Biliverdin-promoted lateral root formation is mediated through heme oxygenase in rice

In this study, we examined the effect of biliverdin (BV), a product of heme oxygenase (HO) catalyzed reaction, on lateral root (LR) formation in rice. Treatment with BV induced LR formation and HO activity. As well, BV, could induce OsHO1 mRNA expression. Zn protoporphyrin IX (the specific inhibitor of HO) reduced LR number, HO activity and OsHO1 mRNA level induced by BV. Our data suggest that HO is required for BV-induced LR formation in rice.     

Cobalt chloride-induced lateral root formation in rice: The role of heme oxygenase

Lateral roots (LRs) perform the essential tasks of providing water, nutrients, and physical support to plants. Therefore, understanding the regulation of LR development is of agronomic importance. Recent findings suggest that heme oxygenase (HO) plays an important role in LR development. In this study, we examined the effect of cobalt chloride (CoCl₂) on LR formation and HO expression in rice. Treatment with CoCl₂ induced LR formation and HO activity. We further observed that CoCl₂ could induce the expression of OsHO1 but not OsHO2. CoCl₂-increased HO activity occurred before LR formation. Zinc protoporphyrin IX (ZnPPIX, the specific inhibitor of HO) and hemoglobin (the carbon monoxide/nitric oxide scavenger) reduced LR formation, HO activity, and OsHO1 expression. Application of biliverdin, a product of HO-catalyzed reaction, to CoCl₂-treated rice seedlings reversed the ZnPPIX-inhibited LR formation and ZnPPIX-decreased HO activity. CoCl₂ had no effect on H₂O₂ content and nitric oxide production. Moreover, application of ascorbate, a H₂O₂ scavenger, failed to affect CoCl₂-promoted LR formation and HO activity. It is concluded that HO is required for CoCl₂-promoted LR formation in rice.     

Nitric oxide mediates strigolactone signaling in auxin and ethylene-sensitive lateral root formation in sunflower seedlings

Strigolactones (SLs) play significant role in shaping root architecture whereby auxin-SL crosstalk has been observed in SL-mediated responses of primary root elongation, lateral root formation and adventitious root (AR) initiation. Whereas GR24 (a synthetic strigolactone) inhibits LR and AR formation, the effect of SL biosynthesis inhibitor (fluridone) is just the opposite (root proliferation). Naphthylphthalamic acid (NPA) leads to LR proliferation but completely inhibits AR development. The diffusive distribution of PIN1 in the provascular cells in the differentiating zone of the roots in response to GR24, fluridone or NPA treatments further indicates the involvement of localized auxin accumulation in LR development responses. Inhibition of LR formation by GR24 treatment coincides with inhibition of ACC synthase activity. Profuse LR development by fluridone and NPA treatments correlates with enhanced [Ca²⁺]_cyt in the apical region and differentiating zones of LR, indicating a critical role of [Ca²⁺] in LR development in response to the coordinated action of auxins, ethylene and SLs. Significant enhancement of carotenoid cleavage dioxygenase (CCD) activity (enzyme responsible for SL biosynthesis) in tissue homogenates in presence of cPTIO (NO scavenger) indicates the role of endogenous NO as a negative modulator of CCD activity. Differences in the spatial distribution of NO in the primary and lateral roots further highlight the involvement of NO in SL-modulated root morphogenesis in sunflower seedlings. Present work provides new report on the negative modulation of SL biosynthesis through modulation of CCD activity by endogenous nitric oxide during SL-modulated LR development.     

Methyl jasmonate-induced lateral root formation in rice: The role of heme oxygenase and calcium

Lateral roots (LRs) play important roles in increasing the absorptive capacity of roots as well as to anchor the plant in the soil. Therefore, understanding the regulation of LR development is of agronomic importance. In this study, we examined the effect of methyl jasmonate (MJ) on LR formation in rice. Treatment with MJ induced LR formation and heme oxygenase (HO) activity. As well, MJ could induce OsHO1 mRNA expression. Zinc protoporphyrin IX (the specific inhibitor of HO) and hemoglobin [the carbon monoxide/nitric oxide (NO) scavenger] reduced LR formation, HO activity and OsHO1 expression. LR formation and HO activity induced by MJ was reduced by the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-oxide. The effects of Ca²⁺ chelators, Ca²⁺-channel inhibitors, and calmodulin (CaM) antagonists on LR formation induced by MJ were also examined. All these inhibitors were effective in reducing the action of MJ. However, Ca²⁺ chelators and Ca²⁺ channel inhibitors induced HO activity when combining with MJ further. It is concluded that Ca²⁺ may regulate MJ action mainly through CaM-dependent mechanism.     

