A review of ^(17)O isotopic labeling techniques for solid-state NMR structural studies of metal oxides in lithium-ion batteries

Recent advances in utilizing ^(17)O isotopic labeling methods for solid-state nuclear magnetic resonance(NMR)investigations of metal oxides for lithium-ion batteries have yielded extensive insights into their structural and dynamic details.Herein,we commence with a brief introduction to recent research on lithium-ion battery oxide materials studied using ^(17)O solid-state NMR spectroscopy.Then we delve into a review of ^(17)O isotopic labeling methods for tagging oxygen sites in both the bulk and surfaces of metal oxides.At last,the unresolved problems and the future research directions for advancing the ^(17)O labeling technique are discussed.

Stable isotope labeling of nanomaterials for biosafety evaluation and drug development

Quantitative information,such as environmental migration,absorption,biodistribution,biotransformation,and elimination,is fundamental and essential for the nanosafety evaluations of nanomaterials.Due to the complexity of biological and environmental systems,it is challenging to develop quantitative approaches and tools that could characterize intrinsic behaviors of nanomaterials in the organisms.The isotopic tracers are ideal candidates to tune the physical properties of nanomaterials while preserving their chemical properties.In this review article,we summarized the stable isotope labeling methods of nanomaterials for evaluating their environmental and biological effects.The skeleton labeling protocols of carbon nanomaterials and metal/metal oxide nanoparticles were introduced.The advantages and disadvantages of stable isotope labeling were discussed in comparison with other quantitative methods for nanomaterials.The quantitative information of nanomaterials in environmental and biological systems was summarized along with the biosafety data.The benefits for drug development of nanomedicine were analyzed based on the targeting effects,persistent accumulation,and safety.Finally,the challenges and future perspectives of stable isotope labeling in nanoscience and nanotechnology were discussed.

Investigation of Nitrite Production Pathway in Integrated Partial Denitrification/Anammox Process via Isotope Labelling Technique and the Relevant Microbial Communities

In this study,the nitrogen removal performance of partial denitrificaiton/anammox(PDA)process was investigated by using an UASB reactor.High total nitrogen(TN)removal efficiency(91.97%)was achieved at an influent nitrogen loading rate of 0.64 kg/(m3·d).Anammox bacteria did execute the function of converting nitrate to nitrite in PDA system according to ^(15)N isotope labeling experiments and the contribution was approximately 36.3%.Candidatus_Brocadia,Candidatus_Kuenenia and Thauera were functional strains for anammox and denitrification process,respectively.Thauera and Candidatus_Brocadia were more important for TN removal at high loading rates(0.64 kg/(m3·d)).This result can provide a theoretical and technical foundation for the application of the PDA process.

Fe-N-C core-shell catalysts with single low-spin Fe(Ⅱ)-N_(4)species for oxygen reduction reaction and high-performance proton exchange membrane fuel cells

Fe-N-doped carbon materials(Fe-N-C)are promising candidates for oxygen reduction reaction(ORR)relative to Pt-based catalysts in proton exchange membrane fuel cells(PEMFCs).However,the intrinsic contributions of Fe-N_(4)moiety with different chemical/spin states(e.g.D1,D2,D3)to ORR are unclear since various states coexist inevitably.In the present work,Fe-N-C core-shell nanocatalyst with single lowspin Fe(Ⅱ)-N_(4)species(D1)is synthesized and identified with ex-situ ultralow temperature Mossbauer spectroscopy(T=1.6 K)that could essentially differentiate various Fe-N_(4)states and invisible Fe-O species.By quantifying with CO-pulse chemisorption,site density and turnover frequency of Fe-N-C catalysts reach 2.4×10^(-9)site g^(-1)and 23 e site~(-1)s^(-1)during the ORR,respectively.Half-wave potential(0.915V_(RHE))of the Fe-N-C catalyst is more positive(approximately 54 mV)than that of Pt/C.Moreover,we observe that the performance of PEMFCs on Fe-N-C almost achieves the 2025 target of the US Department of Energy by demonstrating a current density of 1.037 A cm^(-2)combined with the peak power density of 0,685 W cm^(-2),suggesting the critical role of Fe(Ⅱ)-N_(4)site(D1).After 500 h of running,PEMFCs still deliver a power density of 1.26 W cm^(-2)at 1.0 bar H_(2)-O_(2),An unexpected rate-determining step is figured out by isotopic labelling experiment and theoretical calculation.This work not only offers valuable insights regarding the intrinsic contribution of Fe-N_(4)with a single spin state to alkaline/acidic ORR,but also provides great opportunities for developing high-performance stable PEMFCs.

