With a decade of effort, significant progress has been achieved in the synthesis, processing, and applications of MXenes. Metal ions play many crucial roles, such as in MXene delamination, structure regulation, surface modification, MXene composite construction, and even some unique applications. The different roles of metal ions are attributed to their many interactions with MXenes and the unique nature of MXenes, including their layered structure, surface chemistry, and the existence of multi‐valent transition metals. Interactions with metal ions are crucial for the energy storage of MXene electrodes, especially in metal ion batteries and supercapacitors with neutral electrolytes. This review aims to provide a good understanding of the interactions between metal ions and MXenes, including the classification and fundamental chemistry of their interactions, in order to achieve their more effective utilization and rational design. It also provides new perspectives on MXene evolution and exfoliation, which may suggest optimized synthesis strategies. In this respect, the different effects of metal ions on MXene synthesis and processing are clarified, and the corresponding mechanisms are elaborated. Research progress on the roles metal ions have in MXene applications is also introduced. Interactions with metal ions play crucial roles in the development of MXenes. This review discusses in details the fundamental chemistry of these interactions, including intercalation, crosslinking and etching, and presents how these interactions affect MXene synthesis, processing and applications, which yields the approaches for the rational designs, optimized synthesis and potential applications of MXene‐based materials. Their properties can be effectively changed by altering their composition, interlayer structure, and surface chemistry, which gives them potential use in many fields, such as optoelectronics, catalysis, energy, sensors, and biomedicine.Since graphite was delaminated to form graphene, there has been tremendous research interest on 2D materials in academia and industry, due to its remarkable physical and electronic properties. In recent decades, various other 2D materials besides graphene have been explored including transition metal dichalcogenides (TMDs), metal carbides/nitrides, layered double hydroxides (LDHs), boron nitride (h‐BN), graphene‐like single elements , etc.3, 4 and well defined in 2012. This category of 2D materials shares the general formula Mn+1XnTx (n = 1–3), where M is a transition metal , and X is carbon and/or nitrogen, with surface terminations indicated by T . The presence of single or mixed terminations in MXenes is determined by their synthesis or processing. In general, they are synthesized by selectively etching A layers from MAX phases, where A is a group of 13 or 14 elements , and M and X are the same as for the corresponding MXene. The first and the most studied MXene is Ti3C2Tx , which was first produced in 2011 with a hydrofluoric acid (HF) solution as the etchant. The molten Lewis acid salt route enables other surface groups  to terminate the MXene surface. Further termination substitution by ‐NH, ‐S, ‐Se, ‐Te, or complete removal can be achieved by substitution reactions. Modification of the surface chemistry enables fine‐tuning of the band gap as well as other properties, such as electrical, optical, and optoelectronic. For example, Liu et al. indicated that, compared with the bare surface, ‐O terminations tend to increase the work function of MXenes, while ‐OH terminations tend to cause a decrease. The tendency produced by ‐F terminations depends on the specific structure of the MXenes. A superconducting transition behavior has been reported at low‐temperatures for ‐S  terminated Nb2C, while no such a behavior occurs for Nb2COx. Till now, more than 30 MXenes with different compositions have been discovered experimentally, and over 100 different MXenes have been theoretically predicted. The M sites can also be filled by two or more transition metals to form solid solutions or ordered structures. This unique structure gives MXenes attractive electronic, optical, and magnetic properties. A transition metal carbide core gives MXenes metallic conductivity (20 000 S cm−1 for Ti3C2Tx), and multi‐valent transition metals ensure good redox activity. The abundant surface terminations provide active sites, solution dispersion ability, and the ability to change properties. Through strategic design and controlled synthesis, MXenes have proven to be promising in various applications, such as supercapacitors, rechargeable metal‐ion batteries, catalysis, electromagnetic interference (EMI) shielding, and water treatment.MXenes are a range of new 2D transition metal carbide/nitride materials with a functional structure, which were first discovered in 20117 and +, Na+, K+) are found to be spontaneously or electrochemically intercalated between MXene layers. In the case of spontaneous intercalation, delamination of the MXene and expansion of the interlayer spacing can be achieved. Controlling the interlayer spacing is generally supposed to be an effective way of changing the band gap, Fermi level, conductivity, and other properties of 2D materials. Furthermore, the intercalated metal ions hardly change the lattice structure of MXenes and still maintain high ion mobility. Electrochemical intercalation induces changes in the chemical potential and redox state of MXenes, which plays a significant role in the energy storage in metal ion batteries. Interestingly, some metal ions behave differently and may induce a dramatic structural or lattice change of MXenes. For example, divalent metal ions  can trigger crosslinking to form a 3D network, which is beneficial for ion diffusion. Some metal ions with redox activities  may generate atomic defects, pores, or produce MXene degradation. Because of the redox activities of metal ions and MXenes, MXene sheets with anchored metal atoms or nanoparticles can be synthesized in a one‐step method. The evolution from MAX to MXene also involves metal ions, such as Ax+ oxidized from the A atoms in MAX by etchants, examples being Li+ in the LiF/HCl route and Cu2+ in the molten CuCl2 route. Considering that the quality of MXene sheets highly depends on the preparation process (including the etching reaction and delamination), it is vital to figure out the role of metal ions in MXene synthesis and the interactions between them.The layered structure, abundant surface terminations, and redox activity of MXenes allow them to be altered in a variety of ways, when coupled with different metal ions. Metal ions  and 3D structure. The modification of targeted surface terminations can be achieved by chain effects produced by metal ions, such as ‐Cl terminations produced by etching the MAX precursors in molten MClFigure Over the years of research on MXenes, interactions with metal ions have been discovered and utilized, and have played crucial roles in many aspects, ranging from synthesis and processing to structure design and various applications. However, some scientific questions have not been answered, such as the different effects of metal ions on MXene structure control. Therefore, a timely review of the interaction between metal ions and MXenes is needed. This review classifies the different effects of metal ions on MXenes into three categories: intercalation, crosslinking, and etching Figure 1. The r2π bonds, and strong chemical bonds. The match of their energy levels is the key for the interaction, which is associated with the chemical structure and surface chemistry of the host. For graphite‐like materials with non‐polar surface and delocalization π electrons, both electron acceptors and donors are allowed to create interactions by charge transfer effects, and electrons flow away from or into the their conduction band. TMDs‐like materials only accept electrons to fill the d‐orbitals, and thus electron acceptors do not create interactions. However, clays have fundamentally different behaviors because of their surface functional groups. Electrostatic interactions and coordination interactions with metal cations are the primary forms. For MXene materials, it is well known that they have abundant surface terminations (‐O/‐OH/‐F/‐Cl) like clays, and thus expectedly have spontaneous ion intercalation or exchange behavior due to electrostatic interactions. In addition, MXenes are composed of transition metal atoms like TMDs, so that their various valence states guarantee the possibility of charge transfer or oxidation/reduction reactions. In this case, three main effects of metal ions on MXenes are classified, including intercalation, crosslinking and etching. The roles in the MXene synthesis and processing are introduced, and the corresponding fundamental interaction chemistry is also discussed.In the MXene development, metal ions play key roles in the synthesis and processing, such as MXene delamination, surface modification, structure control, property altering, MXene composite construction. The various roles are attributed to their interactions with MXenes and the unique nature of MXenes. In general, chemical interactions between guest ions and host layer solids include electrostatic interactions, charge transfer effects, the host.27 For2.12.1.1 Additional intercalation is also required to obtain single or few‐layer MXene sheets. The first report of the synthesis of the MXene, Ti3C2Tx, was in 2011, where it was produced by immersing a Ti3AlC2 precursor in a HF solution, where the selective etching of Al layers resulted in a graphene‐like structure, and certain terminations  were introduced on the surface. The obtained MXene (HF‐MXene) could be spontaneously intercalated with organic molecules  and cations . Given this post‐intercalation ability, a one‐step and milder synthesis route was proposed in 2014, using a mixture of hydrochloric acid (HCl) and lithium fluoride salt (LiF) as the etchant. Compared with HF‐MXene, a dramatic change in the c‐lattice parameter  with Li+ intercalation was observed, causing easier delamination and fewer defects.Unlike van der Waals forces between the graphenes in graphite, there are metallic M‐A bonds between the MXene layers in the MAX phase, which are hard to break by mechanical forces. Therefore, chemical  reactions are necessary, and oxidizing etchants that react with the A layers in MAX are generally used.35 AddFigure 2O molecules occupy some of the Ti3C2 interlayer space, after the removal of Al atoms by the HF solution. Only a small number of cations (Mx+) can be intercalated with an uneven distribution , there is only a slight shift of the (0002) peak from its original position at 8.91°. However, when HF/LiCl, HF/NaCl or HF/SnCl4 is the etchant in MXene synthesis, a larger peak shift is observed for M@Ti3C2‐II . The c‐LP value of the obtained MXene is increased from 20.32 Å (Li@Ti3C2‐I) to 23.00 Å (Li@Ti3C2‐II) by Li+ intercalation, and from 20.44 Å (Sn@Ti3C2‐I) to 24.84 Å (Sn@Ti3C2‐II) by Sn4+ intercalation. Moreover, the MXene obtained by in situ intercalation during the etching process  involves ion exchange and swelling, which makes the MXene delamination easier.It has been shown that the cation intercalation during etching process plays a vital role in the synthesis, in addition to the formation of HF. As illustrated in cations Mx+ can  Two different synthesis routes with different molar ratios of LiF and Ti3AlC2 are shown in Figure 3AlC2 is 5:1, and the LiF reacts with HCl to produce HF in etching process. Comparing with Route 1, Route 2 provides excess Li+ for the