PLURIPETALA mediates ROP2 localization and stability in parallel to SCN1 but synergistically with TIP1 in root hairs

Prenylation, the post‐translational attachment of prenyl groups to substrate proteins, can affect their distribution and interactomes. Arabidopsis PLURIPETALA (PLP) encodes the shared α subunit of two heterodimeric protein isoprenyltransferases, whose functional loss provides a unique opportunity to study developmental and cellular processes mediated by its prenylated substrates, such as ROP GTPases. As molecular switches, the distribution and activation of ROPs are mediated by various factors, including guanine nucleotide exchange factors, GTPase activating proteins, guanine nucleotide dissociation inhibitors (RhoGDIs), prenylation, and S‐acylation. However, how these factors together ensure that dynamic ROP signalling is still obscure. We report here that a loss‐of‐function allele of PLP resulted in cytoplasmic accumulation of ROP2 in root hairs and reduced its stability. Consequently, two downstream events of ROP signalling, i.e. actin microfilament (MF) organization and the production of reactive oxygen species (ROS), were compromised. Genetic, cytological and biochemical evidence supports an additive interaction between prenylation and RhoGDI1/SCN1 in ROP2 distribution and stability whereas PLP acts synergistically with the protein S‐acyl transferase TIP GROWTH DEFECTIVE1 during root hair growth. By using root hair growth as a model system, we uncovered complex interactions among prenylation, RhoGDIs, and S‐acylation in dynamic ROP signalling.     

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.     

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.     

AGD1, a class 1 ARF-GAP, acts in common signaling pathways with phosphoinositide metabolism and the actin cytoskeleton in controlling Arabidopsis root hair polarity

The Arabidopsis thaliana AGD1 gene encodes a class 1 adenosine diphosphate ribosylation factor‐gtpase‐activating protein (ARF‐GAP). Previously, we found that agd1 mutants have root hairs that exhibit wavy growth and have two tips that originate from a single initiation point. To gain new insights into how AGD1 modulates root hair polarity we analyzed double mutants of agd1 and other loci involved in root hair development, and evaluated dynamics of various components of root hair tip growth in agd1 by live cell microscopy. Because AGD1 contains a phosphoinositide (PI) binding pleckstrin homology (PH) domain, we focused on genetic interactions between agd1 and root hair mutants altered in PI metabolism. Rhd4, which is knocked‐out in a gene encoding a phosphatidylinositol‐4‐phosphate (PI‐4P) phosphatase, was epistatic to agd1. In contrast, mutations to PIP5K3 and COW1, which encode a type B phosphatidylinositol‐4‐phosphate 5‐kinase 3 and a phosphatidylinositol transfer protein, respectively, enhanced the root hair defects of agd1. Enhanced root hair defects were also observed in double mutants to AGD1 and ACT2, a root hair‐expressed vegetative actin isoform. Consistent with our double‐mutant studies, targeting of tip growth components involved in PI signaling (PI‐4P), secretion (RABA4b) and actin regulation (ROP2), were altered in agd1 root hairs. Furthermore, tip cytosolic calcium ([Ca²⁺]_cyt) oscillations were disrupted in root hairs of agd1. Taken together, our results indicate that AGD1 links PI signaling to cytoskeletal‐, [Ca²⁺]_cyt?, ROP2‐, and RABA4b‐mediated root hair development.     

Arabidopsis PCaP2 modulates the phosphatidylinositol 4,5‐bisphosphate signal on the plasma membrane and attenuates root hair elongation

Phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P₂] serves as a subcellular signal on the plasma membrane, mediating various cell‐polarized phenomena including polar cell growth. Here, we investigated the involvement of Arabidopsis thaliana PCaP2, a plant‐unique plasma membrane protein with phosphoinositide‐binding activity, in PtdIns(4,5)P₂ signaling for root hair tip growth. The long‐root‐hair phenotype of the pcap2 knockdown mutant was found to stem from its higher average root hair elongation rate compared with the wild type and to counteract the low average rate caused by a defect in the PtdIns(4,5)P₂‐producing enzyme gene PIP5K3. On the plasma membrane of elongating root hairs, the PCaP2 promoter‐driven PCaP2–green fluorescent protein (GFP), which complemented the pcap2 mutant phenotype, overlapped with the PtdIns(4,5)P₂ marker 2xCHERRY‐2xPH^PLC in the subapical region, but not at the apex, suggesting that PCaP2 attenuates root hair elongation via PtdIns(4,5)P₂ signaling on the subapical plasma membrane. Consistent with this, a GFP fusion with the PCaP2 phosphoinositide‐binding domain PCaP2^N23, root hair‐specific overexpression of which caused a low average root hair elongation rate, localized more intense to the subapical plasma membrane than to the apical plasma membrane similar to PCaP2–GFP. Inducibly overexpressed PCaP2–GFP, but not its derivative lacking the PCaP2^N23 domain, replaced 2xCHERRY‐2xPH^PLC on the plasma membrane in root meristematic epidermal cells, and suppressed FM4‐64 internalization in elongating root hairs. Moreover, inducibly overexpressed PCaP2 arrested an endocytic process of PIN2–GFP recycling. Based on these results, we conclude that PCaP2 functions as a negative modulator of PtdIns(4,5)P₂ signaling on the subapical plasma membrane probably through competitive binding to PtdIns(4,5)P₂ and attenuates root hair elongation.     

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

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

Genetic variation in ZmTIP1 contributes to root hair elongation and drought tolerance in maize

Drought is a major abiotic stress that threatens maize production globally. A previous genome‐wide association study identified a significant association between the natural variation of ZmTIP1 and the drought tolerance of maize seedlings. Here, we report on comprehensive genetic and functional analysis, indicating that ZmTIP1, which encodes a functional S‐acyltransferase, plays a positive role in regulating the length of root hairs and the level of drought tolerance in maize. We show that enhancing ZmTIP1 expression in transgenic Arabidopsis and maize increased root hair length, as well as plant tolerance to water deficit. In contrast, ZmTIP1 transposon‐insertional mutants displayed the opposite phenotype. A calcium‐dependent protein kinase, ZmCPK9, was identified as a substrate protein of ZmTIP1, and ZmTIP1‐mediated palmitoylation of two cysteine residues facilitated the ZmCPK9 PM association. The results of this research enrich our knowledge about ZmTIP1‐mediated protein S‐acylation modifications in relation to the regulation of root hair elongation and drought tolerance. Additionally, the identification of a favourable allele of ZmTIP1 also provides a valuable genetic resource or selection target for the genetic improvement of maize.     

The Small GTPase ROP10 of Medicago truncatula Is Required for Both Tip Growth of Root Hairs and Nod Factor-Induced Root Hair Deformation

Rhizobia preferentially enter legume root hairs via infection threads, after which root hairs undergo tip swelling, branching, and curling. However, the mechanisms underlying such root hair deformation are poorly understood. Here, we showed that a type II small GTPase, ROP10, of Medicago truncatula is localized at the plasma membrane (PM) of root hair tips to regulate root hair tip growth. Overexpression of ROP10 and a constitutively active mutant (ROP10CA) generated depolarized growth of root hairs, whereas a dominant negative mutant (ROP10DN) inhibited root hair elongation. Inoculated with Sinorhizobium meliloti, the depolarized swollen and ballooning root hairs exhibited extensive root hair deformation and aberrant infection symptoms. Upon treatment with rhizobia-secreted nodulation factors (NFs), ROP10 was transiently upregulated in root hairs, and ROP10 fused to green fluorescent protein was ectopically localized at the PM of NF-induced outgrowths and curls around rhizobia. ROP10 interacted with the kinase domain of the NF receptor NFP in a GTP-dependent manner. Moreover, NF-induced expression of the early nodulin gene ENOD11 was enhanced by the overexpression of ROP10 and ROP10CA. These data suggest that NFs spatiotemporally regulate ROP10 localization and activity at the PM of root hair tips and that interactions between ROP10 and NF receptors are required for root hair deformation and continuous curling during rhizobial infection.     