Nitric oxide and prostaglandin E2 participate in lipopolysaccharide/interferon-gamma-induced heme oxygenase 1 and prevent RAW264.7 macrophages from UV-irradiation-induced cell death

Induction of heme oxygenase (HO)-1 during inflammation has been demonstrated in many cell types, but the contribution of inflammatory molecules nitric oxide (NO) and prostaglandin E(2) (PGE(2)) has remained unresolved. Here we show that NO donors including sodium nitroprusside (SNP) and spermine nonoate (SP-NO), and PGE(2) significantly stimulate HO-1 expression in RAW264.7 macrophages, associated with alternative induction on NO and PGE(2) in medium, respectively. NO donors also show the inductive effect on cyclo-oxygenase 2 protein and PGE(2) production. In the presence of lipopolysaccharide and interferon-gamma (LPS/IFN-gamma), HO-1 protein was induced slightly but significantly, and SNP, SP-NO, and PGE(2) enhanced HO-1 protein induced by LPS/IFN-gamma. L-Arginine analogs N-nitro-L-arginine methyl ester (L-NAME) and N-nitro-L-arginine (NLA) significantly block HO-1 protein induced by LPS/IFN-gamma associated with a decrease in NO (not PGE(2)) production. And, NSAIDs aspirin and diclofenase dose dependently inhibited LPS/IFN-gamma-induced HO-1 protein accompanied by suppression of PGE(2) (not NO) production. PD98059 (a specific inhibitor of MEKK), but not SB203580 (a specific inhibitor of p38 kinase), attenuated PGE(2) (not SP-NO) induced HO-1 protein. Under UVC (100 J/m(2)) and UVB (50 J/m(2)) irradiation, PGE(2) or SP-NO treatment prevents cells from UVC or UVB-induced cell death, and HO-1 inhibitor tin protoporphyrin (SnPP) reverses the preventive effects of PGE(2) and SP-NO. The protective activity induced by PGE(2) on UVC or UVB irradiation-induced cell death was blocked by MAPK inhibitor PD98059 (not SB203580). These results demonstrated that inflammatory molecules NO and PGE(2) were potent inducers of HO-1 gene, and protected cells from UV-irradiation-induced cell death through HO-1 induction.     

Differential heme oxygenase induction by stannous and stannic ions in the heart

Heme oxygenase is the rate-limiting enzyme in heme catabolism, and is induced by oxidative stress, foreign and endogenous chemicals, and many trace elements and heavy metals. This study examined the effect of the oxidative state of the heavy metal tin, on heme oxygenase-1 induction in cardiac tissue. Subcutaneous administration of stannous and stannic chloride failed to induce the enzyme in this tissue. Atomic absorption spectroscopy revealed the absence of tin in the heart cells. Investigation of several metal formulations showed that both stannous and stannic citrate were able to enter the bloodstream from the injection site and into heart tissue. Northern blot analysis revealed that heme oxygenase-1 mRNA was elevated several-fold in rat hearts from animals which received either stannous or stannic citrate, and that mRNA levels corresponded with the increase in enzyme activity. The presence of citrate facilitated the transport of the tin ion into the blood stream and possibly across cardiac cell membrane. The stannous ion was more potent as an inducer of heme oxygenase than was the stannic ion.     

Guanosine stimulates neurite outgrowth in PC12 cells via activation of heme oxygenase and cyclic GMP