intercalation, and twice the amount of HCl is used to satisfy the Al etching. The combined effect improves the reaction kinetics resulting in the complete conversion of Ti3AlC2 to Ti3C2Tx  and a uniform thickness (≈2.7 nm), as shown in Figure  the increased thickness measured by AFM is due to surface adsorbates, such as water molecules. Moreover, the MXene flakes produced using Route 2 show well‐defined and clean edges with a much lower defect concentration in high‐resolution TEM images, resulting in better electronic properties.Large MXene flakes of high quality can also be obtained by controlling the concentration of intercalated metal ions.40 Two Figure  and acce2.1.2 MXenes prepared by the LiF/HCl or HF/LiCl method can be intercalated with various other metal cations, including univalent ions  and multivalent ions . After immersing the MXenes in different salt solutions, intercalants remain in the interlayer space due to electrostatic interactions  intercalated Ti3C2Tx MXene electrodes (Mn+‐MXene) with controllable interlayer spacings. As shown in Figure 2+‐MXene), 5.9° (Mg2+‐MXene), 5.6° (Al3+‐MXene), 5.9° (Ho3+‐MXene), 5.8° (In3+‐MXene), and 5.7° (Be2+‐MXene), corresponding to expanded d‐spacings  of 13.2, 14.7, 15.0, 15.6, 15.3, 15.2, and 15.1 Å, respectively. The change in d‐spacing is explained by the intercalation chemistry and the related hydrated radius of metal ion, which will be explained in the next section. As expected, the Mn+‐MXene electrodes have an almost negligible interfacial resistance with the electrolyte, indicating that the charge‐transfer and ion diffusion are increased by the presence of the intercalants.It is well known that intercalation is an effective way to alter the physical and chemical properties of 2D materials, including the band gap, conductivity, optical and mechanical properties, chemical reactivity, etc. A larger interlayer spacing increases ion diffusion and exposes more active sites, significantly improving the electrochemical performance.4 MXen The introduction of intercalants  is thought to result in a lower conductivity, worse mechanical properties, and instability. Removing the intercalants by cation exchange in an acid solution or annealing can significantly increase the electrical conductivity. However, Osti et al. found that K+ intercalation improved stability and structural homogeneity, and decreased the mobility of water confined between layers. It was also shown by Ding et al. that the intercalation of Al3+ ions improved the stability of a Ti3C2Tx membrane in aqueous solution and there was no structural collapse after long‐time soaking in water. An increase in the tensile strength of a Ti3C2Tx film produced by Al3+ intercalation was reported by Liu et al., while the intrinsic electrical conductivity was maintained. Therefore, more research is needed to clarify the effects of metal ion intercalation on the mechanical properties, electrical conductivity, and stability of MXenes.Apart from the interlayer spacing, the mechanical properties, electrical conductivity, and stability of MXenes are also influenced.45 The+, Na+, K+, and Mg2+) strongly affected the oxidation state of the Ti atoms in Ti3C2Tx MXene after drying in air, while H2SO4 pre‐treatment prevented it. Combining XAS with X‐ray photoemission electron microscopy (X‐PEEM), the Ti oxidation states of Li‐, and Mg‐Ti3C2Tx were found to depend on their thickness, while those of Na‐ and K‐Ti3C2Tx did not. This difference is related to many factors, such as the probing depth, the particle geometry, and the amount of water confined in the MXene layers under different pressures. Overall, the impact of the intercalation of different metal ions on MXene properties differs and this requires further investigation. Furthermore, metal cation intercalation in alkali solutions may lead to a larger d‐spacing than in neutral solutions and to a change in surface terminations. Halim et al. showed that ‐F terminations (from 1.0 to 0.7 moles per mole of Ti3C2Tx) produced by fluoride etchants during MXene synthesis were replaced by ‐O terminations  after NaOH treatment.The effect of intercalation on the electronic structure and surface chemistry has also been also investigated. Using X‐ray absorption spectroscopy (XAS), Al‐Temimy et al. found that cation intercalation  Ti3C2Tx released Li+ and H+ ions and took up K+ and Na+ ions, when immersed in 1 m KCl and 1 m NaCl, respectively. However, no ion exchange occurred in 1 m LiCl, with almost zero driving force , while Mg2+‐MXene responds at ≈10% RH.The mechanism of the intercalation of various metal ions by immersing MXene in different salt solutions has also been investigated. Sharma et al. found by ICP‐MS measurements that the clay‐like  quantified the relative position between ‐O surface termination (OT) and cations. The capacitance at the open circuit potential (OCP) was measured to be in inverse proportion to this cation‐OT distance. Considering the effect of water molecules confined between MXene layers, a modified electric double layer (EDL) model was proposed to explain this relationship. At the same time, the EDL energy storage mechanism was identified in the neutral aqueous electrolytes, which will be discussed later in detail.The specific intercalation states and interfacial chemistry of different metal ions between two MXene sheets have been tracked, which helps provide a deeper understanding of MXene energy storage properties and processes. Gao et al. used ab initio molecular dynamics (AIMD) simulations to investigate the atomic arrangements around Lis Figure .60]60+, N Kajiyama et al. used solid‐state 23Na NMR to reveal the Na+ intercalation mechanism. As presented in Figure + signals can be detected, representing solvated Na+ from the residual electrolyte, solvated Na+ trapped in the SEI layer or adsorbed on the surface, and completely or partially de‐solvated Na+ intercalation. It is believed that de‐solvated Na+ intercalation is the main contributor to energy storage. Furthermore, the solvent effect has also been investigated. Wang et al. demonstrated Li+ intercalation with a complete de‐solvation behavior in a propylene carbonate (PC) system. However, solvent co‐intercalation occurs in acetonitrile (ACN) and dimethylsulfoxide (DMSO) systems. Remarkably, the efficient de‐solvation in PC results in a high volumetric capability. The interaction between intercalated ions and the MXene surface is supposed to determine the electrochemical behavior of MXenes during charging. Regretfully, few studies have focused on intercalation chemistry in a nonaqueous system, and more attention needs to be paid to this subject.Understanding intercalation chemistry in a nonaqueous system is also essential, especially during the electrochemical process. Metal cations, also the charge carriers, intercalate and de‐intercalate the MXene layers to achieve energy storage and release, and the state of the intercalated ions needs to be understood.63 Kaj2.22.2.13C2Tx colloidal suspensions, and flocculation or gelation occurs due to the destruction of electrostatic repulsive forces between the sheets. The treatment with alkali metal hydroxides  can result in Ti3C2Tx nanosheet aggregation and a crumpled structure, with the intercalation of metal ions. A 3D nanoribbon structure can be fabricated using an increased alkali concentration. A high concentration NaCl solution was also found to induce a crumpled morphology. Interestingly, some multivalent metal ions  induce Ti3C2Tx nanosheet crosslinking to form a stable 3D hydrogel, rather than simple flocculation or aggregation . The MXene concentration is a key factor, as only flocculation occurs in low concentration, because of the limited interaction range and fast charge neutralization of metal ions. Following this, Ding et al. fabricated a 3D Ti3C2Tx MXene aerogel by Mg2+ crosslinking, without polymeric binders or templates. Using the thermal contraction of polystyrene (PS) substrate, a crumple‐textured Ti3C2Tx coating (CT‐MXene) was pre‐prepared. This CT‐MXene allows a higher Mg2+ loading, and which induces gelation on the surface after soaking in a Ti3C2Tx dispersion (21 mg mL−1), as illustrated in Figure 2+ ions are intercalated. The aerogel formed is really tough because of the strong crosslinking, so that the pore structure is preserved even after the intercalated Mg2+ is removed by acid rinsing with only a slight change in specific surface areas  than the original  to an acidic MXene or MXene/GO hybrid dispersion, where the metal ions diffuse out and induce gelation. Large pore size and thick lamellas tend to form in a higher ion concentration and a quicker assembly process by pH control. The prepared foams can be loaded with different nanoparticles (SiO2 and Fe3O4).The addition of a metal salt solution can alter the stability of Tig Figure . The str2.2.2 The main reason is the imbalance between electrostatic attraction and electrostatic repulsion. The OH− in alkali metal hydroxides  passivate the positively charged edge sites of Ti3C2Tx flakes, while Na+ is adsorbed on the negatively charged surface sites. The positive Na+ ions attract the adjacent MXene nanosheets, which results in a large number of face‐to‐face interactions between them. In the case of a salt solution, the Debye length decreases with the increased salt concentration, which indicates that the repulsion between the nanosheets is reduced, creating a face‐to‐face interaction between them. This strong interaction causes the nanosheet aggregation and a crumpled structure after drying, which may also account for the nanoribbons formed in high concentration alkali metal hydroxides, as mentioned earlier. The high concentration of OH− may over‐passivate the edge making it negatively charged. Meanwhile, the increased concentration of Na+, which prefers to be absorbed on the surface, attracts the negatively charged edge. Such a plane face‐to‐edge attraction induces the high‐degree of folding and crumping of the MXene sheet, and thus nanoribbons are formed. This process is like rolling a sheet of paper into a tube‐shape from one edge side.Before introducing the crosslinking behavior of metal ions, the aggregation of MXene flakes in alkali metal hydroxides and salt solutions of high concentration will be explained first.77 The2+, Mg2+, Co2+, Ni2+, Mg2+, Al3+) and surface terminations is greater in the crosslinking. Giving the Ti3C2Tx gelation process with Fe2+ ions as an example. Firstly, the electrostatic interactions with Fe2+ ions and negatively charged Ti3C2Tx sheets break the original electrostatic balance among sheets. Then, as linkers, the ions interact with ‐OH/‐O and ‐F groups on MXene surface. In this process, the hydration shells around Fe2+ are destroyed, as obvious Fe‐O and Fe‐F bonds (very low intensity) are detected in in XPS spectra  act as the oxidant to etch the A layer  in the MAX phase. For example, Ti3SiC2 reacts with molten CuCl2 at 750 °C and produces Ti3C2Cl2, Cu clusters, and SiCl4 gas. By coupling different MAX phases with appropriate molten salts (including cations and anions), a broad family of MXene materials can be theoretically synthesized, with different surface chemistries and structures. Similar to the HF route, Ti vacancies are also found, which indicates that the metal ions not only etch A atoms but also Ti atoms. Because of this, it is essential to study the interaction between metal ions and MXene during synthesis in molten salts.The molten salt route is becoming more important in MXene synthesis, in addition to the wet chemical etching route. In this approach, the metal ions  at 1000 °C for 7 h, or in molten CoCl2 . After the reaction, the products were treated with a HCl solution and FeCl3 to remove Cu particles, or only a HCl solution to remove Co particles. The final products had a visible layered structure with a Cu content of 1.8 at% , and a specific surface area of 85 m2 g−1 was achieved.It has been reported by Gu et al. that specific numbers of Ti vacancies are introduced by reacting Tir Figure 7a.222SO4y Figure .