Roothairless5, which functions in maize (Zea mays L.) root hair initiation and elongation encodes a monocot‐specific NADPH oxidase

Root hairs are instrumental for nutrient uptake in monocot cereals. The maize (Zea mays L.) roothairless5 (rth5) mutant displays defects in root hair initiation and elongation manifested by a reduced density and length of root hairs. Map‐based cloning revealed that the rth5 gene encodes a monocot‐specific NADPH oxidase. RNA‐Seq, in situ hybridization and qRT‐PCR experiments demonstrated that the rth5 gene displays preferential expression in root hairs but also accumulates to low levels in other tissues. Immunolocalization detected RTH5 proteins in the epidermis of the elongation and differentiation zone of primary roots. Because superoxide and hydrogen peroxide levels are reduced in the tips of growing rth5 mutant root hairs as compared with wild‐type, and Reactive oxygen species (ROS) is known to be involved in tip growth, we hypothesize that the RTH5 protein is responsible for establishing the high levels of ROS in the tips of growing root hairs required for elongation. Consistent with this hypothesis, a comparative RNA‐Seq analysis of 6‐day‐old rth5 versus wild‐type primary roots revealed significant over‐representation of only two gene ontology (GO) classes related to the biological functions (i.e. oxidation/reduction and carbohydrate metabolism) among 893 differentially expressed genes (FDR <5%). Within these two classes the subgroups ‘response to oxidative stress’ and ‘cellulose biosynthesis’ were most prominently represented.     

Stimulation of root hair elongation in Arabidopsis thaliana by low phosphorus availability

Low phosphorus availability stimulates root hair elongation in many plants, which may have adaptive significance in soil phosphorus acquisition. We investigated the effect of low phosphorus on the elongation of Arabidopsis thaliana root hairs. Arabidopsis thaliana plants were grown in plant culture containing high (1000 mmol m^?3) or low (1 mmol m^?3) phosphorus concentrations, and root hair elongation was analysed by image analysis. After 15d of growth, low-phosphorus plants developed root hairs averaging 0.9 mm in length while high-phosphorus plants of the same age developed root hairs averaging 0.3 mm in length. Increased root hair length in low-phosphorus plants was a result of both increased growth duration and increased growth rate. Root hair length decreased logarithmically in response to increasing phosphorus concentration. Local changes in phosphorus availability influenced root hair growth regardless of the phosphorus status of the plant. Low phosphorus stimulated root hair elongation in the hairless axr2 mutant, exogenously applied IAA stimulated root hair elongation in wild-type high-phosphorus plants and the auxin antagonist CM PA inhibited root hair elongation in low-phosphorus plants. These results indicate that auxin may be involved in the low-phosphorus response in root hairs.     

The Ca2+‐binding protein PCaP2 located on the plasma membrane is involved in root hair development as a possible signal transducer

Plasma membrane‐associated Ca²⁺‐binding protein–2 (PCaP2) of Arabidopsis thaliana is a novel‐type protein that binds to the Ca²⁺/calmodulin complex and phosphatidylinositol phosphates (PtdInsPs) as well as free Ca²⁺. Although the PCaP2 gene is predominantly expressed in root hair cells, it remains unknown how PCaP2 functions in root hair cells via binding to ligands. From biochemical analyses using purified PCaP2 and its variants, we found that the N–terminal basic domain with 23 amino acids (N23) is necessary and sufficient for binding to PtdInsPs and the Ca²⁺/calmodulin complex, and that the residual domain of PCaP2 binds to free Ca²⁺. In mutant analysis, a pcap2 knockdown line displayed longer root hairs than the wild‐type. To examine the function of each domain in root hair cells, we over‐expressed PCaP2 and its variants using the root hair cell‐specific EXPANSIN A7 promoter. Transgenic lines over‐expressing PCaP2, PCaP2^G2A (second glycine substituted by alanine) and ?23PCaP2 (lacking the N23 domain) exhibited abnormal branched and bulbous root hair cells, while over‐expression of the N23 domain suppressed root hair emergence and elongation. The N23 domain was necessary and sufficient for the plasma membrane localization of GFP‐tagged PCaP2. These results suggest that the N23 domain of PCaP2 negatively regulates root hair tip growth via processing Ca²⁺ and PtdInsP signals on the plasma membrane, while the residual domain is involved in the polarization of cell expansion.     

