Hepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitination

Ferroportin exports iron into plasma from absorptive enterocytes, erythrophagocytosing macrophages, and hepatic stores. The hormone hepcidin controls cellular iron export and plasma iron concentrations by binding to ferroportin and causing its internalization and degradation. We explored the mechanism of hepcidin-induced endocytosis of ferroportin, the key molecular event in systemic iron homeostasis. Hepcidin binding caused rapid ubiquitination of ferroportin in cell lines overexpressing ferroportin and in murine bone marrow-derived macrophages. No hepcidin-dependent ubiquitination was observed in C326S ferroportin mutant which does not bind hepcidin. Substitutions of lysines between residues 229 and 269 in the third cytoplasmic loop of ferroportin prevented hepcidin-dependent ubiquitination and endocytosis of ferroportin, and promoted cellular iron export even in the presence of hepcidin. The human ferroportin mutation K240E, previously associated with clinical iron overload, caused hepcidin resistance in vitro by interfering with ferroportin ubiquitination. Our study demonstrates that ubiquitination is the functionally relevant signal for hepcidin-induced ferroportin endocytosis.     

Phosphatidic acid induces leaf cell death in Arabidopsis by activating the Rho-related small G protein GTPase-mediated pathway of reactive oxygen species generation

Phosphatidic acid (PA) level increases during various stress conditions. However, the physiological roles of this lipid in stress response remain largely unknown. In this study, we report that PA induced leaf cell death and elevated the levels of reactive oxygen species (ROS) in the whole leaf and single cells. To further elucidate the mechanism of PA-induced cell death, we then examined whether Rho-related small G protein (ROP) 2, which enhanced ROS production in an in vitro assay, is involved in PA-induced ROS production and cell death. In response to PA, transgenic leaves of Arabidopsis expressing a constitutively active rop2 mutant exhibited earlier cell death and higher levels of ROS than wild type (WT), whereas those expressing a dominant-negative rop2 mutant exhibited later cell death and lower ROS. However, in the absence of exogenous PA, no spontaneous cell death or elevated ROS was observed in constitutively active rop2 plants, suggesting that the activation of ROP GTPase alone is insufficient to activate the ROP-mediated ROS generation pathway. These results suggest that PA modulates an additional factor required for the active ROP-mediated ROS generation pathway. Therefore, PA may be an important regulator of ROP-regulated ROS generation and the cell death process during various stress and defense responses of plants.     

Inhibition of Iron Uptake Is Responsible for Differential Sensitivity to V-ATPase Inhibitors in Several Cancer Cell Lines

Many cell lines derived from tumors as well as transformed cell lines are far more sensitive to V-ATPase inhibitors than normal counterparts. The molecular mechanisms underlying these differences in sensitivity are not known. Using global gene expression data, we show that the most sensitive responses to HeLa cells to low doses of V-ATPase inhibitors involve genes responsive to decreasing intracellular iron or decreasing cholesterol and that sensitivity to iron uptake is an important determinant of V-ATPase sensitivity in several cancer cell lines. One of the most sensitive cell lines, melanoma derived SK-Mel-5, over-expresses the iron efflux transporter ferroportin and has decreased expression of proteins involved in iron uptake, suggesting that it actively suppresses cytoplasmic iron. SK-Mel-5 cells have increased production of reactive oxygen species and may be seeking to limit additional production of ROS by iron.     

Biological effects of mutant ceruloplasmin on hepcidin-mediated internalization of ferroportin

Ceruloplasmin plays an essential role in cellular iron efflux by oxidizing ferrous iron exported from ferroportin. Ferroportin is posttranslationally regulated through internalization triggered by hepcidin binding. Aceruloplasminemia is an autosomal recessive disorder of iron homeostasis resulting from mutations in the ceruloplasmin gene. The present study investigated the biological effects of glycosylphosphatidylinositol (GPI)-linked ceruloplasmin on the hepcidin-mediated internalization of ferroportin. The prevention of hepcidin-mediated ferroportin internalization was observed in the glioma cells lines expressing endogenous ceruloplasmin as well as in the cells transfected with GPI-linked ceruloplasmin under low levels of hepcidin. A decrease in the extracellular ferrous iron by an iron chelator and incubation with purified ceruloplasmin in the culture medium prevented hepcidin-mediated ferroportin internalization, while the reconstitution of apo-ceruloplasmin was not able to prevent ferroportin internalization. The effect of ceruloplasmin on the ferroportin stability was impaired due to three distinct properties of the mutant ceruloplasmin: namely, a decreased ferroxidase activity, the mislocalization in the endoplasmic reticulum, and the failure of copper incorporation into apo-ceruloplasmin. Patients with aceruloplasminemia exhibited low serum hepcidin levels and a decreased ferroportin protein expression in the liver. The in vivo findings supported the notion that under low levels of hepcidin, mutant ceruloplasmin cannot stabilize ferroportin because of a loss-of-function in the ferroxidase activity, which has been reported to play an important role in the stability of ferroportin. The properties of mutant ceruloplasmin regarding the regulation of ferroportin may therefore provide a therapeutic strategy for aceruloplasminemia patients.     