HDPairFinder:A data processing platform for hydrogen/deuterium isotopic labeling-based nontargeted analysis of trace-level amino-containing chemicals in environmental water

The combination of hydrogen/deuterium(H/D)formaldehyde-based isotopic methyl labeling with solid-phase extraction and high-performance liquid chromatography–high resolution mass spectrometry(HPLC-HRMS)is a powerful analytical solution for nontargeted analysis of trace-level amino-containing chemicals in water samples.Given the huge amount of chemical information generated in HPLC-HRMS analysis,identifying all possible H/Dlabeled amino chemicals presents a significant challenge in data processing.To address this,we designed a streamlined data processing pipeline that can automatically extract H/D-labeled amino chemicals from the raw HPLC-HRMS data with high accuracy and efficiency.First,we developed a cross-correlation algorithm to correct the retention time shift resulting from deuterium isotopic effects,which enables reliable pairing of H-and D-labeled peaks.Second,we implemented several bioinformatic solutions to remove false chemical features generated by in-source fragmentation,salt adduction,and natural13C isotopes.Third,we used a data mining strategy to construct the AMINES library that consists of over 38,000 structure-disjointed primary and secondary amines to facilitate putative compound annotation.Finally,we integrated these modules into a freely available R program,HDPairFinder.R.The rationale of each module was justified and its performance tested using experimental H/D-labeled chemical standards and authentic water samples.We further demonstrated the application of HDPairFinder to effectively extract N-containing contaminants,thus enabling the monitoring of changes of primary and secondary N-compounds in authentic water samples.HDPairFinder is a reliable bioinformatic tool for rapid processing of H/D isotopic methyl labeling-based nontargeted analysis of water samples,and will facilitate a better understanding of N-containing chemical compounds in water.

Pyrolysis mechanism of glucose and mannose: The formation of 5-hydroxymethyl furfural and furfural

Fast pyrolysis of biomass will produce various furan derivatives, among which 5-hydroxymethyl furfural（5-HMF） and furfural（FF） are usually the two most important compounds derived from holocellulose. In this study, density functional theory（DFT） calculations are utilized to reveal the formation mechanisms and pathways of 5-HMF and FF from two hexose units of holocellulose, i.e., glucose and mannose. In addition, fast pyrolysis experiments of glucose and mannose are conducted to substantiate the computational results, and the orientation of 5-HMF and FF is determined by 13C-labeled glucoses. Experimental results indicate that C1 provides the aldehyde group in both 5-HMF and FF, and FF is mainly derived from C1 to C5 segment. According to the computational results, glucose and mannose have similar reaction pathways to form 5-HMF and FF with d-fructose（DF） and 3-deoxy-glucosone（3-DG） as the key intermediates. 5-HMF and FF are formed via competing pathways. The formation of 5-HMF is more competitive than that of FF, leading to higher yield of 5-HMF than FF from both hexoses. In addition, compared with glucose,mannose can form 5-HMF and FF via extra pathways because of the epimerization at C2 position. Therefore, mannose pyrolysis results in higher yields of 5-HMF and FF than glucose pyrolysis.