SPIKE1 Activates the GTPase ROP6 to Guide the Polarized Growth of Infection Threads in Lotus japonicus

In legumes, rhizobia attach to root hair tips and secrete nodulation factor to activate rhizobial infection and nodule organogenesis. Endosymbiotic rhizobia enter nodule primordia via a specialized transcellular compartment known as the infection thread (IT). The IT elongates by polar tip growth, following the path of the migrating nucleus along and within the root hair cell. Rho-family ROP GTPases are known to regulate the polarized growth of cells, but their role in regulating polarized IT growth is poorly understood. Here, we show that LjSPK1, a DOCK family guanine nucleotide exchange factor (GEF), interacts with three type I ROP GTPases. Genetic analyses showed that these three ROP GTPases are involved in root hair development, but only LjROP6 is required for IT formation after rhizobia inoculation. Misdirected ITs formed in the root hairs of Ljspk1 and Ljrop6 mutants. We show that LjSPK1 functions as a GEF that activates LjROP6. LjROP6 enhanced the plasma membrane localization LjSPK1 in Nicotiana benthamiana leaf cells and Lotus japonicus root hairs, and LjSPK1 and LjROP6 interact at the plasma membrane. Taken together, these results shed light on how the LjROP6-LjSPK1 module mediates the polarized growth of ITs in L. japonicus.     

The plant beneficial rhizobacterium Achromobacter sp. 5B1 influences root development through auxin signaling and redistribution

Roots provide physical and nutritional support to plant organs that are above ground and play critical roles for adaptation via intricate movements and growth patterns. Through screening the effects of bacterial isolates from roots of halophyte Mesquite (Prosopis sp.) on Arabidopsis thaliana, we identified Achromobacter sp. 5B1 as a probiotic bacterium that influences plant functional traits. Detailed genetic and architectural analyses in Arabidopsis grown in vitro and in soil, cell division measurements, auxin transport and response gene expression and brefeldin A treatments demonstrated that root colonization with Achromobacter sp. 5B1 changes the growth and branching patterns of roots, which were related to auxin perception and redistribution. Expression analysis of auxin transport and signaling revealed a redistribution of auxin within the primary root tip of wild‐type seedlings by Achromobacter sp. 5B1 that is disrupted by brefeldin A and correlates with repression of auxin transporters PIN1 and PIN7 in root provasculature, and PIN2 in the epidermis and cortex of the root tip, whereas expression of PIN3 was enhanced in the columella. In seedlings harboring AUX1, EIR1, AXR1, ARF7ARF19, TIR1AFB2AFB3 single, double or triple loss‐of‐function mutations, or in a dominant (gain‐of‐function) mutant of SLR1, the bacterium caused primary roots to form supercoils that are devoid of lateral roots. The changes in growth and root architecture elicited by the bacterium helped Arabidopsis seedlings to resist salt stress better. Thus, Achromobacter sp. 5B1 fine tunes both root movements and the auxin response, which may be important for plant growth and environmental adaptation.     

