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

Abstract

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.

In 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.⁷

ROPs 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.¹⁰

It 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).

Figure 1.

A 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.

Figure 2.

Changes 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.¹

We 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.

Addendum to: Bloch D, Monshausen G, Singer M, Gilroy S, Yalovsky S. Nitrogen source interacts with ROP signalling in root hair tip-growth. Plant Cell and Environment. 2010;34:76–88. doi: 10.1111/j.1365-3040.2010.02227.x.