High-performance isotope-labeling liquid chromatography mass spectrometry for investigating the effect of drinking Goji tea on urine metabolome profiling

Urine is a human biofluid that is widely used for metabolomics research on disease biomarker discovery.Ideally,the metabolome profiles generated from comparative groups of individuals should mainly consist of the endogenous human metabolites that reflect the healthy states of the individuals.However,external factors,such as diet,may alter the urine metabolome profile by either introducing a significant amount or variety of exogenous metabolites to urine or inducing changes of the metabolome profile.Thus,strict control of the external factors during the sample collection process is critical for urine metabolomics aimed at discovery of disease biomarkers.In this work,we describe a study to determine the effect of drinking Goji tea,which is considered a nutritional supplement drink in some regions of the world,on urine metabolome profile.The purpose of this work is not to determine the nutritional values of Goji tea,but to investigate whether drinking a moderate amount of Goji tea 1-3 h(short-term effect)or 12 h(longer-term effect)before urine collection can cause significant variations of urine metabolome profiles.A highly sensitive dansylation isotope labeling liquid chromatography mass spectrometry(LC-MS)method was used to determine the urine metabolomes before and after drinking Goji tea.From the studies of the short term(<3 h)and longer term(12 h)effects of drinking Goji tea,it is clear that the consumption of a moderate amount of Goji tea does not affect the urine metabolome significantly.Fasting for 12 h should be sufficient to remove any potential interference of Goji metabolites from the human urine metabolome profile.

Proteomic analysis of energy metabolism and signal transduction in irradiated melanoma cells

AIM: To analyze proteomic and signal transduction alterations in irradiated melanoma cells. METHODS: We combined stable isotope labeling with amino acids in cell culture (SILAC) with highly sensitive shotgun tandem mass spectrometry (MS) to create an efficient approach for protein quantification. Protein protein interaction was used to analyze relationships among proteins. RESULTS: Energy metabolism protein levels were significantly different in glycolysis and not significantly different in oxidative phosphorylation after irradiation. Conversely, tumor suppressor proteins related to cell growth and development were downregulated, and those related to cell death and cell cycle were upregulated in irradiated cells. CONCLUSION: Our results indicate that irradiation induces differential expression of the 29 identified proteins closely related to cell survival, cell cycle arrest, and growth inhibition. The data may provide new insights into the pathogenesis of uveal melanoma and guide appropriate radiotherapy.

Triggering in-plane defect cluster on MoS_(2) for accelerated dinitrogen electroreduction to ammonia

Electrochemical nitrogen reduction reaction (eNRR) is an alternative promising manner for sustainable N2 fixation with low-emission. The major challenge for developing an efficient electrocatalyst is the cleaving of the stable Ntriple bondN triple bonds. Herein, we design a new MoS_(2) with in-plane defect cluster through a bottom-up approach for the first time, where the defect cluster is composed of three adjacent S vacancies. The well-defined in-plane defect clusters could contribute to the strong chemical adsorption and activation towards inert nitrogen, achieving an excellent eNRR performance with an ammonia yield rate of 43.4 ± 3 μg h^(−1) mgcat.^(−1) and a Faradaic efficiency of 16.8 ± 2% at −0.3 V (vs. RHE). The performance is much higher than that of MoS_(2) with the edge defect. Isotopic labeling confirms that N atoms of produced NH4+ originate from N2. Furthermore, the in-plane defect clusters realized the alternate hydrogenation of nitrogen in a side-on way to synthesize ammonia. This work provides a prospecting strategy for fine-tuning in-plane defects in a catalyst, and also promotes the progress of eNRR.

Quantitative proteomic determination of diethylstilbestrol action on prostate cancer

Diethylstilbestrol （DES） has a direct cellular mechanism inhibition on prostate cancer. Its action is independent from the oestrogen receptors and is preserved after a first-line hormonal therapy. We aimed to identify proteins involved in the direct cellular inhibition effects of DES on prostate cancer. We used a clonogenic assay to establish the median lethal concentration of DES on 22RV1 cells. 22RV1 cells were exposed to standard and DES-enriched medium. After extraction, protein expression levels were obtained by two-dimensional differential in-gel electrophoresis （2D-DIGE） and isotope labelling tags for relative and absolute quantification （iTRAQ）. Proteins of interest were analysed by quantitative RT-PCR and western blotting. The differentially regulated proteins （P〈0.01） were interrogated against a global molecular network based on the ingenuity knowledge base. The 2D-DIGE analyses revealed DES-induced expression changes for 14 proteins （〉 1.3 fold; P〈0.05）. The iTRAQ analyses allowed the identification of 895 proteins. Among these proteins, 65 had a modified expression due to DES exposure （i.e., 23 overexpressed and 42 underexpressed）. Most of these proteins were implicated in apoptosis and redox processes and had a predicted mitochondrial expression. Additionally, ingenuity pathway analysis placed the OAT and HSBP1 genes at the centre of a highly significant network. RT-PCR confirmed the overexpression of OAT （P=0.006） and HSPB1 （P=0.046）.