A root hairless barley mutant for elucidating genetic of root hairs and phosphorus uptake

This paper reports a new barley mutant missing root hairs. The mutant was spontaneously discovered among the population of wild type (Pallas, a spring barley cultivar), producing normal, 0.8 mm long root hairs. We have called the mutant bald root barley (brb). Root anatomical studies confirmed the lack of root hairs on mutant roots. Amplified Fragment Length Polymorphism (AFLP) analyses of the genomes of the mutant and Pallas supported that the brb mutant has its genetic background in Pallas. The segregation ratio of selfed F₂ plants, resulting from mutant and Pallas outcross, was 1:3 (–root hairs:+root hairs), suggesting a monogenic recessive mode of inheritance.In rhizosphere studies, Pallas absorbed nearly two times more phosphorus (P) than the mutant. Most of available inorganic P in the root hair zone (0.8 mm) of Pallas was depleted, as indicated by the uniform P depletion profile near its roots. The acid phosphatase (Apase) activity near the roots of Pallas was higher and Pallas mobilised more organic P in the rhizosphere than the mutant. The higher Apase activity near Pallas roots also suggests a link between root hair formation and rhizosphere Apase activity. Hence, root hairs are important for increasing plant P uptake of inorganic as well as mobilisation of organic P in soils.Laboratory, pot and field studies showed that barley cultivars with longer root hairs (1.10 mm), extracted more P from rhizosphere soil, absorbed more P in low-P field (Olsen P=14 mg P kg^–1 soil), and produced more shoot biomass than shorter root hair cultivars (0.63 mm). Especially in low-P soil, the differences in root hair length and P uptake among the cultivars were significantly larger. Based on the results, the perspectives of genetic analysis of root hairs and their importance in P uptake and field performance of cereals are discussed.     

FERONIA mutation induces high levels of chloroplast‐localized Arabidopsides which are involved in root growth

The FERONIA (FER) signaling pathway is known to have diverse roles in Arabidopsis thaliana, such as growth, reproduction, and defense, but how this receptor kinase is involved in various biological processes is not well established. In this work, we applied multiple mass spectrometry techniques to identify metabolites involved in the FER signaling pathway and to understand their biological roles. A direct infusion Fourier transform ion cyclotron resonance (FT‐ICR)‐MS approach was used for initial screening of wild‐type and feronia (fer) mutant plant extracts, and Arabidopsides were found to be significantly enriched in the mutant. As Arabidopsides are known to be induced by wounding, further experiments on wounded and non‐wounded leaf samples were carried out to investigate these oxylipins as well as related phytohormones using a quadrupole‐time‐of‐flight (Q‐TOF) MS by direct injection and LC‐MS/MS. In a root growth bioassay with Arabidopside A isolated from fer mutants, the wild‐type showed significant root growth inhibition compared with the fer mutant. Our results therefore implicated Arabidopsides, and Arabidopside A specifically, in FER functions and/or signaling. Finally, matrix‐assisted laser desorption/ionization MS imaging (MALDI‐MSI) was used to visualize the localization of Arabidopsides, and we confirmed that Arabidopsides are highly abundant at wounding sites in both wild‐type and fer mutant leaves. More significantly, five micron high‐spatial resolution MALDI‐MSI revealed that Arabidopsides are localized to the chloroplasts where many stress signaling molecules are made.     

Co-regulation of root hair tip growth by ROP GTPases and nitrogen source modulated pH fluctuations