Cytochrome c Trp65Ser substitution results in inhibition of acetic acid-induced programmed cell death in Saccharomyces cerevisiae

To gain further insight into the role of cytochrome c (cyt c) in yeast programmed cell death induced by acetic acid (AA-PCD), comparison was made between wild type and two mutant cells, one lacking cyt c and the other (W65Scyc1) expressing a mutant iso-1-cyt c in a form unable to reduce cyt c oxidase, with respect to occurrence of AA-PCD, cyt c release, ROS production and caspase-like activity. We show that in W65Scyc1 cells: i. no release of mutant cyt c occurs with inhibition of W65Scyc1 cell AA-PCD shown to be independent on impairment of electron flow, ii. there is a decrease in ROS production and an increase in caspase-like activity. We conclude that cyt c release does not depend on cyt c function as an electron carrier and that when still associated to the mitochondrial membrane, cyt c in its reduced form has a role in AA-PCD, by regulating ROS production and caspase-like activity.     

Reactive oxygen species induced by Streptococcus pyogenes invasion trigger apoptotic cell death in infected epithelial cells

Streptococcus pyogenes (group A streptococcus, GAS), one of the most common pathogens of humans, attaches and invades into human pharyngeal or skin epithelial cells. We have previously reported that induction of apoptosis is associated with GAS invasion, which induces mitochondrial dysfunction and apoptotic cell death. We demonstrate here that GAS‐induced apoptosis is mediated by reactive oxygen species (ROS) production. Both the induction of apoptosis and ROS production markedly increased upon invasion of wild‐type GAS strain JRS4 into HeLa cells; however, the apoptotic response was not observed in fibronectin‐binding protein F1‐disrupted mutant SAM1‐infected cells. In Bcl‐2‐overexpressing HeLa cells (HBD98‐2‐4), the induction of apoptosis, ROS production and mitochondrial dysfunction were significantly suppressed, whereas the numbers of invaded GAS was not different between HeLa (mock cells) and the HeLa HBD98‐2‐4 cells. Whereas Rac1 activation occurred during GAS invasion, ROS production in GAS‐infected cells was clearly inhibited by transfection with the Rac1 mutants (L37 or V12L37), but not by the dominant active mutant (V12L61) or by the dominant negative mutant (N17). These observations indicate that GAS invasion triggers ROS production through Rac1 activation and generated ROS induced mitochondrial dysfunction leading to cellular apoptosis.     

Dickeya dadantii pectic enzymes necessary for virulence are also responsible for activation of the Arabidopsis thaliana innate immune system

Soft‐rot diseases of plants attributed to Dickeya dadantii result from lysis of the plant cell wall caused by pectic enzymes released by the bacterial cell by a type II secretion system (T2SS). Arabidopsis thaliana can express several lines of defence against this bacterium. We employed bacterial mutants with defective envelope structures or secreted proteins to examine early plant defence reactions. We focused on the production of AtrbohD‐dependent reactive oxygen species (ROS), callose deposition and cell death as indicators of these reactions. We observed a significant reduction in ROS and callose formation with a bacterial mutant in which genes encoding five pectate lyases (Pels) were disrupted. Treatment of plant leaves with bacterial culture filtrates containing Pels resulted in ROS and callose production, and both reactions were dependent on a functional AtrbohD gene. ROS and callose were produced in response to treatment with a cellular fraction of a T2SS‐negative mutant grown in a Pels‐inducing medium. Finally, ROS and callose were produced in leaves treated with purified Pels that had also been shown to induce the expression of jasmonic acid‐dependent defence genes. Pel catalytic activity is required for the induction of ROS accumulation. In contrast, cell death observed in leaves infected with the wild‐type strain appeared to be independent of a functional AtrbohD gene. It was also independent of the bacterial production of pectic enzymes and the type III secretion system (T3SS). In conclusion, the work presented here shows that D. dadantii is recognized by the A. thaliana innate immune system through the action of pectic enzymes secreted by bacteria at the site of infection. This recognition leads to AtrbohD‐dependent ROS and callose accumulation, but not cell death.     

