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

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

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.     

Building a hair: tip growth in Arabidopsis thaliana root hairs

The Arabidopsis thaliana root hair is used as a model for studying tip growth in plants. We review recent advances, made using physiological and genetic approaches, which give rise to different, yet compatible, current views of the establishment and maintenance of tip growth in epidermal cells. For example, an active calcium influx channel localized at the tip of Arabidopsis root hairs has been identified by patch-clamp measurements. Actin has been visualized in vivo in Arabidopsis root hairs by using a green-fluorescent-protein-talin reporter and shown to form a dense mesh in the apex of the growing tip. The kojak gene, which encodes a protein similar to the catalytic subunit of cellulose synthase, is needed in the first stages of hair growth. A role for LRX1, a leucine-rich repeat extensin, in determining the morphology of the cell wall of root hairs has been established using reverse genetics. The new information can be integrated into a general and more advanced view of how these specialized plant cells grow.     

ACTIN2 is essential for bulge site selection and tip growth during root hair development of Arabidopsis

Root hairs develop as long extensions from root epidermal cells. After the formation of an initial bulge at the distal end of the epidermal cell, the root hair structure elongates by tip growth. Because root hairs are not surrounded by other cells, root hair formation provides an excellent system for studying the highly complex process of plant cell growth. Pharmacological experiments with actin filament-interfering drugs have provided evidence that the actin cytoskeleton is an important factor in the establishment of cell polarity and in the maintenance of the tip growth machinery at the apex of the growing root hair. However, there has been no genetic evidence to directly support this assumption. We have isolated an Arabidopsis mutant, deformed root hairs 1 (der1), that is impaired in root hair development. The DER1 locus was cloned by map-based cloning and encodes ACTIN2 (ACT2), a major actin of the vegetative tissue. The three der1 alleles develop the mutant phenotype to different degrees and are all missense mutations, thus providing the means to study the effect of partially functional ACT2. The detailed characterization of the der1 phenotypes revealed that ACT2 is not only involved in root hair tip growth, but is also required for correct selection of the bulge site on the epidermal cell. Thus, the der1 mutants are useful tools to better understand the function of the actin cytoskeleton in the process of root hair formation.     

A novel GTPase activated by the small subunit of ribosome

The GTPase activity of Escherichia coli YjeQ, here named RsgA (ribosome small subunit-dependent GTPase A), has been shown to be significantly enhanced by ribosome or its small subunit. The enhancement of GTPase activity was inhibited by several aminoglycosides bound at the A site of the small subunit, but not by a P site-specific antibiotic. RsgA stably bound the small subunit in the presence of GDPNP, but not in the presence of GTP or GDP, to dissociate ribosome into subunits. Disruption of the gene for RsgA from the genome affected the growth of the cells, which predominantly contained the dissociated subunits having only a weak activation activity of RsgA. We also found that 17S RNA, a putative precursor of 16S rRNA, was contained in the small subunit of the ribosome from the RsgA-deletion strain. RsgA is a novel GTPase that might provide a new insight into the function of ribosome.     

The Arabidopsis small G protein ROP2 is activated by light in guard cells and inhibits light-induced stomatal opening

ROP small G proteins function as molecular switches in diverse signaling processes. Here, we investigated signals that activate ROP2 in guard cells. In guard cells of Vicia faba expressing Arabidopsis thaliana constitutively active (CA) ROP2 fused to red fluorescent protein (RFP-CA-ROP2), fluorescence localized exclusively at the plasma membrane, whereas a dominant negative version of RFP-ROP2 (DN-ROP2) localized in the cytoplasm. In guard cells expressing green fluorescent protein-ROP2, the relative fluorescence intensity at the plasma membrane increased upon illumination, suggesting that light activates ROP2. Unlike previously reported light-activated factors, light-activated ROP2 inhibits rather than accelerates light-induced stomatal opening; stomata bordered by guard cells transformed with CA-rop2 opened less than controls upon light irradiation. When introduced into guard cells together with CA-ROP2, At RhoGDI1, which encodes a guanine nucleotide dissociation inhibitor, inhibited plasma membrane localization of CA-ROP2 and abolished the inhibitory effect of CA-ROP2 on light-induced stomatal opening, supporting the negative effect of active ROP2 on stomatal opening. Mutant rop2 Arabidopsis guard cells showed phenotypes similar to those of transformed V. faba guard cells; CA-rop2 stomata opened more slowly and to a lesser extent, and DN-rop2 stomata opened faster than wild-type stomata in response to light. Moreover, in rop2 knockout plants, stomata opened faster and to a greater extent than wild-type stomata in response to light. Thus, ROP2 is a light-activated negative factor that attenuates the extent of light-induced changes in stomatal aperture. The inhibition of light-induced stomatal opening by light-activated ROP2 suggests the existence of feedback regulatory mechanisms through which stomatal apertures may be finely controlled.     