Undifferentiated rat pheochromocytoma (PC12) cells extend neurites when cultured in the presence of nerve growth factor (NGF). Extracellular guanosine synergistically enhances NGF-dependent neurite outgrowth. We investigated the mechanism by which guanosine enhances NGF-dependent neurite outgrowth. Guanosine administration to PC12 cells significantly increased guanosine 3,5-cyclic monophosphate (cGMP) within the first 24 h whereas addition of soluble guanylate cyclase (sGC) inhibitors abolished guanosine-induced enhancement of NGF-dependent neurite outgrowth. sGC may be activated either by nitric oxide (NO) or by carbon monoxide (CO). -Nitro-l-arginine methyl ester (l-NAME), a non-isozyme selective inhibitor of nitric oxide synthase (NOS), had no effect on neurite outgrowth induced by guanosine. Neither nNOS (the constitutive isoform), nor iNOS (the inducible isoform) were expressed in undifferentiated PC12 cells, or under these treatment conditions. These data imply that NO does not mediate the neuritogenic effect of guanosine. Zinc protoporphyrin-IX, an inhibitor of heme oxygenase (HO), reduced guanosine-dependent neurite outgrowth but did not attenuate the effect of NGF. The addition of guanosine plus NGF significantly increased the expression of HO-1, the inducible isozyme of HO, after 12 h. These data demonstrate that guanosine enhances NGF-dependent neurite outgrowth by first activating the constitutive isozyme HO-2, and then by inducing the expression of HO-1, the enzymes responsible for CO synthesis, thus stimulating sGC and increasing intracellular cGMP.     

性激素对血红素氧化酶在大鼠前列腺腹侧叶表达的影响

血红素氧化酶(heme oxygenase,HO)是产生内源性一氧化碳(carbon monoxide,CO)的限速酶,最近发现内源性CO在调节平滑肌张力方面起重要作用。而人的良性前列腺增生(benign prostates hyperplasia,BPH)所致的膀胱出口梗阻与前列腺平滑肌张力有密切关系,但还不清楚内源性HO/CO系统是否介导了前列腺平滑肌的活动。为了观察性激素对大鼠前列腺腹侧叶中血红素氧化酶-1(heme oxygenase-1,HO-1)和血红素氧化酶-2(heme oxygenase-2,HO-2)基因表达的影响,我们采用睾丸切除术建立雄性SD大鼠去势模型,用RT-PCR方法观察HO-1和HO-2的转录水平,应用免疫组织化学结合图像分析技术,观察去势、外源性雄激素和雌激素对前列腺腹侧叶中HO—1和HO-2蛋白水平的影响。结果表明,HO-1和HO-2在正常大鼠前列腺腹侧叶中都有表达,腺上皮细胞和纤维平滑肌间质呈现HO-1的免疫活性,HO-2的免疫染色仅在腺上皮细胞内检测到;去势组HO-1的mRNA和蛋白表达水平显著低于正常对照组(P<0.01):外源性给予雄激素组和雌激素组的HO-1表达水平明显增高(P<0.01),且雌激素主要增加前列腺纤维平滑肌间质的HO-1表达:HO-2在各组间的表达无明显差异(P>0.05)。这些结果提示,性激素对HO-1有诱导作用,但对HO-2无明显的影响,因此推测一氧化碳-血红素氧化酶(CO—HO)     

Escherichia coli heme oxygenase modulates host innate immune responses

Induction of mammalian heme oxygenase (HO)‐1 and exposure of animals to carbon monoxide (CO) ameliorates experimental colitis. When enteric bacteria, including Escherichia coli, are exposed to low iron conditions, they express an HO‐like enzyme, chuS, and metabolize heme into iron, biliverdin and CO. Given the abundance of enteric bacteria residing in the intestinal lumen, our postulate was that commensal intestinal bacteria may be a significant source of CO and those that express chuS and other Ho‐like molecules suppress inflammatory immune responses through release of CO. According to real‐time PCR, exposure of mice to CO results in changes in enteric bacterial composition and increases E. coli 16S and chuS DNA. Moreover, the severity of experimental colitis correlates positively with E. coli chuS expression in IL‐10 deficient mice. To explore functional roles, E. coli were genetically modified to overexpress chuS or the chuS gene was deleted. Co‐culture of chuS‐overexpressing E. coli with bone marrow‐derived macrophages resulted in less IL‐12p40 and greater IL‐10 secretion than in wild‐type or chuS‐deficient E. coli. Mice infected with chuS‐overexpressing E. coli have more hepatic CO and less serum IL‐12 p40 than mice infected with chuS‐deficient E. coli. Thus, CO alters the composition of the commensal intestinal microbiota and expands populations of E. coli that harbor the chuS gene. These bacteria are capable of attenuating innate immune responses through expression of chuS. Bacterial HO‐like molecules and bacteria‐derived CO may represent novel targets for therapeutic intervention in inflammatory conditions.     