[37]s Figure .[42] A Ti3C2Tx MXene hydrogel electrode prepared by Fe2+ crosslinking can achieve an excellent rate performance (≈226 F g−1), and its typical pseudocapacitive behavior is well maintained at 1 V s−1, as shown in Figure  Ding et al. used a Mg2+‐MXene aerogel as electrodes and PVA/H2SO4 as a gel electrolyte to construct quasi‐solid state micro‐supercapacitors. As a result, a high areal‐specific capacitance (409.3 mF cm−2 at 5 mV s−1) was obtained, as well as high areal energy densities (7.4–21.6 μWh cm−2) and high areal power densities (0.1–1.1 µW cm−2). In addition, MXenes are regarded as ideal 2D supports to couple with other pseudocapacitive materials, and their interactions with metal ions help the fabrication of composite electrodes. As illustrated in Figure 2/Ti3C2Tx composite is synthesized by impregnating Ti3C2Tx MXene in a MnSO4 solution, with KMnO4 being reduced. After annealing, the composite electrode had a capacitance of 212.1 F g−1 at 1 A g−1. Similarly, other composite electrodes have been extensively investigated, such as Ti3C2/MoS2, Ti3C2/CuS, Ti3C2/FeOOH quantum dots, Ti3C2/NiCoFe‐LDH, Ti3C2/NiFe‐LDH, Ti3C2/Ni3S2, Ti3C2/Fe3O4, Ti3C2/Ni2CO3(OH)2, etc.A 3D MXene electrode can be fabricated to address the restacking problem of 2D nanosheets and shorten the mass transfer path by using the crosslinking of metal ions.29 A T3.2 MXenes are new materials that can accommodate the insertion of various metal ions between their layers. As electrode materials, they are predicted to provide remarkable theoretical capacities . Given that the metal ions play a crucial role as charge carriers in metal‐ion batteries (MIBs), their interactions with MXenes, especially in electrochemistry, become extremely important.Rechargeable lithium‐ion batteries (LIBs) are one of the most significant energy storage devices, and are widely used in portable electronic devices and the new electric vehicles. The pursuit of higher energy densities and the limited lithium resources have promoted the exploration of new electrode materials and non‐lithium‐ion batteries, including sodium‐ion batteries (SIBs), potassium‐ion batteries (PIBs), calcium‐ion batteries (CIBs), etc.61 MXe2X with a higher effective surface area is expected to provide higher gravimetric capacities than M3X2 and M4X3. That is to say, compared with Ti3C2 and Ti4C3, Ti2C has a higher theoretical gravimetric capacity. In addition, ‐OH and ‐F terminations are believed to block ion diffusion and lower the theoretical capacities, while ‐O functional groups provide additional capacity. For example, once terminated with ‐F or ‐OH, the theoretical capacity is reduced to 130 or 67 mAh g−1 for Li+ storage. The different theoretical specific capacities shown for different metal ions can be understood by referring to the maximum adatom content and the corresponding charge transfer, which determines the electronic properties after ion adsorption.Figure 3C2 and the charge transfer from adatoms to Ti3C2. It is calculated that the amount of charge transfer are 0.21 (Li), 0.40 (Na), 0.47 (K), and 1.31 e− (Ca), with the respective maximum adatom contents being 2.8, 2.2, 1.2, and 1. Moreover, the corresponding diffusion barriers are estimated to be 0.068 (Li), 0.096 (Na), 0.103 (K), and 0.118 (Ca) eV, which are much lower than that on graphite (0.3 eV for Li). These calculations indicate that MXenes are promising electrode materials with a high rate.Given the preferred ion adsorption on the surface, Mand M4X3.129 Th2C powders prepared by 10 wt% HF gives capacities of 225 mAh g−1 at C/25, and 110 mAh g−1 at 1 C. To achieve satisfied performance with rational material designs, the investigation of ion storage mechanism is urgently needed. MXenes store ions by adsorption and intercalation, and generally show distorted rectangular CVs with small pairs of redox peaks. In organic electrolytes, partially or complete de‐solvation of cations occurs at the MXene‐electrolyte interface or in intercalation, due to the lower solvation energy than hydration. The solvents would decompose and solid electrolyte interface (SEI) forms under the wide potential window. In most cases, those cations adsorbed on the MXene surface have solvation shells, and the complete charge separation results in EDL storage. However, those intercalated cations are usually partially or fully de‐solvated, and a donor band forms due to the orbital overlap with a close contact of MXene‐cation. This orbital hybridization allows charge transfer between MXenes and intercalated cations, and enhances the capacity. In 1.0 m LiPF6‐ethylene carbonate/dimethyl carbonate (LiPF6‐EC/DMC), a reversible interlayer spacing change (9.4–9.8 Å) is found, which is caused by the partially de‐solvated Li+ (de‐)intercalation. At the same time, a reversible reduction/oxidation of Ti of Ti2CTx is caused. As mentioned in section + per Ti3C2Tx) leads to a higher capacity. In 1 m NaPF6‐ethylene carbonate/diethyl carbonate (NaPF6‐EC/DEC), the layered Ti3C2Tx has a capacity of 270 mAh g−1 at 20 mA g−1 for the first Na+ intercalation/adsorption and a good cycle stability (100 mAh g−1 over 100 cycles) the first few cycles. Electrolyte decomposition is the main reason for the irreversible capacity in these cycles. A combination of XRD and TEM, shows that the interlayer spacing increases from 0.97 to 1.20 nm during the first sodiation, and remains unchanged in the next de‐sodiation and the second sodiation intercalation of de‐solvated Na+ and result in a stable cycling.Experimentally, when used the anode materials in LIBs, multilayer Ti1 at 1 C.62 To n Figure . This Na Luo et al. used the ion‐exchange behavior and electrostatic interactions between MXenes and metal ions to produce Ti3C2 decorated with Sn4+ ions. As shown in Figure + intercalation and Sn4+ intercalation is achieved by ion exchange, where a surfactant  helps produce a uniform distribution of Sn4+. As a result, an outstanding electrochemical performance was achieved, with an ultrahigh capacity  and excellent cycling stability with a 94.3% capacity retention after 200 cycles (500 mA g−1). Here, the Sn (LixSn) and Ti3C2 (Ti3C2TxLix) both contribute to the capacity. Similarly, Wang et al. produced Sn‐intercalated V2C MXene (c‐LP: 19 Å) which had a stable discharge capacity of 690.8 mAh g−1 at 1 A g−1, and 205.2 mAh g−1 at 20 A g−1, when used as the anode in LIBs.According to the above discussion, the interlayer structure can determine the intercalation kinetics of metal ions and make full use of the active sites.139 Lu N (V)‐modified Ti3C2Tx MXene was prepared by microwave irradiation Ti3C2Tx powders in NH4VO3/EG (ethylene glycol), as shown in Figure  The V atoms were doped into Ti3C2 lattice to occupy Ti vacancies, and bind with ‐O surface groups to increase the interlayer spacing, through the interaction of VO2+ ions with Ti3C2Tx. Meanwhile, the surface was modified by N, through the interaction with NH4+. The co‐doping of V and N increases the capacity from 180.1 to 251.3 mAh g−1 at 0.1 C, and improves the cycling durability. It has been reported that V2C MXene with a controlled interlayer spacing exhibits the Li‐ion capacity to 686.7 mAh g−1 at 0.1 A g−1. The performance is found to be improved to 1117.3 mAh g−1, by intercalation with Co ions. Here, strong interaction between the VO2+ ions and MXene is confirmed, as evidenced by the existence of V‐O‐Co bonding.Doping is also powerful in changing the band structure and improving the electrochemical performance.142 N  As mentioned earlier, alkali metal hydroxides produce a crumpled 3D structure of MXenes, and by using this property, Na+ ion storage can reach a specific capacity of 267 mAh g−1 (25 mA g−1) in the 1st cycle and 170 mAh g−1 in the 2nd cycle after being treated with NaOH. One treated with LiOH still display had a capacity of ≈160 mAh g−1 (0.1 A g−1), after 300 cycles. The 3D assembly of Ti3C2Tx MXene can be achieved with the assistance of GO sheets and ethylenediamine (EDA), and its microstructure could be regulated by the interaction of metal ions with MXene sheets. Zhao et al. designed a 3D Ti3C2Tx network with a local laminated structure, by adding KOH in the assembly process. It showed a excellent long‐term cycling as the anode of SIB, with a capacity retention of 188 mAh g−1 after 1500 cycles (100 mA g−1), as shown in Figure 3D structure design is also frequently used to improve the kinetics and shorten the mass transfer length, thus improving the rate performance, especially in SIBs.144 As For example, through the adsorption of Sn2+ on Ti3C2 sheets, SnOx@Ti3C2 anode was prepared by a hydrothermal reaction, which had a much higher Li‐storage capacity of ≈450 mAh g−1 than SnO (≈63 mAh g−1) and Ti3C2 anodes. As illustrated in Figure 2+, Co2+, and urea to an alkali‐Ti3C2 suspension, followed by hydrothermal treatment. Then Ti3C2/NiCoP composite was then prepared by phosphorization with NaH2PO2. When used as the anode material in SIBs, it retained a specific capacity of 261.7 mAh g−1 at 1 A g−1 after 2000 cycles. Zou et al. used the self‐reduction of Ag+ ions on MXene form a Ti3C2‐Ag hybrid, which had an extraordinary long cycle lifetime (>5000 cycles) with a high Li‐storage capacity of 310 mAh g−1 at 1 C, as well as a good rate performance (150 mAh g−1 at 50 C). Other hybrid electrodes have been fabricated for MIBs, such as Ti3C2/Fe2O3, Ti3C2/W‐doped Nb2O5, Ti3C2/MoO3‐x, and Ti3C2/GeOx for LIBs, and Ti3C2/FeS2, Ti3C2/Sb2O3, Ti3C2/CoNiO2, and Ti3C2/SnS for SIBs, etc.Similar to their use in supercapacitors, the use of MXene hybrid materials is another common way to provide a highly conductive framework for high‐capacity active materials.146 Fo This kind of device is composed of one battery‐type electrode and the other capacitor‐type electrode. In most cases, the battery‐type redox reaction occurs on the anode side, and capacitive reaction occurs on the cathode side. Sometimes, there is a reverse situation, and it depends on the potentials of the two reactions. As introduced before, MXenes demonstrate excellent ion storage performance, so they can be anodes in MICs. Luo et al. fabricated pre‐intercalated Ti3C2 MXene (CTAB‐Sn(IV)@Ti3C2) with cetyltrimethylammonium bromide (CTAB) and Sn4+ pillaring. When coupling the CTAB‐Sn(IV)@Ti3C2 anode and activated carbon (AC) cathode, the lithium‐ion hybrid capacitor (LIC) displayed the specific capacitance of 268 F g−1 at 0.2 A g−1 (based on the anode mass), and good capacity retention of 71.1% after 4000 cycles at 2 A g−1. Fan et al. prepared nitrogen‐doped porous Ti3C2Tx (N‐Ti3C2Tx) by using the template of melamine‐formaldehyde (MF) nanospheres. With the AC cathode, the constructed sodium ion hybrid capacitors (SICs) achieved a gravimetric energy and power density of 101.6 Wh kg−1 and 3269 W kg−1, respectively. Song et al. designed S‐doped Ti3C2Tx aerogels (SMGA) by crosslinking S/Ti3C2Tx composites and GO sheets, with linking agents . The resulting electrodes exhibited excellent Na+ ion storage (1.26 mAh cm−2) with a high mass‑loading (12.3 mg cm−2). The constructed SMGA//AC SICs displayed a capacity retention of 92.9%, and maintained a capacity of 93.7 mAh g−1 (based on the anode mass) after 