The small GTPase AtRAC2/ROP7 is specifically expressed during late stages of xylem differentiation in Arabidopsis

The RAC/ROP family of small GTPases are central regulators of important cellular processes in plants. AtRAC2/ROP7 is an ancient member of the RAC/ROP gene family in Arabidopsis thaliana whose functions are generally unknown. In order to study the spatial expression pattern of the AtRAC2/ROP7 gene, transgenic plants expressing GUS or GFP under the control of the AtRAC2/ROP7 promoter were analysed. Functional analysis of AtRAC2/ROP7 was done using transgenic plants overexpressing wild-type and constitutively activated AtRAC2/ROP7 (Val15Gly), and an AtRAC2/ROP7T-DNA insertion mutant. The AtRAC2/ROP7 promoter directs a highly specific xylem-specific expression in the root, hypocotyl, stem, and leaves. The expression is developmentally limited to the late stages of xylem differentiation, and coincides with the formation of secondary cell walls. Leaf epidermal cells of transgenic plants overexpressing constitutively active AtRAC2/ROP7 exhibited highly impaired lobe formation, suggesting that AtRAC2/ROP7 is able to regulate polar cell expansion. Finally, GFP-AtRAC2/ROP7 fusion proteins were localized to the plasma membrane. The results indicate a role for AtRAC2/ROP7 in the development of secondary cell walls of xylem vessels.     

Salt-induced plasticity of root hair development is caused by ion disequilibrium in Arabidopsis thaliana

Root hair development is controlled by environmental signals. Studies on root hair plasticity in Arabidopsis thaliana have mainly focused on phosphate and iron deficiency. Root hair growth and development and their physiological role in response to salt stress are largely unknown. Here, we show that root epidermal cell types and root hair development are highly regulated by salt stress. Root hair length and density decreased significantly in a dose-dependent manner on both primary roots and junction sites between roots and shoots. The root hair growth and development were sensitive to inhibition by ions but not to osmotic stress. High salinity also alters anatomical structure of roots, leading to a decrease in cell number in N positions and enlargement of the cells. Moreover, analysis of the salt overly sensitive mutants indicated that salt-induced root hair response is caused by ion disequilibrium and appears to be an adaptive mechanism that reduces excessive ion uptake. Finally, we show that genes WER, GL3, EGL3, CPC, and GL2 might be involved in cell specification of root epidermis in stressed plants. Taken together, data suggests that salt-induced root hair plasticity represents a coordinated strategy for early stress avoidance and tolerance as well as a morphological sign of stress adaptation.     

Involvement of the mitogen-activated protein kinase SIMK in regulation of root hair tip growth

Mitogen-activated protein kinases (MAPKs) are involved in stress signaling to the actin cytoskeleton in yeast and animals. We have analyzed the function of the stress-activated alfalfa MAP kinase SIMK in root hairs. In epidermal cells, SIMK is predominantly nuclear. During root hair formation, SIMK was activated and redistributed from the nucleus into growing tips of root hairs possessing dense F-actin meshworks. Actin depolymerization by latrunculin B resulted in SIMK relocation to the nucleus. Conversely, upon actin stabilization with jasplakinolide, SIMK co-localized with thick actin cables in the cytoplasm. Importantly, latrunculin B and jasplakinolide were both found to activate SIMK in a root-derived cell culture. Loss of tip-focused SIMK and actin was induced by the MAPK kinase inhibitor UO 126 and resulted in aberrant root hairs. UO 126 inhibited targeted vesicle trafficking and polarized growth of root hairs. In contrast, overexpression of gain-of-function SIMK induced rapid tip growth of root hairs and could bypass growth inhibition by UO 126. These data indicate that SIMK plays a crucial role in root hair tip growth.     