Growth of plant cells involves tight regulation of the cytoskeleton and vesicle trafficking by processes including the action of the ROP small G proteins together with pH-modulated cell wall modifications. Yet, little is known on how these systems are coordinated. In a paper recently published in Plant Cell and Environment¹ we show that ROPs/RACs function synergistically with NH₄NO₃-modulated pH fluctuations to regulate root hair growth. Root hairs expand exclusively at their apical end in a strictly polarized manner by a process known as tip growth. The highly polarized secretion at the apex is maintained by a complex network of factors including the spatial organization of the actin cytoskeleton, tip-focused ion gradients and by small G proteins. Expression of constitutively active ROP mutants disrupts polar growth, inducing the formation of swollen root hairs. Root hairs are also known to elongate in an oscillating manner, which is correlated with oscillatory H⁺ fluxes at the tip. Our analysis shows that root hair elongation in wild type plants and swelling in transgenic plants expressing a constitutively active ROP11 (rop11^CA) is sensitive to the presence of NH₄⁺ at concentrations higher than 1 mM and on NO₃⁻. The NH₄⁺ and NO₃⁻ ions did not affect the localization of ROP in the membrane but modulated pH fluctuations at the root hair tip. Actin organization and reactive oxygen species distribution were abnormal in rop11^CA root hairs but were similar to wild-type root hairs when seedlings were grown on medium lacking NH₄⁺ and/or NO₃⁻. These observations suggest that the nitrogen source-modulated pH fluctuations may function synergistically with ROP regulated signaling during root hair tip growth. Interestingly, under certain growth conditions, expression of rop11^CA suppressed ammonium toxicity, similar to auxin resistant mutants. In this short review we discuss these findings and their implications.Key words: ROP, RAC, nitrogen, root hair, cell polarity, ammoniumIn Arabidopsis, root hairs grow out at the basal, rootward region (closer to root tip) of specialized root epidermal cells and expand exclusively at their apical end in a strictly polarized manner by a process known as tip growth. Tip growth is facilitated by Rho of Plants (ROP)-regulated processes such as maintenance of longitudinally-oriented actin cables in the shank of the root hair that are required for myosin-mediated organelle transport through the cytoplasm. ROPs also play a role in sustaining fine F-actin structures at the root hair tip, which promote the transport of secretory vesicles to sites of their fusion with the plasma membrane.^2,3 In addition, the polar growth of root hairs involves an oscillatory tip-focused Ca²⁺ gradient⁴ and tip-localized reactive oxygen species (ROS).⁵ Tip growth is also associated with oscillatory fluxes of H⁺ at the apex that correlate with the periodicity of growth.^6,7 These oscillations in extracellular pH and ROS have been shown to modulate tip growth and are predicted to act in a coordinated and complementary mode to regulate root hair elongation. Growth accelerates following reduction of apoplastic pH and slows upon apoplastic ROS increase and a coincident pH increase.⁷ROPs are small G proteins that localize to the plasma membrane at the apex of growing root hairs, where they activate a range of downstream pathways required for tip growth.^8,9 ROP activity is regulated by its cycling between a GTP-bound, active and GDP-bound, inactive state. Ectopic expression of constitutively active mutants of ROPs (dominant mutations in conserved residues that abolish the GTPase activity) depolarizes the growth of root hairs.^8–10 Downstream pathways activated by such ROP GTPases include the regulation of cytoskeletal dynamics and vesicular trafficking, production of ROS, maintenance of intracellular Ca²⁺ gradients and accumulation of signaling lipids, features all related to the regulation of apical growth.^11,12 For example, ectopic expression of constitutively active ROP11 (Atrop11^CA) depolarizes root hair growth, leading to the formation of swollen root hairs. This bulging root hair phenotype was associated with altered actin organization and inhibition of endocytosis.¹⁰It is well known that root hair development is highly plastic and regulated by environmental signals.