Study on Effect of Pesticides on Rice Leaf Photosynthate Export Rate with Tracer Kinetics

Impacts of three pesticides, triazophos, jingganmycin and bisultap, on the export rate of pho-tosynthate of rice leaf were studied with 14C for rice varieties, spraying time and application rate. Dynamics data of photosynthate of labeled rice functional leaves of different treatments were recorded with living plant nutrient detection instrument with multi-probes through tracer method of radioactive nuclide. Compartment analysis model and mathematical equations were constructed using principle and method of kinetics analysis, calculating constant (K) of export rate. The result showed that the export rate of photosynthate of labeled leaf reduced after pesticide treatments. The reduction of the export rate was positively related with the pesticide application rates and mainly attributed to decrease of photosynthetic rate.

Spatial patterns nitrogen transfer models of ectomycorrhizal networks in a Mongolian scotch pine plantation

Ectomycorrhizal(EM)networks provide a variety of services to plants and ecosystems include nutrient uptake and transfer,seedling survival,internal cycling of nutrients,plant competition,and so on.To deeply their structure and function in ecosystems,we investigated the spatial patterns and nitrogen(N)transfer of EM networks usingN labelling technique in a Mongolian scotch pine(Pinus sylvestris var.mongolica Litv.)plantation in Northeastern China.In August 2011,four plots(20 × 20 m)were set up in the plantation.125 ml 5 at.%0.15 mol/LNHNOsolution was injected into soil at the center of each plot.Before and 2,6,30 and 215 days after theN application,needles(current year)of each pine were sampled along four 12 m sampling lines.Needle total N andN concentrations were analyzed.We observed needle N andN concentrations increased significantly over time afterN application,up to 31 and0.42%,respectively.There was no correlation between needle N concentration andN/N ratio(R2=0.40,n=5,P=0.156),while excess needle N concentration and excess needleN/N ratio were positively correlated across different time intervals(R~2=0.89,n=4,P\0.05),but deceased with time interval lengthening.NeedleN/N ratio increased with time,but it was not correlated with distance.NeedleN/N ratio was negative with distance before and 6th day and 30th day,positive with distance at 2nd day,but the trend was considerably weaker,their slop were close to zero.These results demonstrated that EM networks were ubiquitous and uniformly distributed in the Mongolian scotch pine plantation and a random network.We found N transfer efficiency was very high,absorbed N by EM network was transferred as wide as possible,we observed N uptake of plant had strong bias forN andN,namely N fractionation.Understanding the structure and function of EM networks in ecosystems may lead to a deeper understanding of ecological stability and evolution,and thus provide new theoretical approaches to improve conservation practices for the management of the Earth’s ecosystems.

Calcium Carbide:From Elemental Carbon to Isotope-Economic Synthesis of^(13)C_(2)-Labeled Heterocycles

^(13)C-Carbon is the most available source of carbon-13.It is a relatively inexpensive solid material,which can be easily converted to calcium carbide-^(13)C_(2).In current work,Ca^(13)C_(2)was used for in situ generation of^(13)C_(2)-acetylene in 1,3-dipolar cycloaddition and[4+2]cycloaddition reaction.For the first time,1H-1,2,3-triazoles-4,5-^(13)C_(2)and isoxazoles-4,5-^(13)C_(2)were synthesized using calcium carbide-^(13)C_(2).A Diels-Alder type cycloaddition of 3,6-di(pyridin-2-yl)-1,2,4,5-tetrazine and Ca^(13)C_(2)was investigated,and the best way for the synthesis of 3,6-di(pyridin-2-yl)pyridazine-4,5-^(13)C_(2)was proposed for the first time.Here we perform a detailled description of NMR spectra of^(13)C_(2)-labeled triazoles,isoxazoles and 3,6-di(pyridin-2-yl)pyridazine.