保护基因HO在组织细胞中的作用及其机制研究进展

血红素加氧酶(HO)是血红素降解过程中的限速酶,将血红素降解为胆绿素、CO和游离铁。HO有三种同工酶,HO—1为诱导型,而HO—2和HO—3呈结构性表达。HO—1是一种分布广泛的应激蛋白,具有抗炎、抗凋亡、抗增生效应。各组织细胞中HO—1受不同的应激而诱导,通过上调HO—1基因表达来防御由细胞因子诱导的氧化应激和凋亡。HO—1的细胞保护机制目前尚未明确,可能涉及CO、NO等信号分子,抗凋亡基因的表达,以及NF—κB与p38MAPK信号转导途径的介导。本文就HO在组织细胞中的作用及其可能的机制进行综述。     

Induction of heme oxygenase 1 in liver of spontaneously diabetic rats

It has been suggested that diabetes induces an increase in oxidative stress; the increased expression of heme-oxygenase 1 (HO-1) in liver is believed to be a sensitive marker of the stress response. The aim of this study was to examine whether diabetes is able to induce HO-1 expression in liver. The specific mRNA was amplified by RT/PCR and calibrated with amplified β-actin mRNA.

The mRNA HO-1 levels in the liver of spontaneously diabetic rats were increased by 1.8 fold compared with non diabetics; this supports the hypothesis of weak but significant oxidative damage due to chronic hyperglycaemia. This work represents the first in vivo study exploring the semi-quantitative expression of HO-1 in the liver of spontaneously diabetic rats.     

Dissociation and unfolding of inducible nitric oxide synthase oxygenase domain identifies structural role of tetrahydrobiopterin in modulating the heme environment

The oxygenase domain of the inducible nitric oxide synthase, Δ65 iNOSox is a dimer that binds heme, L-Arginine (L-Arg), and tetrahydrobiopterin (H₄B) and is the site for NO synthesis. The role of H₄B in iNOS structure-function is complex and its exact structural role is presently unknown. The present paper provides a simple mechanistic account of interaction of the cofactor tetrahydrobiopterin (H₄B) with the bacterially expressed Δ65 iNOSox protein. Transverse urea gradient gel electrophoresis studies indicated the presence of different conformers in the cofactor-incubated and cofactor-free Δ65 iNOSox protein. Dynamic Light Scattering (DLS) studies of cofactor-incubated and cofactor-free Δ65 iNOSox protein also showed two distinct populations of two different diameter ranges. Cofactor tetrahydrobiopterin (H₄B) shifted one population, with higher diameter, to the lower diameter ranges indicating conformational changes. The additional role played by the cofactor is to elevate the heme retaining capacity even in presence of denaturing stress. Together, these findings confirm that the H₄B is essential in modulating the iNOS heme environment and the protein environment in the dimeric iNOS oxygenase domain. (Mol Cell Boichem xxx: 1–10, 2005) Supported by Calcutta University Research Grants.     

NO和H2O2诱导大豆根尖和边缘细胞耐铝反应的作用

 NO和H₂O₂是参与植物抗非生物胁迫反应的重要信号分子, 为了确定NO和H₂O₂在大豆(Glycine max)根尖和根边缘细胞(root border cells, RBCs)耐铝反应中的作用及其相互关系, 以‘浙春3号’大豆为材料, 研究了铝毒胁迫下大豆根尖内源NO和H₂O₂的变化, 以及外源NO和H₂O₂诱导大豆根尖和RBCs的耐铝反应。结果表明, 50 μmol·L^–1 Al处理48 h显著抑制大豆根的伸长, 提高Al在根尖的积累, 同时显著增加根尖内源NO和H₂O₂含量。施加0.25 mmol·L^–1外源NO供体亚硝基铁氰化钠(Na₂[Fe(CN)₅NO]·2H₂O, sodium nitroprusside, SNP)和0.1 mmol·L^–1H₂O₂, 能有效地缓解Al对大豆根伸长的抑制、根尖Al积累和RBCs 的死亡, 该缓解作用可以被0.05 mmol·L^–1 NO清除剂2-(4- 羧基苯)-4,4,5,5- 四甲基咪唑-1- 氧-3- 氧化物, 钾盐(C₁₄H₁₆N₂O₄·K, carboxy-PTIO, cPTIO)和150 U·mL^–1 H₂O₂清除酶(catalase, CAT)逆转。并且外源NO能够显著促进根尖H₂O₂的积累, 而外源H₂O₂对根尖NO的含量无显著影响。这表明NO和H₂O₂是诱导大豆根尖及RBCs耐铝反应的两种信号分子, NO可能通过调控H₂O₂的形成, 进而诱导大豆根尖及RBCs的耐铝反应。     

Involvement of heme oxygenase as antioxidant defense in soybean nodules

Objective: We have previously demonstrated that the inducible form of heme oxygenase plays a critical role in protecting against oxidative stress in mammals. To gain further insight into the functions of this enzyme in plants, we have tested its activity and expression in soybean nodules subjected to cadmium (Cd) stress.