^13,14 Yet, despite the known function of ROP GTPases and their regulatory proteins in root hair growth there is no data in the literature describing the relationship between ROP signaling and environmental factors in this process. Our results¹ show that induction of root hair swelling by rop11^CA occurs only under specific growth conditions, indicating that there is an interplay between ROP activity and the external environment, particularly nitrogen supply. We demonstrated that high external concentrations of ammonium are essential for the induction of depolarized root hair growth and activation of downstream pathways by rop11^CA. Depletion of ammonium did not affect the membrane localization and expression of GFP-rop11^CA, implying that NH₄⁺ was required in addition to ROP activity to cause root hair swelling. In agreement with this idea, normal actin organization and ROS localization were detected in rop11^CA root hairs when NH₄⁺ was depleted, suggesting that ammonium functions downstream of, or in parallel to ROP signaling (Fig. 1).Open in a separate windowFigure 1A model for regulation of root hair tip growth by ROP GTPases and pH oscillations dependent on nitrogen supply. GTP bound ROPs activate downstream effectors which directly affect actin organization, vesicular trafficking and localized ROS production as well as indirectly affecting the localization of membrane proteins involved in ion/proton fluxes. High concentrations of nitrogen ions in the growth medium increase pH oscillations at the apex of growing root hairs. In turn downstream ROP effectors sense the changes in pH and adjust their function accordingly. pH oscillations affect tip growth independent of ROPs via changes of wall pH and possibly through additional unknown factors. Dashed lines indicate that these effects were not confirmed experimentally.Plants can absorb and use various forms of nitrogen from soils, primarily the inorganic ions ammonium and nitrate. The concentrations of these ions are highly heterogeneous around the plant and can vary across several orders of magnitude among different soils and as a result of seasonal changes.¹⁵ Thus, plants would be expected to display highly plastic, N-regulated developmental responses and to employ a range of nitrogen uptake transport systems to optimize exploitation of local N resources. Transport systems that mediate NH₄ fluxes across the plasma membrane of root cells are divided into two categories: high affinity transport systems (HATS) that mediate uptake from relatively dilute solutions at relatively low rates and low affinity transport systems (LATS) that operate at high rates and higher external concentrations.¹⁶ The HATS are plasma membrane localized NH₄⁺-specific transporters (AMTs) that are most likely proton-coupled and their expression and function are repressed at external ammonium concentrations of 1 mM or higher.^17–19 In contrast, ammonium uptake by LATS is believed to take place through non-specific cation channels.^17,20 The NH₄⁺ concentration in the 0.5× Murashige Skoog (MS) medium is 10.3 mM, exceeding by an order of magnitude the concentration at which the high affinity NH₄⁺ uptake system is repressed. The root hair swelling in Atrop11_CA plants and inhibition of root hair elongation in wild type plants occurred primarily at external ammonium concentrations greater than 1 mM, and thus is most likely associated with uptake by the LATS.As noted above, root hair elongation is associated with oscillations of cytoplasmic and apoplastic pH that have been linked to growth control. Simultaneous fluorescence ratio imaging of internal and external pH revealed that application of 10 mM NH₄NO₃ enhanced the amplitude of these pH oscillations at the extreme apex of wild type root hairs1 and Figure 2. These oscillations are thought to modulate tip growth through altering the extensibility of the wall.⁴ Additional measurements (Fig. 2) show that similar to the effects of NH₄NO₃, addition of NH₄Cl induced increase in the apoplastic pH fluctuations and reduced the pH. However, the effects of NH₄Cl on cytoplasmic pH fluctuations seem subtler compared to the effects of NH₄NO₃. Thus, one possible explanation for the observed swelling of the root hair apex in rop11_CA expressing plants in media containing NH₄NO₃ is that rop11_CA root hairs are affected in their ability to re-establish the normal proton gradient across the plasma membrane in response to ammonium transport. The altered proton gradient would then prevent the normal localized oscillatory changes in pH-dependent wall properties required to restrict expansion to the very tip of the elongating root hair.Open in a separate windowFigure 2Changes in apoplastic and cytoplasmic pH fluctuations, following application of NH₄NO₃, NH₄Cl or KNO₃. (A) Apolplastic pH (pH_ex) following treatments with either NH₄NO₃, NH₄Cl or KNO₃. Note the increase pH fluctuations induced by either NH₄NO₃ and NH₄Cl but not by KNO₃. (B) Cytoplasmic pH (pH_cyt) following treatments as above. Note the changes in pH fluctuations induced by NH₄NO₃ and the subtler effects of NH₄Cl.Concurrent absorption of NH₄⁺ and NO₃⁻- maintains the cation-anion balance within both the rooting medium and the root, and thus potentially has an important function in maintaining intracellular and extracellular pH.