell development. Here, we show that Arabidopsis thaliana ARMADILLO-REPEAT KINESIN1 (ARK1) plays a key role in root hair tip growth by promoting microtubule catastrophe events. This destabilizing activity appears to maintain adequate free tubulin concentrations in order to permit rapid microtubule growth, which in turn is correlated with uniform tip growth. Microtubules in ark1-1 root hairs exhibited reduced catastrophe frequency and slower growth velocities, both of which were restored by low concentrations of the microtubule-destabilizing drug oryzalin. An ARK1-GFP (green fluorescent protein) fusion protein expressed under its endogenous promoter localized to growing microtubule plus ends and rescued the ark1-1 root hair phenotype. Transient overexpression of ARK1-RFP (red fluorescent protein) increased microtubule catastrophe frequency. ARK1-fusion protein constructs lacking the N-terminal motor domain still labeled microtubules, suggesting the existence of a second microtubule binding domain at the C terminus of ARK1. ARK1-GFP was broadly expressed in seedlings, but mutant phenotypes were restricted to root hairs, indicating that ARK1’s function is redundant in cells other than those forming root hairs.     

What are the implications of variation in root hair length on tolerance to phosphorus deficiency in combination with water stress in barley (Hordeum vulgare)?

Background and Aims

Phosphorus commonly limits crop yield and is frequently applied as fertilizer; however, supplies of quality rock phosphate for fertilizer production are diminishing. Plants have evolved many mechanisms to increase their P-fertilizer use efficiency, and an understanding of these traits could result in improved long-term sustainability of agriculture. Here a mutant population is utilized to assess the impact of root hair length on P acquisition and yield under P-deficient conditions alone or when combined with drought.

Methods

Mutants with various root hair phenotypes were grown in the glasshouse in pots filled with soil representing sufficient and deficient P treatments and, in one experiment, a range of water availability was also imposed. Plants were variously harvested at 7 d, 8 weeks and 14 weeks, and variables including root hair length, rhizosheath weight, biomass, P accumulation and yield were measured.

Key Results

The results confirmed the robustness of the root hair phenotypes in soils and their relationship to rhizosheath production. The data demonstrated that root hair length is important for shoot P accumulation and biomass, while only the presence of root hairs is critical for yield. Root hair presence was also critical for tolerance to extreme combined P deficit and drought stress, with genotypes with no root hairs suffering extreme growth retardation in comparison with those with root hairs.

Conclusions

The results suggest that although root hair length is not important for maintaining yield, the presence of root hairs is implicit to sustainable yield of barley under P-deficient conditions and when combined with extreme drought. Root hairs are a trait that should be maintained in future germplasm.     

CHARACTERIZATION OF THE NUCLEAR DNA OF HORDEUM VULGARE ROOT HAIRS: AMPLIFICATION DISAPPEARS UNDER SALT STRESS

In Hordeum vulgare L., the nucleus of differentiating root hairs contains amplified, extrachromosomal DNA sequences. Cytophotometry shows that the nuclei of trichoblasts and root hairs grown under normal conditions contain up to 50% more DNA than those grown in 200 mM salt. Although the root hairs develop and differentiate under salt stress, amplification of their nuclear DNA is suppressed. From this, we conclude that amplification is not necessary for differentiation at the cellular level. Characterization of the amplified nuclear DNA of the root hair is based on the physical/chemical nature of the DNA. The amplified sequences separate as a satellite band when total nucleic acids are centrifuged on CsCl gradients. Enzyme restriction of the satellite and main bands with Msp I and Hpa II followed by agarose gel electrophoresis shows that the satellite band is not more highly methylated than the main band. Restriction of the root hair DNA with EcoRI reveals repetitive DNA sequences not seen in similarly restricted whole root, leaf or salt-stressed root hair preparations. While these unique, repetitive sequences in the 2–6 kb region of the gel do not hybridize with ribosomal, chloroplast, or mitochondrial DNAs, RNA hybridization shows that some of them are transcribed. We believe that the amplified sequences are extrachromosomal based on their selective degradation during root hair senescence, their separation as a satellite band and their restriction patterns.     

Role of root hairs in phosphorus depletion from a macrostructured soil

One rape (Brassica napus cv. Wesroona) plant and four cotton (Gossypium hirsutum cv. Sicot 3) plants were grown in plastic cells containing soil labelled with 407 kBq of³³P g⁻¹ soil. After 5–8 days of growth, the³³P depletion zones of all plants were autoradiographed and³³P uptake by plants was measured. The autoradiographs were scanned with a microdensitometer and the optical densities at several places within the³³P depletion zones of roots were obtained. The volume of soil explored by root hairs was estimated from measurements of root diameters and lengths of roots and root hairs. About half of the total³³P depleted by cotion roots came from outside the root hair cylinder whereas most of³³P taken up by rape was from within the root hair cylinder. Plants grown in a macrostructured soil may have roots growing in voids, within aggregates or on the surfaces of aggregates. The results of this study demonstrate that root hairs have a strong influence on the accessibility of phosphorus to roots in such a soil, and thus on the phosphorus nutrition of plants.     

Contrasting phosphate acquisition of mycorrhizal fungi with that of root hairs using the root hairless barley mutant

Comparisons between plant species or cultivars differing in root hair length have indicated a major impact of root hairs on the mycorrhizal dependency of plants with respect to phosphate (P) uptake. The current study aimed to investigate this relationship by comparing directly the mycorrhizal dependency of a spontaneous root hairless mutant, brb , in Hordeum vulgare cv Pallas and its wild type. Both brb and wild type were grown at different soil P levels in association with different mycorrhizal fungi. P uptake of brb and wild type was similar at high P levels, but P uptake by non-mycorrhizal brb plants at low P levels was substantially lower than that of the non-mycorrhizal wild-type plants. However, P uptake of the mutant was much increased by mycorrhizas and with one fungus, the additional P uptake was effectively translated into increased plant growth. Roots of the mutant contained typical colonization structures and a radioactive tracer confirmed P transport by the extraradical mycelium. This is the first direct evaluation of the relative effectiveness of root hairs and mycorrhizas. Mycorrhizas effectively substituted root hairs in P uptake, whereas the additional P was most often used less effectively in promoting plant growth than P provided by root hairs.