Diphenylene Iodonium as a Prominent Halogen Bond Donor: The Case of Human Monoamine Oxidase B.

Herein we have investigated the formation and interplay of several noncovalent interactions (NCIs) involved in the inhibition of human monoamine oxidase B (MAO B). Concretely, an inspection of the Protein Data Bank (PDB) revealed the formation of a halogen bond (HlgB) between a diphenylene iodonium (DPI) inhibitor and a water molecule present in the active site, in addition to a noncovalent network of interactions (e. g. lone pair-π, hydrogen bonding, OH-π, CH-π and π-stacking interactions) with surrounding protein residues. Several theoretical models were built to understand the strength and directionality features of the HlgB in addition to the interplay with other NCIs present in the active site of the enzyme. Besides, a computational study was carried out using DPI as HlgB donor and several electron rich molecules (CO, H2O, CH2O, HCN, pyridine, OCN-, SCN-, Cl- and Br-) as HlgB acceptors. The results were analyzed using several state-of-the-art computational tools. We expect that our results will be useful for those scientists working in the fields of rational drug design, chemical biology as well as supramolecular chemistry.

Diphenyleneiodonium Treatment Inhibits the Development of Severe Herpes Stromal Keratitis Lesions.

Reactive oxygen species (ROS) play an important role in tissue inflammation. In this study, we measured the intracellular level of ROS in herpes stromal keratitis (HSK) corneas and determined the outcome of manipulating ROS level on HSK severity. Our results showed the predominance of ROS generation in neutrophils but not CD4 T cells in HSK corneas. NADPH oxidase 2 (NOX2) enzyme is known to generate ROS in myeloid cells. Our results showed baseline expression of different NOX2 subunits in uninfected corneas. After corneal herpes simplex virus-1 (HSV-1) infection, an enhanced expression of NOX2 subunits was detected in infected corneas. Furthermore, flow cytometry results showed a higher level of gp91 (Nox2 subunit) protein in neutrophils from HSK corneas, suggesting the involvement of NOX2 in generating ROS. However, no significant decrease in ROS level was noticed in neutrophils from HSV-1-infected gp91-/- mice than in C57BL/6J (B6) mice, suggesting NOX2 is not the major contributor in generating ROS in neutrophils. Next, we used diphenyleneiodonium (DPI), a flavoenzyme inhibitor, to pharmacologically manipulate the ROS levels in HSV-1-infected mice. Surprisingly, the neutrophils from peripheral blood and corneas of the DPI-treated group exhibited an increased level of ROS than the vehicle-treated group of infected B6 mice. Excessive ROS is known to cause cell death. Accordingly, DPI treatment resulted in a significant decrease in neutrophil frequency in peripheral blood and corneas of infected mice and was associated with reduced corneal pathology. Together, our results suggest that regulating ROS levels in neutrophils can ameliorate HSK severity. IMPORTANCE Neutrophils are one of the primary immune cell types involved in causing tissue damage after corneal HSV-1 infection. This study demonstrates that intracellular ROS production in the neutrophils in HSK lesions is not NOX2 dependent. Furthermore, manipulating ROS levels in neutrophils ameliorates the severity of HSK lesions. Our findings suggest that excessive intracellular ROS in neutrophils disrupt redox homeostasis and affect their survival, resulting in a decrease in HSK lesion severity.

The development of diphenyleneiodonium analogs as GPR3 agonists.

G protein-coupled receptor 3 (GPR3) is an orphan receptor potentially involved in many important physiological processes such as drug abuse, neuropathic pain, and anxiety and depression related disorders. Pharmacological studies of GPR3 have been limited due to the restricted number of known agonists and inverse agonists for this constitutively active receptor. In this medicinal chemistry study, we report the discovery of GPR3 agonists based off the diphenyleneiodonium (DPI) scaffold. The most potent full agonist was the 3-trifluoromethoxy analog (32) with an EC50 of 260 nM and 90% efficacy compared to DPI. Investigation of a homology model of GPR3 from multiple sequence alignment resulted in the finding of a binding site rich in potential π-π and π-cation interactions stabilizing DPI-scaffold agonists. MMGBSA free energy analysis showed a good correlation with trends in observed EC50s. DPI analogs retained the same high receptor selectivity for GPR3 over GPR6 and GPR12 as observed with DPI. Collectively, the DPI analog series shows that order of magnitude improvements in potency with the scaffold were attainable; however, attempts to replace the iodonium ion to make the scaffold more druggable failed.