Regulation of mitochondrial NAD pool via NAD+ transporter 2 is essential for matrix NADH homeostasis and ROS production in Arabidopsis

Reactive oxygen species(ROS) play a crucial role in numerous biological processes in plants, including development, responses to environmental stimuli, and programmed cell death(PCD). Deficiency in MOSAIC DEATH 1(MOD1), a plastid-localized enoyl-ACP reductase essential for de novo fatty acid biosynthesis in Arabidopsis thaliana, leads to the increased malate export from chloroplasts to mitochondria, and the subsequent accumulation of mitochondria-generated ROS and PCD. In this study, we report the identification and characterization of a mod1 suppressor, som592. SOM592 encodes mitochondrion-localized NAD~+ transporter 2(NDT2). We show that the mitochondrial NAD pool is elevated in the mod1 mutant. The som592 mutation fully suppressed mitochondrial NADH hyper-accumulation, ROS production, and PCD in the mod1 mutant, indicating a causal relationship between mitochondrial NAD accumulation and ROS/PCD phenotypes. We also show that in wild-type plants, the mitochondrial NAD+uptake is involved in the regulation of ROS production in response to continuous photoperiod. Elevation of the alternative respiration pathway can suppress ROS accumulation and PCD in mod1, but leads to growth restriction. These findings uncover a regulatory mechanism for mitochondrial ROS production via NADH homeostasis in Arabidopsis thaliana that is likely important for growth regulation in response to altered photoperiod.     

Gelsolin, but not its cleavage, is required for TNF-induced ROS generation and apoptosis in MCF-7 cells

The tumor necrosis factor (TNF) can induce apoptosis in many cells including MCF-7 cells. To identify the genes responsible for TNF-induced apoptosis, we generated a series of TNF-resistant MCF-7 cell lines by employing retrovirus insertion-mediated random mutagenesis. In one of the resistant lines, gelsolin was found to be disrupted by viral insertion. Exogenous expression of gelsolin in this mutant cell line (Gel^mut) restored the sensitivity to TNF-induced cell death and knock-down of gelsolin by siRNA conferred MCF-7 cells with resistance to TNF, indicating that gelsolin is required for TNF-induced apoptosis. Interestingly, the resistance of Gel^mut cells to apoptosis induction is selective to TNF, since Gel^mut and wild-type cells showed similar sensitivity to other death stimuli that were tested. Furthermore, TNF-induced ROS production in Gel^mut cells was significantly decreased, demonstrating that gelsolin-mediated ROS generation plays a crucial role in TNF-induced apoptosis in MCF-7 cells. Importantly, caspase-mediated gelsolin cleavage is dispensable for TNF-triggered ROS production and subsequent apoptosis of MCF-7 cells. Our study thus provides genetic evidence linking gelsolin-mediated ROS production to TNF-induced cell death.     

Diesel exhaust particles are mutagenic in FE1-MutaMouse lung epithelial cells

The particulate phase of diesel engine exhaust is likely carcinogenic. However, the mechanisms of diesel exhaust particles (DEPs) induced mutagenicity/carcinogenicity are still largely unknown. We determined the mutant frequency following eight repeated 72 h incubations with 37.5 or 75 μg/ml DEP (NIST SRM 1650) in the FE1-Muta™Mouse lung epithelial cell line. We measured DEP-induced acellular and intracellular production of reactive oxygen species (ROS) and compared with ROS production induced by carbon black, which we have previously shown is mutagenic in this cell line [N.R. Jacobsen, A.T. Saber, P. White, P. Moller, G. Pojana, U. Vogel, S. Loft, J. Gingerich, L. Soper, G.R. Douglas, H. Wallin. Increased mutant frequency by carbon black, but not quartz, in the lacZ and cII transgenes of muta(TM)mouse lung epithelial cells, Environ. Mol. Mutagen. 48(6) (2007) 451–461]. The mutant frequency was marginally elevated in cells treated with 37.5 μg/ml DEP (1.29-fold [95% CI: 0.96–1.60], p = 0.08) and significantly increased in cells treated with 75 μg/ml DEP (1.55-fold [95% CI: 1.23–1.87], p < 0.001). ROS production from DEP was low both within cells and in acellular systems when compared to carbon black. These results show that DEP are mutagenic in a mammalian cell line in vitro and that additional pathways besides ROS production, such as those involving the presence of polycyclic aromatic hydrocarbons, likely are involved in the mutagenesis.     