Materials and methods: Four-weeks-old soybean nodulated plants were treated with different cadmium chloride concentrations (0, 50 and 200 μM) during 48 h. Oxidative stress parameters such as TBARS content, GSH levels and antioxidant enzyme activities were measured as well as heme oxygenase activity and expression. Besides, the effect of biliverdin and Zn-protophorphyrin IX were analized.

Results: Treatment with 200 μM Cd during 48 h caused a 67% increase in TBARS content, whereas GSH decreased 44%, and total superoxide dismutase, gluthatione reductase and guaiacol peroxidase were also inhibited 54, 20 and 60%, respectively. A total of 200 μM Cd produced the overexpression of heme oxygenase-1, as well as a 10-fold enhancement of its activity. Co-administration of biliverdin (10 μM) completely prevented the effects caused by Cd. Treatment with Zn protoporphyrin IX, a strong inhibitor of heme oxygenase, expectedly decreased heme oxygenase-1 activity to half. When the inhibitor was given together with Cd, completely prevented the enzyme induction and oxidative stress parameters were significantly enhanced.

Conclusion: Taking together, these results are indicating that heme oxygenase plays a protective role against oxidative cell damage in soybean nodules.     

Cadmium stress suppresses lateral root formation by interfering with the root clock

A biological clock activated by oscillating signals, known as root clock, has been linked to lateral root (LR) formation and is essential for regular LR spacing along the primary root. However, it remains unclear how this internal mechanism is influenced by environmental factors known to affect the LR pattern. Here, we report that excessive cadmium (Cd) inhibits LR formation by disrupting the lateral root cap (LRC)‐programmed cell death (PCD)‐regulated root clock. Cd restricts the frequency of the oscillating signal rather than its amplitude. This could be attributed to the inhibition on meristematic activity by Cd, which resulted in decreased LRC cell number and LRC‐PCD frequency. Genetic evidence further showed that LRC cell number is positively correlated with root resistance to Cd. Our study reveals root cap dynamics as a novel mechanism mediating root responses to Cd, providing insight into the signalling pathways of the root clock responding to environmental cues.     

上调血红素氧合酶1对糖尿病心肌梗死大鼠心功能的远期影响

目的：研究上调血红素氧合酶1（HO-1）的表达对糖尿病心肌梗死大鼠心功能的影响及其机制。方法：将60只成年Wistar雄性大鼠随机分为5组（n=12）,分别为假手术组、糖尿病组、模型组、诱导剂组、抑制剂组。心梗造模后次日开始给药,1次／周,持续6周,术后28周,应用心脏超声、经颈动脉左心室内插管术等方法观察HO-1诱导剂钴原卟啉（CoPP）及HO活性抑制剂锡中卟啉（SnMP）干预后对心室重构及心功能各项指标的远期影响;测定血糖（GLU）、总胆红素（TB）、C反应蛋白（CRP）、血清肌酐（cr）、转氨酶（ALT）等指标;采用ELISA测定白介素-6（IL-6）、肿瘤坏死因子（TNE）、一氧化氮（NO）、前列环素（PG12）、脂联素、超敏CRP（HsCRP）等水平。结果：应用CoPP上调HO-1水平,能够改善糖尿病心梗大鼠左心室压力最大变化速率、左室射血分数、左室短轴缩短率,缩小左室舒张末期内径,升高血清胆红素、一氧化氮和前列环素水平,提高心肌组织磷酸化的内皮型一氧化氮合酶（peNOS）、磷酸化的活化蛋白激酶（pAkt）、磷酸化的腺苷活化的蛋白激酶（pAMPK）的表达,降低血清TNF-α、hs-CRP水平。使用SnMP后,能够阻断CoPP的上述作用。结论：上调HO-1通过peNOS、pAMPK途径能够长期地改善血管内皮功能,抑制炎症反应,提升血清胆红素等,有效抑制心室重塑,改善梗死后糖尿病大鼠远期的心功能。     

Curcumin-induced fibroblast apoptosis and in vitro wound contraction are regulated by antioxidants and heme oxygenase: implications for scar formation