A nonsymbiotic root hair tip growth phenotype in NORK-mutated legumes: implications for nodulation factor-induced signaling and formation of a multifaceted root hair pocket for bacteria

The Medicago truncatula Does not Make Infections (DMI2) mutant is mutated in the nodulation receptor-like kinase, NORK. Here, we report that NORK-mutated legumes of three species show an enhanced touch response to experimental handling, which results in a nonsymbiotic root hair phenotype. When care is taken not to induce this response, DMI2 root hairs respond morphologically like the wild type to nodulation factor (NF). Global NF application results in root hair deformation, and NF spot application induces root hair reorientation or branching, depending on the position of application. In the presence of Sinorhizobium meliloti, DMI2 root hairs make two-dimensional 180 degrees curls but do not entrap bacteria in a three-dimensional pocket because curling stops when the root hair tip touches its own shank. Because DMI2 does not express the promoter of M. truncatula Early Nodulin11 (ENOD11) coupled to beta-glucuronidase upon NF application, we propose a split in NF-induced signaling, with one branch to root hair curling and the other to ENOD11 expression.     

Effects of secondary mutation in det2-1 on root growth and development in Arabidopsis

Brassinosteroids (BRs) are plant steroidal hormones that regulate a wide range of developmental processes. Most BR mutants display impaired growth and responses to developmental and environmental stimuli. Here, we found a BR-deficient mutant det2-1 that displayed exceedingly short roots and agravitropic growth, which were not present in other BR mutants. By back-crossing det2-1 with the wild type, we isolated a secondary mutation named det2-1 phenotype modifier 1 (dpm1) and demonstrated that those aberrant phenotypes in the original det2-1 were independent of the BR deficiency. Phenotypic analysis showed that impaired root growth of dpm1 appeared in BR-deficient condition, but not in a normal condition. In the light condition, the mutant showed enhanced shoot growth which was suppressed in the det2-1 background. Starch granules in the columella cells of the root tip were highly accumulated and expanded in dpm1. Agravitropic roots and the expanded starch granules of dpm1 could not be recovered by BR. Taken together, these results suggest that DPM1 is required for gravitropic growth, and that its functions on root and shoot growth are BR-dependent.     

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.     

A class I ADP-ribosylation factor GTPase-activating protein is critical for maintaining directional root hair growth in Arabidopsis

Membrane trafficking and cytoskeletal dynamics are important cellular processes that drive tip growth in root hairs. These processes interact with a multitude of signaling pathways that allow for the efficient transfer of information to specify the direction in which tip growth occurs. Here, we show that AGD1, a class I ADP ribosylation factor GTPase-activating protein, is important for maintaining straight growth in Arabidopsis (Arabidopsis thaliana) root hairs, since mutations in the AGD1 gene resulted in wavy root hair growth. Live cell imaging of growing agd1 root hairs revealed bundles of endoplasmic microtubules and actin filaments extending into the extreme tip. The wavy phenotype and pattern of cytoskeletal distribution in root hairs of agd1 partially resembled that of mutants in an armadillo repeat-containing kinesin (ARK1). Root hairs of double agd1 ark1 mutants were more severely deformed compared with single mutants. Organelle trafficking as revealed by a fluorescent Golgi marker was slightly inhibited, and Golgi stacks frequently protruded into the extreme root hair apex of agd1 mutants. Transient expression of green fluorescent protein-AGD1 in tobacco (Nicotiana tabacum) epidermal cells labeled punctate bodies that partially colocalized with the endocytic marker FM4-64, while ARK1-yellow fluorescent protein associated with microtubules. Brefeldin A rescued the phenotype of agd1, indicating that the altered activity of an AGD1-dependent ADP ribosylation factor contributes to the defective growth, organelle trafficking, and cytoskeletal organization of agd1 root hairs. We propose that AGD1, a regulator of membrane trafficking, and ARK1, a microtubule motor, are components of converging signaling pathways that affect cytoskeletal organization to specify growth orientation in Arabidopsis root hairs.     