1600 cycles. Zhao et al. designed a 3D K+‐pre‐intercalated Ti3C2Tx MXene by alkali treatments to store K+ ions. The assembled 3D K+‐Ti3C2Tx//AC potassium ion hybrid capacitors (PICs) achieved a high energy density of 163 Wh kg−1 and a long‐term cycling (up to 10 000 cycles), with the formation of a thin and inorganic‐rich SEI in 4 m KFSI electrolytes. Owing to their fast redox kinetics, MXenes can also be used as cathodes of MICs. For instance, Li et al. designed Sn4+ pre‐intercalated Ti2CTx MXene (Sn4+‐Ti2CTx) cathodes with enlarged interlayer spacing for aqueous zinc ion hybrid capacitors (ZICs), with zinc metal anodes. To further shorten the diffusion length, the Sn4+‐Ti2CTx was aligned on the carbon spheres. As expected, a specific capacity of 138 mAh g−1 at 0.1 A g−1 and ultralong cycling of 12 500 cycles have been achieved.In recent years, MXene based metal ion hybrid capacitors (MICs) are attracting increasing research attention, due to the advantages of both high energy density and power density.160 Th3.3 Producing a large number of active catalysis sites on MXene is a widely adopted strategy to increase its performance as a catalyst, where its interactions with different metal ions are made full use of.Recently, MXenes have attracted great attention in catalysis due to their high surface area, good electronic conductivity, and metal atom cores.173 Pr Zhao et al. synthesized a Pt1/Ti3‐xC2Ty single‐atom catalyst by mixing a [PtCl6]2− solution with a MXene (Ti3‐xC2Ty) suspension, and no additional reduction operation was needed  in producing CH3OH in electrocatalytic CO2 reduction (CO2RR). Ramalingam et al. prepared Ru SACs@MXene by thermal annealing, after mixing a Ti3C2Tx, Ru3+ solution with thiourea. The Ru3+ ions interact with the ‐O/‐OH terminations on the MXene surface, and are then reduced by an annealing treatment. For the hydrogen evolution reaction (HER), a low overpotential (76 mV) was achieved at a current density of 10 mA cm−2.First, MXene‐supported single metal atom catalysts (SACs@MXene) can be synthesized by anchoring metal atoms in cation vacancies or surface groups.178 Zhe Figure  was achi, Figure  as well  As shown in Figure 3O4@MXene, SnO2@MXene, MnTiO3@MXene, Pt@MXene, and Ag@MXene catalysts were prepared by the aerosol spray drying of MXene colloids dissolved in the corresponding metal salts, such as Co(OAC)2·4H2O, SnCl4, Mn(CH3COO)2·4H2O, H2PtCl6·6H2O, or AgNO3. The metal ions are distributed uniformly on the 3D MXene matrix with strong interactions, and the salt decomposes into the corresponding metal oxide or metal. Among them, Co3O4@MXene can be converted into CoP@MXene  for OER and 128 mV for HER in 1 m KOH  have been prepared using the reactive metal‐support interactions on Pt/MXene catalysts. For instance, Ti3C2Tx was first impregnated with a Pt(NH3)4(NO3)2 solution to form a fresh Pt/Ti3C2 catalyst and then reduced at 550 °C in 5% H2/N2 for at least 0.5 h. In this process, Pt atoms are not simply embedded in the Ti3C2Tx lattice, but form a bimetallic alloy with surface Ti atoms, which exhibits a superior HER performance in acidic media to give a high *H adsorption strength. Pt3Nb supported on Nb2CTx was prepared in a similar way, and has been reported to provide a higher H2O activation than the original catalyst or pure Nb2CTx. However, Pt atomically thin nanolayers (ATNLs) rather than Pt‐Mo alloys are formed on the Mo2TiC2Tx surface, which is explained by the preferential formation of interfacial Mo‐Pt bonds and the lower interface formation energy of a Pt nanolayer than a nanoparticle. The Pt ATNLs on Mo2TiC2Tx show a stable conversion (7%) and a high selectivity (>98%) for C2 products in the non‐oxidative methane coupling reaction.Recently, ordered surface‐supported intermetallic compounds  have long‐term negative effects on water quality and threaten human health. Therefore, water/wastewater treatment is of great importance to maintain water resources.190 Th Generally, the concentration of toxic HMIs  in water is far lower than that of light metal ions . This means that the interactions of HMIs with MXenes are strong enough to break the shielding effect of water molecules or coordinating anions, with the formation of strong bonds. As illustrated in Figure 3C2Tx due to the formation energy of Ti3C2(O2H2‐2mPbm) being lower than that of Pb(NO3)2. Moreover, alkali‐MXene has a better efficiency for replacing F groups and metal ion  intercalation. Experimentally, a high Pb(II) ion uptake capacity (140 mg g−1) is achieved, with a high selectivity when other cations  and Mg(II)) are also present. Similarly, alkali MXene has a Ba(II) adsorption capacity of 46.46 mg g−1, three times that of the non‐modified one. It also has a ≈99% removal efficiency in a complex solution containing other elements  contained , Cr(VI), U(VI),) and some oxidants 6], KMnO4, AuCl4−) can also be removed. The residual concentration of Cr(VI) can be reduced to 5 ppb after removal by Ti3C2Tx sheets, which meets the standard for drinking water. The removal capacity is as high as 250 mg g−1 and is pH‐dependent. In low pH or neutral solutions, Cr(VI) is reduced to Cr(III), which is anchored on the MXene surface  with the strong absorption and reducing ability.201 Ged Figure . Based oe Figure . Meanwhih Figure  by a RGO3C2Tx laminates show excellent water flux and good ion sieving activity. This was first reported by Ren et al., who showed that a Ti3C2Tx film had a water flux of 37.4 L Bar−1 h−1 m−2 with different ion permeation rates for Na+, Li+, K+, Ca2+, Ni2+, Mg2+ and Al3+. Given that ions permeate through the interlayer in the membranes, ions can be adsorbed on both surfaces of MXene interlayer walls, if the radius of hydrated ions are small. In this case, larger interlayer spacing can be obtained by Na+ ion permeation than Ca2+ and Al3+ ions, as the ions with smaller hydrated radius and lower charges are able to form double layer adsorption. Moreover, the electrostatic attraction between MXene sheets and highly charged ions may cause the interlayer contraction. As a result, the permeation rate of Na+ is about 25 times that of Al3+ and 7 times that of Ca2+. Pandey et al. fabricated an Ag@MXene membrane by self‐reduction of Ag+ ions on MXene sheets. Compared to pure MXene membrane, an increased water flux is achieved, with 92.32% and 79.93% rejection for methyl green and rhodamine B. Additionally, bacteria growth was inhibited by the Ag particles. To prevent swelling and improve the mechanical stability, MXene membranes have been intercalated with cations and crosslinking. Wang et al. prepared an Al3+ intercalated‐MXene membrane  was obtained. MXene foams produced by Zn2+ crosslinking, as discussed earlier, had a shielding performance of 51.0 dB at a thickness of only 85 µm. Satheeshkumar et al. used the self‐reduction of specific metal ions on MXene, and synthesized Ag, Au, and Pd@MXene hybrids by one‐step solution processing, as illustrated in Figure  Metal nanoparticles were uniformly distributed on the surface, and a surface enhanced Raman scattering (SERS) effect was demonstrated, which can be used in sensors, catalysis, and the biomedine. For instance, methylene blue (MB) is used as a chemical indicator, dye, biological stain, and medicine. The signal of adsorbed MB on a NP@MXene surface can be unequivocally detected in Raman spectra, even when its concentration of MB is only 10−6m  ions in water can be pre‐concentrated and self‐reduced to Cd metal on Ti3C2Tx MXene. Then the re‐oxidation signal can be measured in differential pulse voltammetry (DPV) technology, and the limit of detection was low as 0.94 × 10−9m.In addition to the above applications, MXenes and MXene hybrids have been used in electromagnetic interference shielding, as sensors, and in therapeutic biomedicine. Liu et al. used the intercalation of Alstrength.50 Them Figure . Zhang e Dai et al. prepared MnOx/Ti3C2 hybrids by the in situ reduction of MnO4− ions on a Ti3C2 surface. As shown in Figure 3C2 acts as the photothermal‐transducing agent for tumor ablation due to the high photothermal‐conversion efficiency of MXene. MnOx acts as magnetic resonance (MR) and photoacoustic (PA) imaging contrast agents to determine tumor sites and monitor therapeutic process. In vitro and in vivo PA‐imaging of MnOx/Ti3C2 are shown in Figure Applications in therapeutic biomedicine are promising and may be achieved with the rational designs of MXene‐based composites.216 Da4x+/M) of MClx salts. The energy level differences between them determine the degree of charge transfer. The quality of MXene highly depends on the choice of molten salts. In addition, the Ti vacancies can anchor reduced M atoms to form single‐atom metals on MXene lattices.This review has summarized progress on the roles of metal ions in MXene synthesis, processing and applications. Based on the way they change the MXene structure, three classifications are defined: intercalation, crosslinking, and etching. The understanding of the fundamental mechanisms has also been discussed. In the case of intercalation and crosslinking, electrostatic attraction between the metal cations and negatively charged surface groups is the driving force, and this ion exchange behavior enables the insertion of other metal ions. The hydration of metal cations plays a crucial role in improving the exfoliation kinetics and changing the interlayer spacing. Hydrated metal ions intercalate the MXene layers, rather than bare metal ions, and a large hydrated radius tends to produce a large c‐LP. Metal ions with high charge tend to form strong chemical or ionic bonds with the surface groups on adjacent MXene sheets. This crosslinking by metal ions induces MXene gelation to form 3D structures, with a sufficiently high MXene concentration. In the case of etching, the charge transfer seems to be the principal reason for the formation of in‐plane defects (Ti vacancies and in‐plane pores), due to the redox activities of the transition metal in MXenes. In the molten salt route to synthesize MXene, Ti atoms in MAX may also be etched, similar to the Al atoms, due to the higher redox potential  is crucial to open the MXene interlayers but seems to hinder charge transfer, which may reduce the capacitance/capacity in energy storage. A deeper understanding of their relationships is required to explore the potential of MXenes as electrode materials. Third, the MXene crosslinking by metal ions is innovative and effective for producing 3D structure. However, relevant research is limited, and more effort is needed to regulate structural parameters, including pore size and layer thickness. Fourth, in MXene evolution from MAX phases, the kinetics of etching reactions is a crucial issue to obtain MXenes of high quality and few defects. For etching Ti3AlC2 in HF/LiCl, this is addressed by the strong bonding of Al‐F and hydrated Li+ intercalation. However, the strategy of Li+ intercalation seems to play a limited role in other MXene syntheses, such as MoC2, Cr2C, or some o‐MXenes , which cannot be obtained in HF/LiCl. As such, there may be other important factors, and new synthesis routes for MXenes need to be investigated. Considering our current knowledge of the importance of etching and intercalation in the synthesis process, may be other ways of regulating the structure.Research on interactions between metal ions and MXenes is of great importance in MXene synthesis, processing, and applications. However, there are still many issues and scientific questions to be resolved. First, research to date has mostly been focused on the TiThe authors declare no conflict of interest.