Fibroblast apoptosis plays a crucial role in normal and pathological scar formation and therefore we studied whether the putative apoptosis-inducing factor curcumin affects fibroblast apoptosis and may function as a novel therapeutic. We show that 25-μM curcumin causes fibroblast apoptosis and that this could be inhibited by co-administration of antioxidants N -acetyl- l -cysteine (NAC), biliverdin or bilirubin, suggesting that reactive oxygen species (ROS) are involved. This is supported by our observation that 25-μM curcumin caused the generation of ROS, which could be completely blocked by addition of NAC or bilirubin. Since biliverdin and bilirubin are downstream products of heme degradation by heme oxygenase (HO), it has been suggested that HO-activity protects against curcumin-induced apoptosis. Interestingly, exposure to curcumin maximally induced HO-1 protein and HO-activity at 10–15 μM, whereas, at a concentration of >20-μM curcumin HO-1-expression and HO-activity was negligible. NAC-mediated inhibition of 25-μM curcumin-induced apoptosis was demonstrated to act in part via restored HO-1-induction, since the rescuing effect of NAC could be reduced by inhibiting HO-activity. Moreover pre-induction of HO-1 using 5-μM curcumin protected fibroblasts against 25-μM curcumin-induced apoptosis. On a functional level, fibroblast-mediated collagen gel contraction, an in vitro wound contraction model, was completely prevented by 25-μM curcumin, while this could be reversed by co-incubation with NAC, an effect that was also partially HO-mediated. In conclusion, curcumin treatment in high doses (>25 μM) may provide a novel way to modulate pathological scar formation through the induction of fibroblast apoptosis, while antioxidants, HO-activity and its effector molecules act as a possible fine-tuning regulator.     

水稻无侧根突变体生理生化变化的研究

对水稻无侧根突变体RM109,原品种大力及杂交后代F1三者的部分生理指标进行了比较。包括抗氧化酶(SOD、POD、CAT)和琥珀酸脱氢酶(SDH)的活性及叶绿素、丙二醛(MDA)、脯氨酸(Pro)等的含量,以及可溶性蛋白质的电泳分析。结果表明:RM109超氧化物歧化酶(SOD)活性为大力的50%,F1为大力的72%;RM109过氧化物酶(POD)活性比大力高125%,F1的比大力低3%;RM109过氧化氢酶(CAT)活性为大力的56%,F1为大力的11%;RM109琥珀酸脱氢酶活性为大力的60%,差异都极显著。揭示了基因突变后引发的生理生化的变化,从中探讨突变机理。     

RLF,a cytochrome b5‐like heme/steroid binding domain protein,controls lateral root formation independently of ARF7/19‐mediated auxin signaling in Arabidopsis thaliana

Lateral root (LR) formation is important for the establishment of root architecture in higher plants. Recent studies have revealed that LR formation is regulated by an auxin signaling pathway that depends on auxin response factors ARF7 and ARF19, and auxin/indole‐3‐acetic acid (Aux/IAA) proteins including SOLITARY‐ROOT (SLR)/IAA14. To understand the molecular mechanisms of LR formation, we isolated a recessive mutant rlf (reduced lateral root formation) in Arabidopsis thaliana. The rlf‐1 mutant showed reduction of not only emerged LRs but also LR primordia. Analyses using cell‐cycle markers indicated that the rlf‐1 mutation inhibits the first pericycle cell divisions involved in LR initiation. The rlf‐1 mutation did not affect auxin‐induced root growth inhibition but did affect LR formation over a wide range of auxin concentrations. However, the rlf‐1 mutation had almost no effect on auxin‐inducible expression of LATERAL ORGAN BOUNDARIES‐DOMAIN16/ASYMMETRIC LEAVES2‐LIKE18 (LBD16/ASL18) and LBD29/ASL16 genes, which are downstream targets of ARF7/19 for LR formation. These results indicate that ARF7/19‐mediated auxin signaling is not blocked by the rlf‐1 mutation. We found that the RLF gene encodes At5g09680, a protein with a cytochrome b₅‐like heme/steroid binding domain. RLF is ubiquitously expressed in almost all organs, and the protein localizes in the cytosol. These results, together with analysis of the genetic interaction between the rlf‐1 and arf7/19 mutations, indicate that RLF is a cytosolic protein that positively controls the early cell divisions involved in LR initiation, independent of ARF7/19‐mediated auxin signaling.