ASC1/RAS2 suppresses the growth defect on glycerol caused by the atp1-2 mutation in the yeast Saccharomyces cerevisiae

To better define the regulatory role of the F(1)-ATPase alpha-subunit in the catalytic cycle of the ATP synthase complex, we isolated suppressors of mutations occurring in ATP1, the gene for the alpha-subunit in Saccharomyces cerevisiae. First, two atp1 mutations (atp1-1 and atp1-2) were characterized that prevent the growth of yeast on non-fermentable carbon sources. Both mutants contained full-length F(1)alpha-subunit proteins in mitochondria, but in lower amounts than that in the parental strain. Both mutants exhibited barely measurable F(1)-ATPase activity. The primary mutations in atp1-1 and atp1-2 were identified as Thr(383) --> Ile and Gly(291) --> Asp, respectively. From recent structural data, position 383 lies within the catalytic site. Position 291 is located near the region affecting subunit-subunit interaction with the F(1)beta-subunit. An unlinked suppressor gene, ASC1 (alpha-subunit complementing) of the atp1-2 mutation (Gly(291) --> Asp) restored the growth defect phenotype on glycerol, but did not suppress either atp1-1 or the deletion mutant Deltaatp1. Sequence analysis revealed that ASC1 was allelic with RAS2, a G-protein growth regulator. The introduction of ASC1/RAS2 into the atp1-2 mutant increased the F(1)-ATPase enzyme activity in this mutant when the transformant was grown on glycerol. The possible mechanisms of ASC1/RAS2 suppression of atp1-2 are discussed; we suggest that RAS2 is part of the regulatory circuit involved in the control of F(1)-ATPase subunit levels in mitochondria.     

A protein kinase target of a PDK1 signalling pathway is involved in root hair growth in Arabidopsis

Here we report on a lipid-signalling pathway in plants that is downstream of phosphatidic acid and involves the Arabidopsis protein kinase, AGC2-1, regulated by the 3'-phosphoinositide-dependent kinase-1 (AtPDK1). AGC2-1 specifically interacts with AtPDK1 through a conserved C-terminal hydrophobic motif that leads to its phosphorylation and activation, whereas inhibition of AtPDK1 expression by RNA interference abolishes AGC2-1 activity. Phosphatidic acid specifically binds to AtPDK1 and stimulates AGC2-1 in an AtPDK1-dependent manner. AtPDK1 is ubiquitously expressed in all plant tissues, whereas expression of AGC2-1 is abundant in fast-growing organs and dividing cells, and activated during re-entry of cells into the cell cycle after sugar starvation-induced G1-phase arrest. Plant hormones, auxin and cytokinin, synergistically activate the AtPDK1-regulated AGC2-1 kinase, indicative of a role in growth and cell division. Cellular localisation of GFP-AGC2-1 fusion protein is highly dynamic in root hairs and at some stages confined to root hair tips and to nuclei. The agc2-1 knockout mutation results in a reduction of root hair length, suggesting a role for AGC2-1 in root hair growth and development.     

RelA functionally suppresses the growth defect caused by a mutation in the G domain of the essential Der protein

A unique bacterial GTPase, Der, containing two tandem GTP-binding domains, is essential for cell growth and plays a crucial role in a large ribosomal subunit in Escherichia coli. The depletion of Der resulted in accumulation of both large and small ribosomal subunits and also affected the stability of large ribosomal subunits. However, its exact cellular function still remains elusive. Previously, we have shown that two G domain mutants, DerN118D and DerN321D, cannot support cell growth at low temperatures, suggesting that both GTP-binding domains are indispensable. In this study, we show that both Der variants are defective in ribosome biogenesis. Genetic screening of an E. coli genomic library was performed to identify the genes which, when expressed from a multicopy plasmid, can restore the growth defect of the DerN321D mutant at restrictive temperatures. Among seven suppressors isolated, four were located at 62.7 min on the E. coli genomic map, and the gene responsible for the suppression of DerN321D was identified as the relA gene which encodes a ribosome-associated (p)ppGpp synthetase. The synthetic activity of RelA was found to be essential for its DerN321D suppressor activity. Overexpression of RelA in a suppressor strain did not affect the expression of DerN321D but suppressed the polysome defects caused by the DerN321D mutant. This is the first demonstration of suppression of impaired function of Der by a functional enzyme. A possible mechanism of the suppression of DerN321D by RelA overproduction is discussed.