Tetraphenylphosphonium Chloride-Enhanced Ionization Coupled to Orbitrap Mass Spectrometry for Sensitive and Non-targeted Screening of Polyhalogenated Alkyl Compounds from Limited Serum.

Although many types of halogenated compounds are known to bioaccumulate in humans, few are routinely biomonitored and many have remained uncharacterized in human exposome studies due to a lack of high-sensitivity and high-resolution analytical methods. In this study, we discovered tetraphenylphosphonium chloride (Ph4PCl, 10 µM) as a simple additive to the mobile phase, which enhanced the ionizations of polyhalogenated alkyl compounds (such as organochlorinated pesticides [OCPs], chlorinated paraffins [CPs], dechlorane plus [DPs], and some brominated flame retardants [BFRs]) in the form [M + Cl]- and boosted mass spectrometry responses by an average of 1-3 orders of magnitude at a resolution of 140,000. Ph4PCl-enhanced ionization coupled with a halogenation-guided screening process was used to establish a sensitive and non-targeted method that required only single-step sample preparation and identified Cl- and/or bromine-containing alkyl compounds. The method enabled the identification of ∼700 polyhalogenated compounds from 200 µL of human serum, 240 of which were known compounds: 33 short-chain CPs, 52 median-chain CPs, 97 long-chain CPs, 22 very short-chain CPs (vSCCPs), 19 OCPs, 13 DPs, and 4 BFRs. We also identified 325 emerging contaminants (34 unsaturated CPs, 285 chlorinated fatty acid methyl esters [CFAMEs], and 6 chloro-bromo alkenes) and 130 new contaminants (114 oxygen-containing CPs, 2 hexachlorocyclohexane structural analogs, and 11 amino-containing and 3 nitrate-containing chlorinated compounds). The full scan results highlighted the dominance of CPs, CFAMEs, vSCCPs, and dichlorodiphenyltrichloroethanes in the serum samples. Ph4PCl-enhanced ionization enabled the sensitive and non-targeted identifications of polyhalogenated compounds in small volumes of biological fluid.

Combined Levo-tetrahydropalmatine and diphenyleneiodonium chloride enhances antitumor activity in hepatocellular carcinoma.

Metabolic dysregulation is a hallmark of hepatocellular carcinoma (HCC). AMPK is a crucial hub of metabolic regulation during cancer progression. We show that phytochemical Levo-tetrahydropalmatine (THP) activates AMPK-dependent autophagy to downregulate the mitochondrial respiration and glycolysis. Consequently, THP significantly decreased cell viability in two HCC cell lines, BEL-7402 and SMMC-7721. Similarly, NOX4 inhibitor diphenyleneiodonium chloride (DPI) induces concomitant downregulation of the mitochondrial and glycolytic metabolism. In contrast to THP, cells are less sensitive to proliferation inhibition induced by DPI treatment as compared to THP treatment did. Combined treatment of THP and DPI was found to be more efficacious in killing cancer cells than either of the agents treated individually. Indeed, the co-operative effect by the THP-DPI combination improves the pro-apoptotic activity in response to the energy depletion as outlined by a drastic decrease in ATP levels. Therapeutic regime significantly reduced the tumor growth in mice. Importantly, this is realized without causing systemic toxicity to other organs. Collectively, our work shows that the combinatorial therapy of autophagy activator THP and NOX4 inhibitor DPI may be considered as a therapeutic avenue against HCC.

Metal-Free Regioselective N2-Arylation of 1H-Tetrazoles with Diaryliodonium Salts.

We describe a simple, metal-free regioselective N2-arylation strategy for 5-substituted-1H-tetrazoles with diaryliodonium salts to access 2-aryl-5-substituted-tetrazoles. Diaryliodonium salts with a wide range of both electron-rich and previously challenged electron-deficient aryl groups are applicable in this method. Diversely functionalized tetrazoles are tolerable also. We have devised a one-pot system to synthesize 2,5-diaryl-tetrazoles directly from nitriles. The synthetic utility of this method is furthered extended to late-stage arylation of two biologically active molecules.

Metal-free S-arylation of 5-mercaptotetrazoles and 2-mercaptopyridine with unsymmetrical diaryliodonium salts.