Compounds 1–5 (4=[Ru(tbbpy)2(tpphz)Ru(tpy)Cl](PF6)3, 5=[Ru(bpy)2(tpphz)Ru(tpy)(C3H8OS)/(H2O)](PF6)3, tbbpy=4,4′‐di‐tert‐butyl‐2,2′‐bipyridine) are tested for their ability to run water oxidation catalysis (WOC) using cerium(IV) as sacrificial oxidant. The WOC experiments suggest that the stability of monodentate (chloride) ligand strongly correlates to catalytic performance, which follows the trend 1>2>5≥3>4. This is also substantiated by quantum chemical calculations, which indicate a stronger binding for the chloride ligand based on the extended π‐systems in compounds 2 and 3. Additionally, a theoretical model of the mechanism of the oxygen evolution of compounds 1 and 2 is presented; this suggests no differences in the elementary steps of the catalytic cycle within the bpy to the dppz complex, thus suggesting that differences in the catalytic performance are indeed based on ligand stability. Due to the presence of a photosensitizer and a catalytic unit, binuclear complexes 3 and 4 were tested for photocatalytic water oxidation. The bridging ligand architecture, however, inhibits the effective electron‐transfer cascade that would allow photocatalysis to run efficiently. The findings of this study can elucidate critical factors in catalyst design.The substitution behavior of the monodentate Cl ligand of a series of ruthenium(II) terpyridine complexes (terpyridine (tpy)=2,2′:6′,2′′‐terpyridine) has been investigated.  The correlation between the stability of a monodentate chloride ligand and the catalytic water oxidation performance of a set of ruthenium(II) terpyridine complexes based on π‐extension of a bipyridine is investigated. The findings are supported by theoretical calculations and show a significant dependence of the stability of the chloride ligand on the π‐extension and, hence, a decrease in the catalytic turnover number and turnover frequency. Compounds 1–4 were characterized by 1H NMR and UV‐vis spectroscopy as well as mass spectrometry . The peak assignment of the 1H NMR spectra was done by literature reported spectra of similar dinuclear RuPt, RuPd and RuRh complexes. sphere[3 and 4 shows no observable emission for compound 3 and very weak (within the Raman bands) emission for compound 4 indicating that quenching of the emission of the [Ru(R2bpy)2(tpphz)] moiety by the [Ru(tpy)(tpphz)Cl] is very efficient (data not shown).Figure observed.35, 36tBu) to the oxidation potential RuII/RuIII on both the [Ru(R2bpy)2(tpphz)] (shift of ca. 200 mV) and the [Ru(tpy)(tpphz)Cl] (shift of ca. 100 mV) moiety of the complex . The potential is anodically shifted upon introduction of a dppz ligand, manifesting in a shift of the potential of the RuIV/RuIII wave from 0.64 V versus Fc+/Fc0 in 1 to 0.93 versus Fc+/Fc0 in 2. For binuclear complexes 3 a and 4, the waves cannot easily be evaluated due to overlap of waves attributed to both of the metal centers. It seems to be shifted even more anodically for compound 4 to 1.22 V versus Fc+/Fc0. For compound 3 a, no assessment of the wave was possible. However, these results indicate, that the driving force for water oxidation of the system is also altered by extension of the π‐system and hence an effect on the turnover numbers (TONs) cannot be excluded. Nevertheless, theoretical calculations imply that the general mechanism and associated energies are not changed in the aquo coordinated Ru(tpy) moieties irrespective of the actual bidentate ligand . It has to be noted, that the described experiments were conducted in the absence of water and for the chloride coordinated original complexes. The situation might drastically change upon substitution of the chloride ligand with an aquo ligand in aqueous media.Electrochemical characterization also revealed that there is a significant influence of the substituents on the bipyridine ligand ; Figure Ke) derived from the 1H NMR kinetic experiments show values of 3.2, 2.7 and 1.3 for 1, 2 and 3 b, respectively. As was suggested by other studies, replacement of this ligand has to be done prior to catalysis to render the systems active.[Based on the structure of the [Ru(tpy)Cl] moiety, the chloride ligand lies in the same plane as the bidentate ligand  and therefore the Hs Figure . The hal1, 2 and 3 b, 4, 5 were tested towards their capability of water oxidation using cerium(IV)ammonium nitrate (CAN) as sacrificial oxidant. To this end, the complexes were dissolved in water and CAN was added. The solution was stirred in the dark and oxygen evolution was detected with an in operando sensor setup developed in our group earlier.2 generated a moderate TON of 300 after 20 h compared to the well investigated compound 1 with 400 after 20 h. This fact strongly supports the data acquired by the 1H NMR study investigating the kinetics of the chloride substitution and suggests that the stability of the ruthenium chloride bond limits the efficiency of WOC in compound 2. Additionally, significantly lower TONs were observed for the binuclear complexes 3 b and 4 of 120 and 55, respectively, compared to 1 under identical conditions. Due to the even slower substitution of the chloride ligand in these systems, it was assumed that the substitution is limiting the activity of the catalyst. Therefore, replacing the chloro‐ligand in 3 b by a 2‐(methylthio)ethan‐1‐ol, which was immediately replaced by an aquo‐ligand within the catalytic mixture, was done prior to catalysis. As a result, the TON almost doubled from 80 to 150 after 10 h  significantly increased by prior removal of the monodentate chloride ligand in compound 5 (L)(H2O)]  in solution is formed over a longer period of time, limiting catalysis in the initial phase, as compared to 1. As compounds 3 and 4 exhibit a catalytic and a sensitizer moiety, their ability towards photocatalytic water oxidation was tested. Both systems are incapable of oxidizing water photochemically . This indicates that the bridging ligand architecture might not allow the necessary electron‐transfer cascade to enable WOC. However, this might not be true for an intermolecular system in which photocatalysis might still be observed.Compound 5 Figure . This be1 to 2) considerably reduces the obtainable TON. Even though the ligand exchange step could decrease the overall efficiency of the catalytic behavior, a priori it is not possible to discard that the ligand alone could not affect the reaction barriers during the water oxidation reactions and, hence, be responsible of the experimental difference in the TON. Therefore, we used density functional theory (DFT) to assess the impact of the ligands on both the catalytic water oxidation cycle and the ligand exchange step. Based on previous studies on complex 1[II(tpy)(bpm)(OH2)]2+ (bpm=2.2’‐bipyrimidine),[II‐OH2]2+ is generated. Subsequently, the catalytic cycle starts with two proton‐coupled‐electron‐transfer (PCET) steps and an additional oxidation that leads to the [RuV=O]3+ species. This is subject to the water nucleophilic attack step (labeled “WNA”), responsible for the important O−O bond formation that leads to [RuIII‐OOH]2+. After a subsequent PCET step, the final oxygen release (“O2 release” step) operates regenerating the active catalyst, thus completing the catalytic cycle. While the oxidation and PCET steps are considered barrierless,2 release steps involve a transition state and therefore an associated kinetic reaction barrier. Hence, depending on the ligands, WNA or the O2 release have the potential to be the rate limiting step. In order to elucidate, we computed the reaction barriers associated to both steps for complexes 1 and 2. Figure G≠298,H2Oof about 11.0 kcal/mol for both compounds 1 and 2. Likewise, for the O2 release  versus 10.7 kcal/mol (2). Overall, these values are in excellent agreement with previously computed reaction barriers using minimum free energy paths with explicit solvent (QM/MM–MFEP method)II(tpy)(bpm)(OH2)]2+ complex.++H2O substitution reaction for the compounds 1, 2 and 3. As shown in Figure 1,2,3TS. Taking into account also the possible associated reactants and products, the calculated reaction barriers are ΔG≠298,H2O=24.6, 25.8, and 26.0 kcal/mol for 1–3, respectively. The energy differences, despite small, are sufficient to account for the different half‐lives experimentally observed for the considered compounds. Table 1 as a reference. One can see that the general trend is satisfactorily reproduced. If for complex 1 we consider the stable associated reactant, which leads to a higher apparent activation energy , the qualitative trends of the half‐lives still hold. The trend observed in the reaction barriers can be rationalized looking to the Ru−Cl bond strengths along the different compounds. Indeed, at the equilibrium geometries of the reactants the Ru−Cl distance decreases with the size of the π ligand , which can be correlated with higher barriers. Additionally, we calculated Ru−Cl bond dissociation unrelaxed scans starting from the reactant geometries .In any case, our calculations indicate that the WNA step is the rate‐limiting for both complexes, but with nearly identical barriers according to which one would expect similar behavior of the oxygen evolution for both catalysts. Furthermore, if in complex s Figure a, a high1 (bpy), 30.6 kcal/mol in complex 2 (dppz) and 31.1 kcal/mol in complex 3 (tpphz). In conclusion, our calculations demonstrate that the ligand‐exchange step has indeed the largest impact on the experimentally observed TON and the catalytic performance. This result is in line with previous experiments on 1,Taking the asymptotic limit (5 Å), one can also see that the binding energies are larger with increasing ligand size: 29.8 kcal/mol in complex 1H NMR spectroscopy, the substitution of this monodentate halide ligand is substantially decelerated by the introduction of larger π‐systems, for example, a dppz ligand. The observed effect also alters the catalytic performance of the complexes towards thermal water oxidation with cerium(IV) as the oxidant, due to reduction of the amount of available active aquo‐substituted catalyst during the catalysis. Quantum chemical calculations show that π‐extension in the periphery does not alter the reaction barriers to the water nucleophilic attack or oxygen release steps (comparing 1 and 2). Instead, the computed Gibbs free energy profiles for the ligand substitution reaction reveal that there is an increase in the energy barrier when dppz is used instead of bpy. The second metal coordination in complex 3 increases this barrier further. The calculated half‐lives of the individual complexes are in accordance with the kinetic data derived from NMR‐spectroscopy and underline the slower ligand exchange in complexes bearing an extended π‐system in the bidentate ligands. We think that this study can give further insight into understanding catalytic performance and builds a foundation for specific optimization when implementing the use of catalysts for more complex architectures, such as polymeric structures.The influence of π‐conjugation on the substitution of the monodentate Cl ligand in a ruthenium terpyridine water oxidation catalyst has been assessed. Based on Experimental procedures, information regarding the used measurement setup and additional data for the performed experiments are available in the Supporting Information.Computational details: All structures have been subjected to an unrestricted DFT optimization using the PBE0[G298”) at 298.15 K. All energies are reported in kcal/mol. The conductor‐like polarizable continuum model CPCM[3). The QM region used GPW‐DFTT=2600 K to force the ligand exchange and sample the Gibbs free energy surface. The Ru−O coordination number and the Ru−Cl coordination number have been chosen as collective variables, and bias potentials with a full‐width‐at‐half‐maximum of 0.05 and a height of 0.5 kJ/mol were added every 25 ps. During the runtime of 30 ps, no ligand dissociation and exchange could be observed.Sampling of the Gibbs energy surface was attempted with well‐tempered molecular meta‐dynamics2 release steps, guesses for geometry optimizations were built inspired in previous publications.[2–xTB level of theory.[2 release and ligand exchange steps are collected in Tables S5‐S7.For the WNA and Oications.41, 45Reaction barriers can be straightforwardly linked to half‐life times using the expression:t1/2 is the half‐life and k is the rate constant. This rate constant can be obtained from Eyring's equation that read as:where G≠ is the Gibbs energy of activation, kB is the Boltzmann constant, h is Planck's constant and K is the transmission coefficient, which, according to the so‐called classical formulation of the transition state theory,where ΔThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


This page is designed to help you to test your own understanding of the concepts covered in this issue, and to reflect on what you have learnt.www.cehjournal.orgWe hope that you will also discuss the questions with your colleagues and other members of the eye care team, perhaps in a journal club. To complete the activities online – and get instant feedback – please visit Question 1 Visual Impairmenta Visual impairment is defined as <6/18 to 6/60 in the better eye at presentation.□ b The major cause of visual impairment in the world is uncorrected refractive error.□ c In 2015 there were an estimated 253 million visually impaired people in the world.□ d There are more visually impaired men than women, approximately 55% to 45%.□ e The prevalence of vision impairment increased in the world from 1990 to 2015.□ Question 2 Cataract Indicatorsa Cataract Surgical Coverage (CSC) is a measure of the quality of a cataract operation.□ b The main risk factor for incidence of visually impairing cataract is age.□ c Cataract Surgical Rate (CSR) depends mainly on the number of ophthalmologists per million population.□ d A poor outcome after cataract surgery (defined as <6/60 vision at presentation 6 weeks after surgery) is nearly always due to surgical complications.□ e The useful indicators in monitoring a cataract service are cataract surgical rate, cataract surgical coverage and cataract outcome.□ Question 3 Childhood eye diseasea Vitamin A deficiency is no longer a public health problem in the world.□ b Retinopathy of prematurity occurs only in high-income countries.□ c Rubella is a preventable cause of congenital cataract.□ d Uncorrected hypermetropia is the commonest cause of visual acuity <6/12 in school children.□ e Low vision services and aids are often needed after surgery for cataract in children.□ Question 4 Infectious eye diseasesa The treatment for onchocerciasis is Mectizan (ivermectin) given annually to communities where onchocerciasis is endemic.□ b Trachoma is caused by Chlamydia trachomatis serotypes A, B and C.□ c In communities where the prevalence of TF in children aged 1–9 years is 15%, azithromycin treatment to the community is recommended annually for 3 years.□ d Trichiasis surgery for TT can be performed in health clinics by eye assistants trained in TT surgery.□ e The number of blind people from both onchocerciasis and trachoma has decreased between 1990 and 2015.□ b and c are TRUE. Visual impairment is defined as <6/18 to no light perception in the better eye at presentation; it includes moderate and severe VI and blindness. It is estimated that 55% of all visually impaired people in the world are women. The prevalence of VI is estimated to have decreased from 4.58% to 3.38% between 1990 and 2015.b and e are TRUE. CSC is a measure of the coverage, that is quantity of cataract services. The CSR depends on a number of factors including barriers to access e.g. cost, distance; and the indication for surgery. If there are insufficient ophthalmologists then the CSR may be low, however the number of ophthalmologists is not the MAIN factor. Some countries in Latin America and Eastern Europe have many ophthalmologists but relatively low CSR. Poor outcome can be due to uncorrected refractive error; co-existing eye pathology e.g. glaucoma; or surgical complications. It is not true to say poor outcome is “nearly always due to surgical complications”.c and e are TRUE. Vitamin A deficiency remains a public health problem in some countries and is a preventable cause of childhood mortality and visual loss. ROP is seen in Latin America, Eastern Europe and urban centres of middle income countries in Asia and Africa being associated with increased survival of low birth weight babies in neonatal units. Uncorrected MYOPIA is the major cause of decreased acuity in school children.All are TRUE.