Tim18, a component of the mitochondrial translocator, mediates yeast cell death induced by arsenic

Evidence is presented that Tim18, a mitochondria translocase, plays a role in the previously described apoptosis induced by arsenite in Saccharomyces cerevisiae. Tim18 deletion mutant exhibited resistance to arsenite. After arsenite treatment, both the wild type and Tim18-deficient cells showed reactive oxygen species (ROS) production. Arsenite induced the higher expression of tim18 in wild type yeast cells. We found that the tim18 deletion mutant also exhibited resistance to other apoptotic stresses such as acetic acid, H2O2, and hyperosmotic stress. These results suggest that Tim18 is important for yeast cell death induced by arsenic, and it may act downstream of ROS production.     

Phosphatidylinositol 3-kinase-dependent membrane recruitment of Rac-1 and p47phox is critical for alpha-platelet-derived growth factor receptor-induced production of reactive oxygen species

Platelet-derived growth factor (PDGF) plays a critical role in the pathogenesis of proliferative diseases. NAD(P)H oxidase (Nox)-derived reactive oxygen species (ROS) are essential for signal transduction by growth factor receptors. Here we investigated the dependence of PDGF-AA-induced ROS production on the cytosolic Nox subunits Rac-1 and p47(phox), and we systematically evaluated the signal relay mechanisms by which the alphaPDGF receptor (alphaPDGFR) induces ROS liberation. Stimulation of the alphaPDGFR led to a time-dependent increase of intracellular ROS levels in fibroblasts. Pharmacological inhibitor experiments and enzyme activity assays disclosed Nox as the source of ROS. alphaPDGFR activation is rapidly followed by the translocation of p47(phox) and Rac-1 from the cytosol to the cell membrane. Experiments performed in p47(phox)(-/-) cells and inhibition of Rac-1 or overexpression of dominant-negative Rac revealed that these Nox subunits are required for PDGF-dependent Nox activation and ROS liberation. To evaluate the signaling pathway mediating PDGF-AA-dependent ROS production, we investigated Ph cells expressing mutant alphaPDGFRs that lack specific binding sites for alphaPDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase (PI3K), phospholipase Cgamma, and SHP-2). Lack of PI3K signaling (but not Src, phospholipase Cgamma, or SHP-2) completely abolished PDGF-dependent p47(phox) and Rac-1 translocation, increase of Nox activity, and ROS production. Conversely, a mutant alphaPDGFR able to activate only PI3K was sufficient to mediate these subcellular events. Furthermore, the catalytic PI3K subunit p110alpha (but not p110beta) was identified as the crucial isoform that elicits alphaPDGFR-mediated production of ROS. Finally, bromodeoxyuridine incorporation and chemotaxis assays revealed that the lack of ROS liberation blunted PDGF-AA-dependent chemotaxis but not cell cycle progression. We conclude that PI3K/p110alpha mediates growth factor-dependent ROS production by recruiting p47(phox) and Rac-1 to the cell membrane, thereby assembling the active Nox complex. ROS are required for PDGF-AA-dependent chemotaxis but not proliferation.     

The iron exporter ferroportin/Slc40a1 is essential for iron homeostasis

Ferroportin (SLC40A1) is an iron transporter postulated to play roles in intestinal iron absorption and cellular iron release. Hepcidin, a regulatory peptide, binds to ferroportin and causes it to be internalized and degraded. If ferroportin is the major cellular iron exporter, ineffective hepcidin function could explain manifestations of human hemochromatosis disorders. To investigate this, we inactivated the murine ferroportin (Fpn) gene globally and selectively. Embryonic lethality of Fpn(null/null) animals indicated that ferroportin is essential early in development. Rescue of embryonic lethality through selective inactivation of ferroportin in the embryo proper suggested that ferroportin has an important function in the extraembryonic visceral endoderm. Ferroportin-deficient animals accumulated iron in enterocytes, macrophages, and hepatocytes, consistent with a key role for ferroportin in those cell types. Intestine-specific inactivation of ferroportin confirmed that it is critical for intestinal iron absorption. These observations define the major sites of ferroportin activity and give insight into hemochromatosis.     