Herein, we demonstrate the application of unsymmetrical iodonium salts towards S-arylation of heterocyclic thiols (especially tetrazole-5-thiols and pyridine-2-thiol) under metal-free conditions, affording a diverse range of di(hetero)aryl thioethers in moderate to good yields. A detailed study on the effects of counter-anions and the auxiliary of iodonium salts was conducted. Suitable auxiliary selection of the unsymmetrical iodonium salt offers flexibility for a wide range of aryl moieties and its incorporation into S-arylation. The DFT study supports the experimental observations of chemoselective arylation.

Tetramethylpyrazine inhibits neutrophil extracellular traps formation and alleviates hepatic ischemia/reperfusion injury in rat liver transplantation.

Hepatic ischemia/reperfusion injury (IRI) is an adverse effect for liver transplantation which is characterized by immune response mediated inflammation. Recent studies report that neutrophil extracellular traps (NETs) are implicated in hepatic IRI. The aim of this study was to explore the mechanism of action of tetramethylpyrazine (TMP), the main chemical composition of Ligusticum chuanxiong in treatment of ischemic related diseases. Data showed that hepatic IRI increases the leak of alanine aminotransferase (ALT) and aspartate transaminase (AST), and stimulates formation of NETs. Extracellular DNA/NETs assay, hematoxylin-eosin (HE) staining, immunofluorescence assay, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and Western blot assay, showed that TMP significantly reduces formation of NETs and alleviates hepatic IRI. Moreover, TMP and Diphenyleneiodonium (DPI) suppressed ROS production in neutrophils. In addition, analysis showed that activation of NADPH oxidase plays a role in formation of NETs triggered by hepatic IRI. Notably, TMP inhibited formation of NETs though inhibition of NADPH oxidase. Additionally, Combination treatment using TMP and DPI was more effective compared with monotherapy of either of the two drugs. These findings show that combination therapy using TMP and DPI is a promising method for treatment hepatic IRI.

Diphenyleneiodonium efficiently inhibits the characteristics of a cancer stem cell model derived from induced pluripotent stem cells.

Diphenyleneiodonium (DPI) has long been evaluated as an anticancer drug inhibiting NADPH oxidase, the IC50 in several cancer cell lines was reported 10 µM, which is too high for efficacy. In this study, we employed miPS-Huh7cmP cells, which we previously established as a cancer stem cell (CSC) model from induced pluripotent stem cells, to reevaluate the efficacy of DPI because CSCs are currently one of the main foci of therapeutic strategy to treat cancer, but generally considered resistant to chemotherapy. As a result, the conventional assay for the cell growth inhibition by DPI accounted for an IC50 at 712 nM that was not enough to define the effectiveness as an anticancer drug. Simultaneously, the wound-healing assay revealed an IC50 of approximately 500 nM. Comparatively, the IC50 values shown on sphere formation, colony formation, and tube formation assays were 5.52, 12, and 8.7 nM, respectively. However, these inhibitory effects were not observed by VAS2780, also a reputed NADPH oxidase inhibitor. It is noteworthy that these three assays are evaluating the characteristic of CSCs and are designed in the three-dimensional (3D) culture methods. We concluded that DPI could be a suitable candidate to target mitochondrial respiration in CSCs. We propose that the 3D culture assays are more efficient to screen anti-CSC drug candidates and better mimic tumor microenvironment when compared to the adherent monolayer of 2D culture system used for a conventional assay, such as cell growth inhibition and wound-healing assays.

Metal-Free C-H Functionalization via Diaryliodonium Salts with a Chemically Robust Dummy Ligand.

A two-step strategy for the transition-metal-free C-H functionalization of arenes using unsymmetrical iodonium salts as versatile synthetic linchpins is presented. The key to the success of this strategy is the identification of the 3,5-dimethyl-4-isoxazolyl (DMIX) group as a superior dummy ligand, which enables not only site-selective C-H functionalization to afford unsymmetrical iodonium salts, but also highly selective aryl transfer during the subsequent metal-free coupling reaction. Both electron-rich and moderately electron-deficient arenes can be converted into the iodonium salts through C-H functionalization, allowing for diverse structural elaboration by metal-free C-N, C-C, C-S, and C-O coupling.

Catalyst-Controlled Regiodivergence in Rearrangements of Indole-Based Onium Ylides.