The IPCC's scientific assessment of the timing of net‐zero emissions and 2030 emission reduction targets consistent with limiting warming to 1.5°C or 2°C rests on large scenario databases. Updates to this assessment, such as between the IPCC's Special Report on Global Warming of 1.5°C (SR1.5) of warming and the Sixth Assessment Report (AR6), are the result of intertwined, sometimes opaque, factors. Here we isolate one factor: the Earth System Model emulators used to estimate the global warming implications of scenarios. We show that warming projections using AR6‐calibrated emulators are consistent, to within around 0.1°C, with projections made by the emulators used in SR1.5. The consistency is due to two almost compensating changes: the increase in assessed historical warming between SR1.5 (based on AR5) and AR6, and a reduction in projected warming due to improved agreement between the emulators' response to emissions and the assessment to which it is calibrated. Emulators used in IPCC Special Report on warming of 1.5°C and Sixth Assessment Report are remarkably consistent, despite their entirely new calibrationsThe consistency is due to two compensating factors: change in assessed historical warming and improvements to emulator calibration methods However, emulator behavior is the result of the emulator structure (captured by its name and version) and how it has been calibrated that is, both components are required to uniquely identify how the emulator was run. Ignoring the importance of calibration has caused considerable confusion in the past when discussing emulators  in the SR1.5 database contribute to the uncertainty as to when exactly temperature will return back below the 1.5°C limit if temporarily overshot. As a result, it may be more robust to discuss the decade of peak warming and the decadal rate of temperature reduction thereafter rather than the year in which temperatures return back below 1.5°C  than MAGICC6 Figure .The difference can arise from changes in any of the steps  along the cause‐effect chain from emissions to atmospheric concentrations to effective radiative forcing to warming. We first observe that MAGICC6 generally has lower effective radiative forcing than AR5‐like MAGICCv7.5.3 . As AR5‐like MAGICCv7.5.3 is in line with, it not a bit higher than, the AR5 TCR assessment . Similarly, MAGICCv7.5.3 implies a net zero CO2 year of 2050 (2038–2075) and FaIRv1.6.2 implies a net zero CO2 year of 2052 (2042–2070).The relatively small differences in climate projections lead to small changes in key mitigation milestones describing scenario categories, such as the year of net zero CO2 Figure  or 2030 The importance of these changes for policy and economic transition is a separate question, but they may not be seen as zero in all contexts . These differences in mitigation milestones arise even though climate science has remained remarkably consistent (differences of 0.05°C in the median). A key point from SR1.5 also remains relevant, “because of numerous geophysical uncertainties and model dependencies […] absolute temperature characteristics of the various pathway categories are more difficult to distinguish than relative features”  different climate outcomes  across ESMs . Updates to the design of scenarios . Thus, while decisive mitigation efforts this decade will be crucial in determining whether we shoot beyond 1.5°C, adaptation actions will have to be taken on the basis of a minimal warming level around 1.5°C.Supporting Information S1Click here for additional data file.


Camelina sativa is an oil crop with low input costs and resistance to abiotic and biotic stresses. The presence of glucosinolates, plant metabolites with adverse health effects, restricts the use of camelina for human and animal nutrition. Cas9 endonuclease‐based targeted mutagenesis of the three homeologs of each of the glucosinolate transporters CsGTR1 and CsGTR2 caused a strong decrease in glucosinolate amounts, highlighting the power of this approach for inactivating multiple genes in a hexaploid crop. Mutagenesis of the three homeologs of each of the transcription factors CsMYB28 and CsMYB29 resulted in the complete loss of glucosinolates, representing the first glucosinolate‐free Brassicaceae crop. The oil and protein contents and the fatty acid composition of the csgtr1csgtr2 and csmyb28csmyb29 mutant seeds were not affected. The decrease and elimination of glucosinolates improves the quality of the oil and press cake of camelina, which thus complies with international standards regulating glucosinolate levels for human consumption and animal feeding. Camelina sativa has gained increasing interest as sustainable oil crop for food and non‐food applications  and the model plant Arabidopsis thaliana, camelina is a member of the Brassicaceae family. The oil content of camelina seeds is in the range of 30%–49% with a high proportion (30%–43%) of the essential ω‐3 fatty acid α‐linolenic acid, while the amount of the undesired erucic acid is below 3%  glucosinolates in the leaves and silique walls. The two transporters are plasma membrane‐localized and expressed in vascular tissues, while AtGTR1 is additionally expressed in adjacent mesophyll cells. Glucosinolates are transported in the phloem and xylem over long distances from leaves to seeds and between leaves and roots  = N‐OSO3−, is formed. Finally, the side chain of the glucosinolates can be modified in further reactions. Glucosinolate biosynthesis is regulated by R2R3‐type MYB transcription factors, in particular by six members of the subgroup 12 , aromatic  or indole‐derived (tryptophan) glucosinolates  the down‐regulation of glucosinolate biosynthesis by targeted mutagenesis of the MYB28 and MYB29 transcription factor genes. In contrast to Arabidopsis, camelina harbours a hexaploid genome, which increases the complexity of the Cas9‐based gene disruption approach. After transformation of camelina plants with the cas9/gRNA constructs, T‐DNA‐free homozygous or heteroallelic knockout mutants were obtained via segregation. Analyses of seeds and roots revealed that the mutant lines contained strongly and differentially reduced contents of glucosinolates in the seeds, in some lines with a decrease down to zero, without affecting the oil and protein contents of the seeds.To reduce the glucosinolate levels in camelina seeds, we embarked on a Cas9‐mediated mutagenesis approach by pursuing two strategies: (i) the reduction in glucosinolate transport into the seeds by targeting the transporter genes atgtr1atgtr2 seeds lack glucosinolates , GTR2 , MYB28  and MYB29 .The two glucosinolate transporters AtGTR1 and AtGTR2 are involved in the translocation and accumulation of glucosinolates in Arabidopsis seeds. As a consequence, the Arabidopsis CsGTR1, CsGTR2 and CsMYB28 homeologs in different tissues, RNA was extracted from green seeds and roots of wild type (WT), and expression levels were determined by quantitative real‐time PCR (qPCR) using homeolog‐specific primers. All homeologs were more highly expressed in roots compared with the moderate expression in the seeds  in the roots was about 10‐fold higher, and more than 100‐fold higher for CsGTR1‐A and B. Therefore, CsGTR1‐A and CsGTR1‐B represent the predominating activities in the roots. The expression of the three homeologous CsMYB28 genes in the seeds was also lower by a factor of ~10 compared with roots.To study the expression of the CsGTR1 and CsGTR2, and three TSs for the mutagenesis of CsMYB28 and CsMYB29  were selected to mutagenize the homeologs of  Figures . All TSs Figures , and mos‐A Table . The PCR Figures .Tsp45I restriction sites of CsGTR1‐A, and eight lines with mutations in CsGTR2‐C  of the restriction analyses is shown in Figure GTR1 and GTR2) were single‐bp insertions of T (~60%) or A (~20%). Besides, a few events featured other insertions (+C or +27 bps) or short deletions . No mutations were found in TS2, TS3 or TS4 in these plants suggesting a low efficiency of the corresponding guide RNAs. Based on these results, three independent T‐DNA‐free lines designated G1, G2 and G3, which are descendants of lines 4, 15 and 16, respectively, were selected. The mutations in these lines are homozygous, and only the locus CsGTR1‐A in line G2 was biallelic with A or T insertions  or 137 , single T insertions cause one stop codon after amino acids 136 (CsGTR1‐A) or 138 , T2 offspring seeds were germinated to obtain plants with homozygous or heteroallelic mutations in one, two or all three CsMYB28 homeologs. Five T‐DNA‐free T3 mutant plants (designated M1–M5) were selected. The plants M1 (line 24) and M3 (7) are single mutants of CsMYB28‐B and C, respectively, M2 (8) is a double mutant of CsMYB28‐A and B, and M4 (7) and M5 (24) represent triple mutants in all three homeologs . Mutations in TS6/TS6* were mostly single nucleotide insertions , the remaining lines carried WT alleles. For TS7, no mutation was found in these plants, indicating low mutagenesis efficiency. Next, the mutations in TS5*, which differs to TS5 by one mismatch, were analysed for the three loci of CsMYB29 in the plants M1–M5 ; Figure et al., csgtr1csgtr2 mutations. Similar to the seeds, roots contained the aliphatic glucosinolates GL9, GL10 and GL11, but with a different composition of 43.5, 52.8 and 3.6 mol% of total glucosinolates in WT roots, respectively, compared with 31.3, 60.5 and 8.3 mol% in seeds. Therefore, the level of GL9 is elevated in roots at the expense of GL10 and GL11  involved in aliphatic glucosinolate biosynthesis . This result is in line with the Arabidopsis mutant atmyb28atmyb29 which shows