^21,22 In agreement, application of these ions affected the amplitude of pH oscillations¹ and Figure 2. Interestingly, treatments of WT seedlings with 10 mM NH₄NO₃ causes increase in root hair pH oscillations and often tip bursting. Yet, prolonged exposure of WT root hairs to NH₄NO₃ is accompanied by adaptation (our unpublished data). This adaptation does not occur in rop11_CA mutants, suggesting that cycling of ROPs between active and inactive states maybe important in adaptation to changing environment. These data strongly suggest that NH₄⁺-dependent root hair swelling in the plants expressing activated ROP resulted from physiological changes in ion balance rather than a direct effect of ammonium on enzymatic activities required for root hair growth (Fig. 1). Application of NH₄⁺ and NO₃⁻, in the absence of other ions, induced formation of additional growth tips, in which the membrane localized GFP-rop11^CA was concentrated. This observation suggests that interplay between the regulation of ROP localization and activity and the regulation of nitrogen fluxes may have an important function in the maintenance of unidirectional growth. As root hair elongation is coupled to spatially distinct regulation of extracellular pH oscillations and ROS production,⁷ it seems likely that there is a mechanism that can adjust the fluxes of nitrogen ions relative to these pH fluxes. This system would then maintain the oscillations in pH such that polarized growth is continued. One possible mechanism for this coordination is through the highly localized ROP cycling between active and inactive states that has an important role in the spatial activation of cell polarization machinery.^23–27 Due to the function of ROP GTPases in vesicle trafficking, actin organization and maintenance of ROS and Ca²⁺ gradients,^{2,8,9,23,24,28–33} expression of activated ROP11 may indirectly influence cell wall properties by altering the localization and/or recycling of cation and anion transporters/channels or plasma membrane H⁺-ATPases delivered to the growing tip of the hair and in this way affect the maintenance of the proton gradients. In agreement with a possible effect of activated ROPs on localization and/or recycling of membrane transporters we discovered that rop11^CA plants were resistant to ammonium toxicity when grown in the presence of NH₄NO₃ and several micronutrients.¹We propose a model (Fig. 1) in which spatial regulation of ROP activity creates a positive feedback loop with pH oscillations around the growing apex of root hairs. According to this model ROP cycling between active and inactive states spatially and temporally activates the downstream signaling cascades essential for the tip-growth of root hairs. At the same time, localization of membrane proteins involved in maintenance of normal nitrogen fluxes across the plasma membrane is indirectly affected by ROP signaling. Alternatively, ROP signaling is modulated to adapt to altered nitrogen fluxes. NH₄⁺ fluxes increase the amplitude of pH oscillations at the root hair apex and in turn affect cell-wall properties. Thus, when the ROP activity is upregulated by dominant mutations, the synergistic effects of pH changes and constant activation of ROP downstream effectors result in the uncontrolled cell expansion seen as root hair bulging. Previous studies have suggested that feedback between oscillatory pH change and ROS distribution is required to support tip growth.⁷ However, the factors that may integrate these processes are unknown. Our results suggest that spatial regulation of ROP activity in response to changing environments is one of the key elements that may coordinate the pH and ROS oscillations during the root hair tip growth.It will be interesting to examine whether ROP function is coordinated with apoplastic pH fluctuation in other cell types. Recently, it has been suggested that the effects of auxin on pavement cell structure in leaf epidermis require Auxin Binding Protein 1 (ABP1) dependent ROP activation.³⁴ It is well known that auxin induces changes in apoplastic pH. Possibly, like nitrogen source in root hairs, auxin dependent apolplastic pH fluctuations in the leaf epidermis may function coordinately with ROP in the regulation of cell growth. Consistent with this idea, it has been shown that auxin inhibits clathrin-dependent endocytosis through ABP1 reinforcing a possible role in modulating membrane flux/membrane properties.³⁵ Some auxin resistant mutants also display resistance to ammonium toxicity³⁶ further suggesting a link between auxin and membrane transport. Hence, auxin and ROPs may indeed function synergistically to modulate plasma membrane properties, in turn affecting ion balance in the apoplast and so modulating cell wall properties and growth.