We have developed catalyst-controlled regiodivergent rearrangements of onium-ylides derived from indole substrates. Oxonium ylides formed in situ from substituted indoles selectively undergo [2,3]- and [1,2]-rearrangements in the presence of a rhodium and a copper catalyst, respectively. The combined experimental and density functional theory (DFT) computational studies indicate divergent mechanistic pathways involving a metal-free ylide in the rhodium catalyzed reaction favoring [2,3]-rearrangement, and a metal-coordinated ion-pair in the copper catalyzed [1,2]-rearrangement that recombines in the solvent-cage. The application of our methodology was demonstrated in the first total synthesis of the indole alkaloid (±)-sorazolon B, which enabled the stereochemical reassignment of the natural product. Further functional group transformations of the rearrangement products to generate valuable synthetic intermediates were also demonstrated.

Understanding the Effect of Solvent Environment on the Interaction of Hydronium Ion with Biomass Derived Species: A Molecular Dynamics and Metadynamics Investigation.

The addition of aprotic solvents results in higher reactivities and selectivities in many key aqueous phase biomass reactions, including the acid-catalyzed conversion of fructose to 5-hydroxyl methyl furfural (HMF). The addition of certain co-solvents inhibits the formation of humins via preferential solvation of key functional groups and can alter reaction kinetics. An important factor in this context is the relative stability of the hydronium ion (the catalyst) in the vicinity of the biomass moiety as compared to that in bulk, as it could determine its efficacy in the protonation step. Hence, in the present work, molecular dynamics (MD) simulations of HMF (the model product) and fructose (the model reactant) in acidic water and water-DMSO mixtures are performed to analyze their interaction with the hydronium ions. We show that the presence of DMSO favors the interaction of the hydronium ion with fructose, whereas it has a detrimental effect on the interaction of hydronium ion with HMF. Well-tempered metadynamics (WT-MTD) simulations are performed to determine the relative stability of the hydronium ion in the immediate vicinity of fructose and HMF, as compared to that in the bulk solvent phase, as a function of solvent composition. We find that DMSO improves the stabilization of the hydronium ions in the first solvation shell of fructose compared to that in the bulk solvent. On the other hand, hydronium ions become less stable in the immediate vicinity of HMF, as the concentration of DMSO increases.

NADPH-Oxidase 2 Promotes Autophagy in Spinal Neurons During the Development of Morphine Tolerance.

Repeated morphine administration results in analgesic tolerance. However, the underlying mechanism of morphine analgesic tolerance remains unclear. NADPH-oxidase 2 (NOX2) is the first discovered NADPH oxidase, which mainly functions to produce reactive oxygen species. Its specific role in morphine tolerance has not been fully investigated. In this work, we found that chronic morphine administration significantly increased the expression of NOX2 in spinal cord. Pretreatment of NOX2 inhibitor blocked the upregulation of NOX2 and autophagy markers, including LC3B and P62, and consequently the development of morphine tolerance. NOX2 and LC3B were both colocalized with NeuN in spinal dorsal horn in morphine-tolerant rats. Our results suggest that the increased autophagy activity in spinal neurons promoted by NOX2 activation contributes to the development of morphine tolerance. NOX2 may be considered as a new therapeutic target for morphine tolerance.

Diphenyleneiodonium enhances P2X7 dependent non-opsonized phagocytosis and suppresses inflammasome activation via blocking CX43-mediated ATP leakage.

The beneficial effects of antioxidants against oxidative stress have been well described. However, the pharmacological impacts of antioxidants other than inhibiting the production of reactive oxygen species (ROS) remain less understood. This study demonstrated that diphenyleneiodonium (DPI), a canonical NADPH oxidase 2 (NOX2) inhibitor, effectively promoted non-opsonized bacterial phagocytosis. Indeed, DPI abrogated the elevation in the extracellular ATP level of Escherichia coli (E. coli) -infected murine peritoneal macrophages, thereby restoring the association of the purinergic receptor P2X7 with non-muscle myosin heavy chain 9 (MYH9) to upregulate the P2X7 -dependent phagocytosis of E. coli. DPI also suppressed inflammasome activation and reduced necroptosis in E. coli-infected macrophages by decreasing extracellular ATP levels. Mechanistically, DPI upregulated p38 MAPK phosphorylation to suppress the expression and activity of the hemichannel protein connexin 43 (CX43), leading to the inhibition of CX43-mediated ATP efflux in E. coli-infected macrophages. In a murine E. coli infection model, DPI effectively reduced ATP release, decreased bacterial load and inhibited inflammasome activation, thereby improving survival and ameliorating organ injuries in model mice. In summary, our study demonstrates a previously unknown function of DPI in conferring protection against bacterial infection and suggests a putative antimicrobial strategy of modulating CX43 -dependent ATP leakage.