Impact of D181V and A69T on the function of ferroportin as an iron export pump and hepcidin receptor

Mutations in the only known mammalian iron exporter ferroportin cause a rare iron overload disorder termed ferroportin disease. Two distinct clinical phenotypes are caused by different disease mechanisms: mutations in ferroportin either cause loss of iron export function or gain of function due to resistance to hepcidin, the peptide hormone that normally downregulates ferroportin. The aim of the present study was to examine the disease mechanisms of the thus far unclassified A69T and D181V ferroportin mutations. We overexpressed wild-type and mutant ferroportin fused to green fluorescent protein in human embryonic kidney cells and used a ⁵⁹Fe-assay, intracellular ferritin concentrations, confocal microscopy and flow cytometry to study iron export function, subcellular localization and the responsiveness to hepcidin. While the A69T ferroportin mutation seems not to affect the iron export function it causes dose-dependent hepcidin resistance. We further found that D181V mutated ferroportin is iron export defective and hepcidin resistant, similar to the loss of function mutations A77D and C367X. This indicates that intact iron export might be necessary for hepcidin-induced downregulation of ferroportin. This hypothesis was investigated by studying the hepcidin response under modulation of iron availability. Incubation of wild-type ferroportin overexpressing cells with holo-transferrin increases the hepcidin effect whereas chelating extracellular ferrous iron causes hepcidin resistance. In this study we present data that postulates to classify the D181V ferroportin mutation as loss of function and the A69T mutation as dose-dependent hepcidin resistant and outline a possible causal link between iron export function and the hepcidin effect.     

Effects of inhibiting CoQ10 biosynthesis with 4-nitrobenzoate in human fibroblasts

Coenzyme Q(10) (CoQ(10)) is a potent lipophilic antioxidant in cell membranes and a carrier of electrons in the mitochondrial respiratory chain. We previously characterized the effects of varying severities of CoQ(10) deficiency on ROS production and mitochondrial bioenergetics in cells harboring genetic defects of CoQ(10) biosynthesis. We observed a unimodal distribution of ROS production with CoQ(10) deficiency: cells with <20% of CoQ(10) and 50-70% of CoQ(10) did not generate excess ROS while cells with 30-45% of CoQ(10) showed increased ROS production and lipid peroxidation. Because our previous studies were limited to a small number of mutant cell lines with heterogeneous molecular defects, here, we treated 5 control and 2 mildly CoQ(10) deficient fibroblasts with varying doses of 4-nitrobenzoate (4-NB), an analog of 4-hydroxybenzoate (4-HB) and inhibitor of 4-para-hydroxybenzoate:polyprenyl transferase (COQ2) to induce a range of CoQ(10) deficiencies. Our results support the concept that the degree of CoQ(10) deficiency in cells dictates the extent of ATP synthesis defects and ROS production and that 40-50% residual CoQ(10) produces maximal oxidative stress and cell death.     

Serum hepcidin / ferroportin levels in bipolar disorder and schizophrenia

BackgroundDespite several alternatives for cellular iron influx, the only mechanism for cellular iron efflux is ferroportin mediated active transport. In cases of ferroportin dysfunction, iron accumulates in the cell and causes ferroptosis. Hepcidin suppresses ferroportin levels and inflammatory activation increases hepcidin production. Mild inflammation in schizophrenia and bipolar disorder may alter hepcidin and ferroportin.MethodsThe study included a total of 137 patients aged 18–65 years, 57 diagnosed with schizophrenia and 80 with bipolar disorder, according to the DSM-IV diagnostic criteria, and a control group (HC) of 42 healthy individuals. Biochemical analyses, thyroid function tests, hemogram, serum iron level, iron-binding capacity, and ferritin levels were examined. Serum levels of hepcidin and ferroportin were measured with enzyme-linked immunosorbent assay (ELISA) method.ResultsA statistically significant difference was determined between the groups in terms of the serum ferroportin levels (F = 15.69, p < 0.001). Post-hoc analyses showed that the schizophrenia group had higher ferroportin levels than in the bipolar group (p < 0.001) and HCs (p < 0.001). Hepcidin levels did not differ between the groups. Chlorpromazine equivalent doses of antipsychotics correlated with ferroportin levels (p = 0.024).ConclusionFerroportin levels were increased in the schizophrenia group, although iron and hepcidin levels were within normal ranges. Antipsychotics may alter the mechanisms which control ferroportin levels. Further studies are needed to examine the relationships between antipsychotics and iron metabolism for determination of causal relationship.     