a complete loss of aliphatic glucosinolates  carrying mutations in one or more CsMYB28 and CsMYB29 homeologs. Close inspection of the glucosinolate contents in the individual lines revealed that the CsMYB28 and CsMYB29 homeologs contribute to different extents to the accumulation of glucosinolates. While the glucosinolate content in seeds of the mutant M1 disrupted in CsMYB28‐B and CsMYB29‐C was not affected, line M3, which is mutagenized in CsMYB28‐C, CsMYB29‐A (heterozygous), B and C (both homozygous), showed a decrease in glucosinolate levels to 14.3 nmol/mg . In contrast, line M2, which is disrupted in the two homeologs CsMYB28‐A and B, was less severely affected with a glucosinolate content of 18.5 nmol/mg (82.8% of WT). In the roots, more pronounced decreases in the glucosinolate levels were observed. The roots of lines M1, M2 and M3 contained 4.6, 4.7 and 2.4 nmol/mg glucosinolates, respectively, corresponding to 36.6%, 37.5% and 19.4% of WT level (12.5 nmol/mg). The glucosinolate composition in seeds and roots of all lines was very similar to WT , et al., et al., Because rapeseed and other atgtr1atgtr2 that lacks glucosinolates, seeds of the camelina csgtr1csgtr2 mutants are still accumulating reduced amounts of glucosinolates  contribute to the accumulation of glucosinolates in the seeds of camelina.The presence of glucosinolates in the embryo may be derived from alternative import mechanisms. In fact, diffusion between maternal (integuments) tissue and the embryo has previously been ruled out  resulted in the complete loss of aliphatic glucosinolates. The various CsMYB28 and CsMYB29 homeologs presumably contribute to different extents to the synthesis of glucosinolates in camelina roots and seeds. The plant M2 only carries mutations in CsMYB28‐A and B and shows a minor reduction in glucosinolates in seeds, while root glucosinolate content is decreased to <40%. Therefore, CsMYB28‐A and B are highly relevant for glucosinolate production in the roots.The transcription factors AtMYB28, AtMYB29 and AtMYB76 are positive regulators of aliphatic glucosinolate synthesis in Arabidopsis, with overlapping functions but with distinct roles and lack of capability of complementing each other for the loss of glucosinolates in the respective mutants (Beekwilder BoMYB28 gene in Brassica oleracea (broccoli) caused a decrease in glucosinolate contents. The leaves and florets of the broccoli plants carrying mutations in the BoMYB28 homeologs C2 and C9 showed strong decreases in aliphatic glucosinolates, but indole derived‐glucosinolates were not significantly affected  have raised concerns about increased susceptibility to plant diseases  were sown on soil . Plants were grown in pots in the greenhouse under ambient light. During the winter period, additional light of 600 μmol s−1 m−2 for 16 h per day was applied.Seeds of http://n2t.net/addgene:71287; RRID:Addgene_71 287) was obtained from Qi‐Jun Chen . This vector contains an egg cell‐specific promoter for expression of the cas9 endonuclease, an AtU6‐26 promoter‐driven gRNA scaffold for insertion of target‐specific sequences, and a hygromycin B resistance cassette  leaves with the primer pair bn3308/bn3309, introducing 5′‐SpeI and 3′‐KpnI restriction sites (for oligonucleotides see Table XbaI restriction site) by the PcUbi promoter (with 3′‐KpnI) in pHEE401E required the re‐amplification of the cas9 sequence with the primers bn3310/bn3311 (5′‐KpnI/3′‐EcoRI). The PCR products of the PcUbi promoter and of cas9 were sub‐cloned into pJet1.2 , resulting in pJ‐PcUbi and pJ‐Cas9, respectively. The PcUbi sequence was released with KpnI, BglI  and inserted in pJ‐Cas9 linearized with KpnI and BglI, resulting in pJ‐PcUbi‐Cas9. The cloning of the DsRed marker required the removal of a BsaI restriction sequence from DsRed, because this site is also present in the cloning site for gRNA sequences of pHEE401E. Thus, the CaMV35S:DsRed sequence was amplified as two overlapping PCR products. Because of lacking suitable cloning sites in pHEE401E, additional amplification of sequences adjacent to the hygromycin B resistance cassette in pHEE401E was necessary. The 5′‐ and 3′‐adjacent sequences are intended to flank the 5′‐ and 3′‐ends (left and right flanking sequences) of the mutagenized CaMV35S:DsRed sequence, respectively, to allow the insertion into pHEE401E to replace the former hygromycin B resistance gene. These two adjacent sequences  were amplified as PCR products overlapping with CaMV35S:DsRed at its 5′‐ (left) and 3′‐ (right) ends with the primer pairs bn3312/bn3313 (EcoRI‐left‐pcr) and bn3318/bn3319 (right‐SacII‐pcr), respectively, with pHEE401E as template. The primers bn3312 and bn3319 contain overhangs with EcoRI and a SacII restriction sites, respectively. The two overlapping CaMV35S:DsRed sequences were amplified from the plant expression vector pBinGlyRed1‐IKU2  by amplification with the primer pair bn3312/bn3319 to obtain the final PCR fragment EcoRI‐left‐DsRed‐right‐SacII, which was sub‐cloned in pJet1.2 resulting in pJ‐EcoRI‐left‐DsRed‐right‐SacII. Finally, the sub‐cloned sequences in pJ‐PcUbi‐Cas9 and pJ‐EcoRI‐left‐DsRed‐right‐SacII were released with SpeI, EcoRI and EcoRI, SacII, respectively, and inserted in one step in pHEE401E which had been linearized with SpeI, SacII, to produce pHEE‐PcUCas9‐35SDsRed. The Golden Gate entry site in pHEE‐PcUCas9‐35SDsRed allows the cloning of several gRNAs. The assembly of gRNA‐scaffolds, TSs and promoters was achieved by PCR following a published protocol  in CsMYB29, diminishing the chances for off‐target effects. For mutagenesis of the homeologs of the CsGTR1 and CsGTR2 genes, the respective target‐specific sequences were added as overhangs to the oligonucleotides , BSA (B9000S), T4 DNA ligase buffer and BsaI‐HF . After 2 h of incubation at 37 °C, 1 μL of T4 DNA ligase (M0202S) was added followed by incubation at 18 °C for 2 h, 37 °C for 2 h and finally 50 °C for 5 min and 80 °C for 10 min to stop the reactions. The final construct, pHEE‐PcUCas9‐35SDsRed‐GTR1‐GTR2, was transferred into camelina accession WT139 by vacuum infiltration of the flowers with Agrobacterium tumefaciens C58  with ceramic beads in a Precellys homogenizer . The sample was centrifuged and the glucosinolates in the supernatant purified via ion exchange chromatography. The sample was loaded on a column containing 0.1 g Sephadex (DEAE A25120) soaked before overnight in 2 mL 0.5 m acetic acid buffer (pH 5 with NaOH). The resin with the bound glucosinolates was washed five times with one column volume of water and three times with 0.02 m acetic acid buffer (pH 5 with NaOH). Subsequently, 500 μL of a solution containing 1 U of sulfatase  in 0.02 m acetic acid buffer  was loaded on the column, the column sealed after the flow of three drops of buffer and incubated overnight. The desulfoglucosinolates were eluted with 2 mL of water, dried in a vacuum concentrator, dissolved in 200 μL of water and measured by HPLC. This protocol was also applied for glucosinolate extraction from embryo and seed coat samples.Glucosinolates were extracted following a published protocol with modifications  they were freeze dried and ground in a mortar. About 250–400 mg of ground material was subjected to extraction with 80% methanol at 60 °C for 4 h in the presence of 25 μL 4 mm sinigrin). Sephadex resin (1.0 g) was soaked overnight in 10 mL acetic acid buffer (0.5 m), centrifuged and the buffer was removed. Glucosinolates were bound to the Sephadex resin and purified in a batch procedure in 50 mL Falcon tubes. The resin was washed with 30 mL of water or acetic acid buffer (0.02 m) as described above, and water or buffer were removed after each step by centrifugation. Finally, the Sephadex resin with the bound glucosinolates was incubated overnight with 5 mL of a solution containing 3 U of sulfatase (see above). The desulfoglucosinolates were eluted in 30 mL of water followed by centrifugation to remove the Sephadex resin. The sample was concentrated in a rotary evaporator and the desulfoglucosinolates dissolved with 200 μL of water.Glucosinolates were extracted from roots with 40 mL of 80% methanol at 80 °C for 12 h in the presence of the internal standard  and detected by UV absorption at 229 nm. The flow rate was 0.4 mL/min with 5 μL injection volume  were performed after converting the lipids into fatty acid methyl esters which were quantified by GC  and fully developed green embryos using the QIAzol reagent . After digestion of genomic DNA with RNase‐free DNase I (Thermo Fisher Scientific), cDNA was synthesized by reverse transcription using the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). α‐Tubulin from camelina was used as housekeeping gene  csgtr1csgtr2 , and (b) csmyb28csmyb29 (M1–M5) mutant lines compared to wild type (WT).Table S1 Oligonucleotides used for the amplification of the region containing the target sequences of the three homeologs  each of CsGTR1, CsGTR2, CsMYB28 and CsMYB29.Table S2 Oligonucleotides used for the amplification of DNA fragments for modification of pHEE401E.Table S3 Oligonucleotides used for the PCR amplification of the homeologs  each of CsGTR1, CsGTR2 and CsMYB28, and for sequencing of the WT genes in accession WT139.Table S4 Oligonucleotides used for the amplification of the gRNA sequences containing the target sequences.Table S5 Oligonucleotides used for qPCR analysis of the homeologs  each of CsGTR1, CsGTR2, CsMYB28 and CsMAM1.Sequences S1 Sequence analyses of the three homeologous genes of each CsGTR1, CsGTR2, CsMYB28 and CsMYB29 in Camelina sativa accession WT139.Click here for additional data file.