Mitochondrial toxins potentiate hydroxyl radical production in rat striatum during carbon monoxide poisoning.

Hydroxyl radical (•OH) production in the rat striatum during carbon monoxide (CO) poisoning, which inhibits complex IV, was enhanced synergistically by malonate, a mitochondrial complex II inhibitor, but not N-methyl-4-phenylpyridinium or NaCN, complex I and IV inhibitors, respectively. No such enhancement appeared in the case of NaCN combined with malonate. Intrastriatal dopamine, which is involved in •OH production by malonate, did not synergistically enhance CO-induced •OH production. Diphenyleneiodonium, a nonselective NADPH oxidase inhibitor, partly suppressed the potentiation of CO-induced •OH production by malonate. Impairment of mitochondrial functions might potentiate oxidative stress and intensify CO toxicity in the brain.

The effect of NADPH oxidase inhibitor diphenyleneiodonium (DPI) and glutathione (GSH) on Isatis cappadocica, under Arsenic (As) toxicity.

The present work was conducted to assess the effects of arsenic (As, 1000 µM), diphenyleneiodonium (DPI, 10 µM) and reduced glutathione (GSH, 500 µM) on Isatis cappadocica. As treatment decreased plant growth and fresh and dry weight of shoot and root and also enhanced the accumulation of As. As stress also enhanced the oxidative stress biomarkers, hydrogen peroxide (H2O2) and malondialdehyde (MDA) content. However, the application of GSH decreased the content of H2O2 and MDA by 43% and 55%, respectively, as compared to As treatment. The antioxidants like superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR) and glutathione S-transferase (GST) also enhanced with As stress. NADPH oxidase inhibitor, the DPI, enhances the effect of As toxicity by increasing the accumulation of As, H2O2, MDA. DPI also enhances the activity of antioxidant enzymes except GR and GST, However, the application GSH increased the plant growth and biomass yield, decreases accumulation of As, H2O2 and MDA content in As as well as As + DPI treated plants. The thiols content [total thiol (TT), non-protein thiol (NPT) protein thiols (PT), and glutathione (GSH)] were decreased in the As + DPI treatment but supplementation of GSH enhanced them. Novelty statement: The study reveals the beneficial role of GSH in mitigating the deleterious effects of Arsenic toxicity through its active involvement in the antioxidant metabolism, thiol synthesis and osmolyte accumulation. Apart from As, We provided the plants NADPH oxidase inhibitor, the diphenyleneiodonium (DPI), which boosts the As toxicity. At present, there is dearth of information pertaining to the effects of DPI on plants growth and their responses under heavy metal stress.GSH application reversed the effect of diphenyleneiodonium (DPI) under As stress preventing the oxidative damage to biomolecules through the modulation of different antioxidant enzymes. The application of GSH for As stressed soil could be a sustainable approach for crop production.

Oxonium Ion Guided Analysis of Quantitative Proteomics Data Reveals Site-Specific O-Glycosylation of Anterior Gradient Protein 2 (AGR2).

Developments in mass spectrometry (MS)-based analyses of glycoproteins have been important to study changes in glycosylation related to disease. Recently, the characteristic pattern of oxonium ions in glycopeptide fragmentation spectra had been used to assign different sets of glycopeptides. In particular, this was helpful to discriminate between O-GalNAc and O-GlcNAc. Here, we thought to investigate how such information can be used to examine quantitative proteomics data. For this purpose, we used tandem mass tag (TMT)-labeled samples from total cell lysates and secreted proteins from three different colorectal cancer cell lines. Following automated glycopeptide assignment (Byonic) and evaluation of the presence and relative intensity of oxonium ions, we observed that, in particular, the ratio of the ions at m/z 144.066 and 138.055, respectively, could be used to discriminate between O-GlcNAcylated and O-GalNAcylated peptides, with concomitant relative quantification between the different cell lines. Among the O-GalNAcylated proteins, we also observed anterior gradient protein 2 (AGR2), a protein which glycosylation site and status was hitherto not well documented. Using a combination of multiple fragmentation methods, we then not only assigned the site of modification, but also showed different glycosylation between intracellular (ER-resident) and secreted AGR2. Overall, our study shows the potential of broad application of the use of the relative intensities of oxonium ions for the confident assignment of glycopeptides, even in complex proteomics datasets.