Rac1-NADPH oxidase-regulated generation of reactive oxygen species mediates glutamate-induced apoptosis in SH-SY5Y human neuroblastoma cells

Reactive oxygen species (ROS) are known to play an important role in glutamate-induced neuronal cell death. In the present study, we examined whether NADPH oxidase serves as a source of ROS production and plays a role in glutamate-induced cell death in SH-SY5Y human neuroblastoma cells. Stimulation of the cells with glutamate (100 mM) induced apoptotic cell death and increase in the level of ROS, and these effects of glutamate were significantly suppressed by the inhibitors of the NADPH oxidase, diphenylene iodonium, apocynin, and neopterine. In addition, RT-PCR revealed that SH-SY5Y cells expressed mRNA of gp91^phox, p22^phox and cytosolic p47^phox, p67^phox and p40^phox, the components of the plasma membrane NADPH oxidase. Treatment with glutamate also resulted in activation and translocation of Rac1 to the plasma membrane. Moreover, the expression of Rac1N17, a dominant negative mutant of Rac1, significantly blocked the glutamate-induced ROS generation and cell death. Collectively, these results suggest that the plasma membrane-bound NADPH oxidase complex may play an essential role in the glutamate-induced apoptotic cell death through increased production of ROS.     

The mammalian Golgi regulates numb signaling in asymmetric cell division by releasing ACBD3 during mitosis

Mammalian neural progenitor cells divide asymmetrically to self-renew and produce a neuron by segregating cytosolic Numb proteins primarily to one daughter cell. Numb signaling specifies progenitor over neuronal fates but, paradoxically, also promotes neuronal differentiation. Here we report that ACBD3 is a Numb partner in cell-fate specification. ACBD3 and Numb proteins interact through an essential Numb domain, and the respective loss- and gain-of-function mutant mice share phenotypic similarities. Interestingly, ACBD3 associates with the Golgi apparatus in neurons and interphase progenitor cells but becomes cytosolic after Golgi fragmentation during mitosis, when Numb activity is needed to distinguish the two daughter cells. Accordingly, cytosolic ACBD3 can act synergistically with Numb to specify cell fates, and its continuing presence during the progenitor cell cycle inhibits neuron production. We propose that Golgi fragmentation and reconstitution during cell cycle differentially regulate Numb signaling through changes in ACBD3 subcellular distribution and represent a mechanism for coupling cell-fate specification and cell-cycle progression.     

NADPH oxidase-dependent reactive oxygen species formation required for root hair growth depends on ROP GTPase

Reactive oxygen species (ROS) production by an NADPH oxidase (NOX) encoded by AtrbohC/RHD2 is required for root hair growth in Arabidopsis thaliana. ROP (RHO of plants) GTPases are also required for normal root hair growth and have been proposed to regulate ROS production in plants. Therefore, the role of ROP GTPase in NOX-dependent ROS formation by root hairs was investigated. Plants overexpressing wild-type ROP2 (ROP2 OX), constitutively active (CA-rop2), or dominant negative (DN-rop2) rop2 mutant proteins were used. Superoxide formation by root hairs was detected by superoxide dismutase-sensitive nitroblue tetrazolium reduction, and ROS production in the root hair differentiation zone was detected by dihydrofluorescein diacetate oxidation. Both probes showed that ROS production was increased in ROP2 OX and CA-rop2 plants, and decreased in DN-rop2 plants, relative to wild-type plants. When CA-rop2 was expressed in the NOX loss-of-function rhd2-1 mutant, ROS formation and root hair growth were impaired, suggesting that RHD2 is required for this ROP2-dependent ROS formation.