RhOn+(n=1–4), we identify RhO3+as the 1A1'trioxidorhodium(VII) cation, the first chemical species to contain rhodium in the +7 oxidation state, which is the third‐highest oxidation state experimentally verified among all elements in the periodic table.Although the highest possible oxidation states of all transition elements are rare, they are not only of fundamental interest but also relevant as potentially strong oxidizing agents. In general, the highest oxidation states are found in the electron‐rich late transition elements of groups 7–9 of the periodic table. Rhodium is the first element of the 4d transition metal series for which the highest known oxidation state does not equal its group number of 9, but reaches only a significantly lower value of +6 in exceptional cases. Higher oxidation states of rhodium have remained elusive so far. In a combined mass spectrometry, X‐ray absorption spectroscopy, and quantum‐chemical study of gas‐phase RhO3+as a 1A1'trioxido rhodium(VII) cation in D3hpoint group symmetry, the first chemical species with rhodium in the +7 oxidation state.Teaming up with technetium and ruthenium, rhodium is the third element of the 4d series to form the rare +7 oxidation state, the third‐highest oxidation state among all elements. Here, gas‐phase X‐ray absorption spectroscopy, combined with quantum‐chemical calculations, are used to elucidate  The formal concept of oxidation states is of fundamental importance in chemistry to characterize properties of elements in compounds.1, 2 Td transition elements, ruthenium, in the form of tetraoxido ruthenium(VIII),[IrO4]+, represents the only known species with an element in oxidation state +9.4]2+,[PtO2]+.d orbitals increases for higher oxidation states.[4]+ and RhNO3, it has also been pointed out that highly covalent bonds in these species might lead to reconfiguration into isomers with rhodium in a lower oxidation state,RhO4+by oxygen elimination.[η1-O2RhO2]or dioxido‐peroxido η2-O2RhO2species with oxidation states of +5 and +6 instead of the tetraoxido isomer, RhO4,−, [BF4]−, or [AsF6]−, it might be possible to also stabilize RhO3+, based on its electron affinity as shown, e.g., for the stabilization of [IrO4]+.In summary, we have characterized the highest oxidation state of rhodium observed so far, rhodium(VII) in The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


The four cPu were undetectable in the mtDNA both in defective cells and in the wt counterparts (CSA and CSB), contrary to their detection in tDNA, indicating a nonappearance of hydroxyl radical (HO•) reactivity within the mtDNA. In order to assess the HO• reactivity towards purine nucleobases in the two genetic materials, we performed γ–radiolysis experiments coupled with the 8–oxo–Pu and cPu quantifications on isolated mtDNA and tDNA from wtCSB cells. In the latter experiments, all six purine lesions were detected in both of the DNA, showing a higher resistance to HO• attack in the case of mtDNA compared with tDNA, likely due to their different DNA helical topology influencing the relative abundance of the lesions.Mitochondrial (mt) DNA and nuclear (n) DNA have known structures and roles in cells; however, they are rarely compared under specific conditions such as oxidative or degenerative environments that can create damage to the DNA base moieties. Six purine lesions were ascertained in the mtDNA of wild type (wt) CSA (CS3BE–wtCSA) and wtCSB (CS1AN–wtCSB) cells and defective counterparts CS3BE and CS1AN in comparison with the corresponding total (t) DNA (t = n + mt). In particular, the four 5′,8–cyclopurine (cPu) and the two 8–oxo–purine (8–oxo–Pu) lesions were accurately quantified by LC–MS/MS analysis using isotopomeric internal standards after an enzymatic digestion procedure. The 8–oxo–Pu levels were found to be in the range of 25–50 lesions/10 It. It2•−),d breaks . It has d breaks . Indeed,d breaks . Consistd breaks ,32.• radicals with genetic material via C5′ radical chemistry of the purine moieties [R and 5′S diastereoisomeric forms  the model reactivity of mtDNA and tDNA in an “isolated” context, using the reaction of genetic material with HO• radicals under biomimetic conditions [• radical reactivity estimating the contribution of different helical topology in distinct genetic pools such as mtDNA and nDNA.cPu, as transcriptional blocking lesions, have been identified as molecular defects in neurodegenerative processes . In partnditions  and measCSA and CSB SV40–transformed cell lines were established and cultured as previously described . More pr2O) with a concentration of 0.5 mg/mL; in particular, 33.5 μg of tDNA was suspended in 67 μL and 45.9 μg of mt–DNA was suspended in 91.8 μL. The solution was placed in a glass vial of 2 mL containing a 300 µL glass insert, flushed with N2O for 10 min and exposed to gamma rays at room temperature (22 ± 2 °C) using a 60Co–Gammacell apparatus at different doses (dose rates: 1.44 Gy/min). The exact absorbed radiation dose was determined with the Fricke chemical dosimeter, by taking G(Fe3+) 1.61 μmol J−1 [Each sample of mtDNA and tDNA from CSA1N–wtCSB was dissolved in double distilled water , the samples were filtered off by centrifugation through a 3 kDa microspin filter, and were cleaned up and enriched by an HPLC–UV system coupled with a sample collector and injected into the LC–MS/MS system. The quantification of the modified nucleosides was carried out by a triple–stage quadrupole mass spectrometer  using positive electrospray ionization (ESI) following a gradient program  and the detection was executed in multiple reaction monitoring mode (MRM) using the two most intense and characteristic precursor/product ion transitions for each lesion [The purine DNA lesions levels were quantified as described previously ,36,46,54h lesion ,56.t-test was used for the statistical analysis and a two–tailed p-value < 0.05 and p-value < 0.01 were considered to indicate a statistically significant difference.All of the measurements were performed in triplicate and the data were expressed as mean ± standard deviation (SD). The unpaired p = 0.011).The mtDNA from wtCSA (CS3BE–wtCSA), wtCSB (CS1AN–wtCSB), and defective counterparts CS3BE and CS1AN cell lines cultivated under standard atmospheric oxygen tension have been isolated. After hydrolysis of the genetic material to single nucleosides by an enzymatic cocktail containing nucleases, analysis by liquid chromatography with tandem mass spectrometry (LC–MS/MS) was performed for the determination of the modified nucleosides (four cPu and two 8–oxo–Pu), in accordance with a recently optimized protocol ,46,55,56Comparing the 8–oxo–dG and 8–oxo–dA levels of mtDNA  with the• radicals towards mtDNA. The presence of 8–oxo–Pu in the absence of cPu should be informative of the occurrence of the molecular rather than the radical reactivity. Indeed, similar to HO• radicals, other oxidizing species such as H2O2 or ONOO− are also able to generate 8–oxo–Pu as DNA lesions [2O2 scavenger, has shown that high levels of H2O2 are present in CSA defective cells [The absence of cPu lesions in the mtDNA in both defective cells and in the wt counterparts (CSA and CSB) may indicate the absence of reactivity with HO lesions ,37. It ive cells . Moreoveve cells ,58.In humans, mitochondrial DNA represents about 1–10% of total cellular DNA . In contrast with the invariable copy number of the nuclear genome (diploid), a single cell can contain many copies of mtDNA dependent on different processes involved in the mitochondrial homeostasis, such as mitochondrial replication, mitochondrial dynamics, and mitophagy. It has been estimated that each human cell contains from hundreds to thousand mitochondria . Cells wR–diastereoisomers of both cdA and cdG cause greater distortion of the DNA backbone and are better substrates of NER than the corresponding S ones [• reactivity towards DNA helical topology better, we selectively generated HO• radicals by ionizing irradiations in the presence of isolated mtDNA or tDNA samples from wtCSB cells and carried out the quantification of the six purine lesions as described in the next section.Previous work in human HeLa cell extracts indicated that cdA and cdG lesions are excised with a similar efficiency by NER and that the g S ones ,61. Howeg S ones . In orde• radicals are known for their reactivity and ability to cause chemical modifications to DNA, the site of attack being both the base moieties (85–90%) and the 2–deoxyribose units [• radicals generated by irradiation.The findings in the cell cultures motivated our interest to investigate the reactivity of mtDNA and tDNA taken out of their biological contexts. HOse units . Therefoaq−, HO•, and H• as shown in Reaction 1, together with H+ and H2O2. The values in parentheses represent the radiation chemical yields (G) in units of μmol J−1. In an N2O–saturated solution (~0.02 M of N2O), eaq− are converted into the HO• radical via Reaction 2 (k2 = 9.1 × 109 M−1 s−1), with G(HO•) = 0.55 μmol J−1, i.e., HO• radicals and H• atoms account for 90 and 10%, respectively, of the reactive species [• radicals and H• atoms with DNA (Reactions 3 and 4) have been reported to be ca. 2.5 × 108 M−1 s−1 and 6 × 107 M−1 s−1, respectively [Radiolysis of neutral water leads to the reactive species e species ,64. The ectively ,63.H2O R–cdG, 5′R–cdA, 5′S–cdG, and 5′S–cdA in the function of irradiation doses in both tDNA and mtDNA . Furthermore, the slope of the obtained lines represents the number of lesions formed per Gy, as summarized in 7 nu/Gy, the ratios 8–oxo–dG/8–oxo–dA were 6.9 and 4.8 in tDNA and mtDNA, respectively. It is worth mentioning that the same ratio in calf–thymus DNA was found to be 7.7 under similar experimental conditions, although the number of lesions/Gy was four to five times higher with respect to tDNA [• radical with ds–oligonucleotide [• to the C8 position of the guanine moiety accounts for a minor percentage (~10%), whereas the main yield of 8–oxo–dG is produced by a one–electron oxidation reaction involving DNA radicals [•–adducts of guanine and adenine moieties afforded a variety of products, including 8–oxo–dG and 8–oxo–dA [From the analysis of the data reported in  to tDNA . The meccleotide ,56 and ccleotide ,66 has bradicals ,66. The 8–oxo–dA ,67. It iR–cdG, 5′R–cdA, 5′S–cdG, and 5′S–cdA were also formed in the same fashion, but in lower yields , similar to the case of 8–oxo–dG and 8–oxo–dA . The observed differences between tDNA and mtDNA in the reaction with HO• were likely due to the different topology of the nuclear and mitochondrial genome [Intramolecular cyclization products 5′r yields . The slo product  [33,34,3l genome .7 nu) in both the tDNA and mtDNA of CS1AN–wtCSB cells irradiated at different doses. The scale referring to tDNA  the formation 8–oxo–Pu was twice that of cPu, indicating a true competition of the two pathways in 2O2 generated from the irradiation of water (see reaction (1)), and (ii) the values of both 8–oxo–Pu and cPu in tDNA were ~8 times higher than in mtDNA, revealing a different accessibility of HO• to mtDNA and nDNA structures. The structure of mtDNA is arranged in a loop, which is loosely supercoiled [• on tertiary DNA helical forms of supercoiled (SC), open circular (OC), and linear (L) conformation was known [From the reaction of the HOercoiled . The comas known . Purine R and 5′S, for each 2′–deoxyadenosine and 2′–deoxyguanosine moieties. The diastereomeric ratio (R/S) attracts interest as it can inform on mechanistic issues that are related to structural conformations of both isomers in association with their abundance. R/S ratios for cdG and cdA lesions of irradiated tDNA and mtDNA samples. The reported data indicate that the ratios are similar within tDNA or mtDNA and independent of the irradiation dose, i.e., the R/S average ratios are 0.52 for cdG and 1.75 for cdA in tDNA, whereas in mtDNA they are 1.35 for cdG and 1.74 for cdA  [R/S cdA was always higher that the cdG ratio (approximately 1.3-1.6 times). Moreover, in various tissues  of prdx1–/– mice, the R/S cdG was found to be 0.23, 0.14, and 0.40 while the R/S cdA was 0.11, 0.23, and 0.08 [R form is always more abundant in both cdG and cdA. In this respect, it is worth recalling that the R/S ratios of 8.3 for cdG and 6 for cdA were obtained in water upon the irradiation of free nucleosides  [R/S ratios can be used to support further biological implications in the formation and/or repair of these lesions, although a clear scenario for the R/S formation is still missing [It is worth underlining that in previous studies using extracted tDNA from various animal tissues, the e S form , this isrocesses  are simi5 times)  similar 5 times) ; in the and 0.08 . As Figubundant) ,74, indi missing . The str missing . We are • radical reactivity and that the amount of 8–oxo–Pu in mtDNA was consistent with the contribution of DNA reactivity with oxidizing species, such as H2O2 or ONOO−. Moreover, a suggestive hypothesis to explain the absent accumulation of cPu adducts in the mtDNA of CS cells could play a crucial role in specific mechanisms devoted to the maintenance of the mitochondrial genome integrity. In particular, in mammalian cells, a degradation mechanism of damaged mtDNA molecules has been recently discovered [• radicals with isolated tDNA and mtDNA, the values of all six purine lesions in the tDNA are ~8 times higher than in mtDNA. We evidenced a different accessibility of HO• into mtDNA and nDNA structures which were associated with different helical topologies.In this work, we measured the purine lesions in mtDNA of four cellular lines, i.e., wtCSA, wtCSB, and their defective counterparts, and compared with their analogous data of tDNA. The 8–oxo–Pu lesions were comparable in mtDNA and tDNA, although they were found to be constantly higher in mtDNA for all four cellular lines. The cPu lesions were undetectable in mtDNA, suggesting at least a 100 times lower level than in tDNA. We evidenced, for the first time, the DNA damage scenario as a contribution of two distinctive pathways, i.e., the molecular ROS and the radical ROS species, which can be clearly distinguished by comparing our results in the cell cultures with the cPu levels of the irradiation experiments. Indeed, we evaluated that the absence of cPu lesions in the mtDNA of the cellular experiment may indicate a nonappearance of HOscovered . Moreove