Involvement of Reactive Oxygen Species in ABA-Induced Increase in Hydraulic Conductivity and Aquaporin Abundance.

The role of reactive oxygen species (ROS) in ABA-induced increase in hydraulic conductivity was hypothesized to be dependent on an increase in aquaporin water channel (AQP) abundance. Single ABA application or its combination with ROS manipulators (ROS scavenger ascorbic acid and NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI)) were studied on detached roots of barley plants. We measured the osmotically driven flow rate of xylem sap and calculated root hydraulic conductivity. In parallel, immunolocalization of ABA and HvPIP2;2 AQPs was performed with corresponding specific antibodies. ABA treatment increased the flow rate of xylem, root hydraulic conductivity and immunostaining for ABA and HvPIP2;2, while the addition of antioxidants prevented the effects of this hormone. The obtained results confirmed the involvement of ROS in ABA effect on hydraulic conductivity, in particular, the importance of H2O2 production by ABA-treated plants for the effect of this hormone on AQP abundance.

Ethidium Binding to Salmonella enterica ser. Typhimurium Cells and Salmon Sperm DNA.

Bacterial resistance to antibiotics due to increased efficiency of the efflux is a serious problem in clinics of infectious diseases. Knowledge of the factors affecting the activity of efflux pumps would help to find the solution. For this, fast and trustful methods for efflux analysis are needed. Here, we analyzed how the assay conditions affect the accumulation of efflux indicators ethidium (Et+) and tetraphenylphosphonium in Salmonella enterica ser. Typhimurium cells. An inhibitor phenylalanyl-arginyl-ß-naphtylamide was applied to evaluate the input of RND family pumps into the total efflux. In parallel to spectrofluorimetric analysis, we used an electrochemical assessment of Et+ concentration. The results of our experiments indicated that Et+ fluorescence increases immediately after the penetration of this indicator into the cells. However, when cells bind a high amount of Et+, the intensity of the fluorescence reaches the saturation level and stops reacting to the accumulated amount of this indicator. For this reason, electrochemical measurements provide more trustful information about the efficiency of efflux when cells accumulate high amounts of Et+. Measurements of Et+ interaction with the purified DNA demonstrated that the affinity of this lipophilic cation to DNA depends on the medium composition. The capacity of DNA to bind Et+ considerably decreases in the presence of Mg2+, Polymyxin B or when DNA is incubated in high ionic strength media.

Salicylic acid promotes quiescent center cell division through ROS accumulation and down-regulation of PLT1, PLT2, and WOX5.

Salicylic acid (SA) plays a crucial role in plant immunity. However, its function in plant development is poorly understood. The quiescent center (QC), which maintains columella stem cells (CSCs) in the root apical meristem and typically exhibits low levels of cell division, is critical for root growth and development. Here, we show that the Arabidopsis thaliana SA overaccumulation mutant constitutively activated cell death 1 (cad1), which exhibits increased cell division in the QC, is rescued by additional mutations in genes encoding the SA biosynthetic enzyme SALICYLIC ACID INDUCTION DEFFICIENT2 (SID2) or the SA receptor NONEXPRESSER OF PR GENES1 (NPR1), indicating that QC cell division in the cad1 mutant is promoted by the NPR1-dependent SA signaling pathway. The application of exogenous SA also promoted QC cell division in wild-type plants in a dose-dependent manner and largely suppressed the expression of genes involved in QC maintenance, including those encoding the APETALA2 (AP2) transcription factors PLETHORA1 (PLT1) and PLT2, as well as the homeodomain transcription factor WUSCHEL-RELATED HOMEOBOX5 (WOX5). Moreover, we showed that SA promotes reactive oxygen species (ROS) production, which is necessary for the QC cell division phenotype in the cad1 mutant. These results provide insight into the function of SA in QC maintenance.