Glycoside-specific metabolomics combined with precursor isotopic labeling for characterizing plant glycosyltransferases

Glycosylation by uridine diphosphate-dependent glycosyltransferases(UGTs)in plants contributes to the complexity and diversity of secondary metabolites.UGTs are generally promiscuous in their use of acceptors,making it challenging to reveal the function of UGTs in vivo.Here,we described an approach that combined glycoside-specific metabolomics and precursor isotopic labeling analysis to characterize UGTs in Arabidopsis.We revisited the UGT72E cluster,which has been reported to catalyze the glycosylation of monolignols.Glycoside-specific metabolomics analysis reduced the number of differentially accumulated metabolites in the ugt72e1e2e3 mutant by at least 90%compared with that from traditional untargeted metabolomics analysis.In addition to the two previously reported monolignol glycosides,a total of 62 glycosides showed reduced accumulation in the ugt72e1e2e3 mutant,22 of which were phenylalanine-derived glycosides,including 5-OH coniferyl alcohol-derived and lignan-derived glycosides,as confirmed by isotopic tracing of[^(13)C_(6)]-phenylalanine precursor.Our method revealed that UGT72Es could use coumarins as substrates,and genetic evidence showed that UGT72Es endowed plants with enhanced tolerance to low iron availability under alkaline conditions.Using the newly developed method,the function of UGT78D2 was also evaluated.These case studies suggest that this method can substantially contribute to the characterization of UGTs and efficiently investigate glycosylation processes,the complexity of which have been highly underestimated.

Stable isotope labelled mass spectrometry for quantification of the relative abundances for expressed proteins induced by PeaT1

The protein elicitor from the mycelium of Alternaria tenuissima has been isolated.The elicitor triggered resistance to the tobacco mosaic virus in tobacco by inducing relative oxygen species,but without causing hypersensitive necrosis.The elicitor is reported to impart resistance against Verticillium dahliae and to increase yield in cotton,but its mechanism is not yet clear.In this study,the stable isotope labelled mass spectrometry method was used to quantify the relative abundances of protein expression induced by PeaT1 in Arabidopsis.A significant difference in the relative abundances for the expression of different proteins related to metabolism,modification,regulatory,defense,stress and antioxidation was found in Arabidopsis.

Electrosynthesis of ^(15)N-labeled amino acids from ^(15)N-nitrite and ketonic acids

^(15)N isotope-labeled amino acids(^(15)N-amino acids)are crucial in the fields of biology,medicine,and chemistry.^(15)N-amino acids are conventionally synthesized through microbial fermentation and chemical reductive amination of ketonic acids methodologies,which usually require complicated procedures,high temperatures,or toxic cyanide usage,causing energy and environmental concerns.Here,we report a sustainable pathway to synthesize ^(15)N-amino acids from readily available ^(15)N-nitrite(^(15)NO_(2)-)and biomass-derived ketonic acids under ambient conditions driven by renewable electricity.A mechanistic study demonstrates a ^(15)N-nitrite→^(15)NH_(2)OH→^(15)N-pyruvate oxime→^(15)N-alanine reaction pathway for ^(15)N-alanine synthesis.Moreover,this electrochemical strategy can synthesize six ^(15)N-amino acids with 68%–95%yields.Furthermore,a ^(15)N-labeled drug of ^(15)N-tiopronin,the most commonly used hepatitis treatment drug,is fabricated using ^(15)N-glycine as the building block.Impressively,^(15)N sources can be recycled by the electrooxidation of ^(15)NH4^(+) to ^(15)NO_(2)-with a method economy.This work opens an avenue for the green synthesis of ^(15)N-labeled compounds or drugs.

Single-cell Raman and functional gene analyses reveal microbial P solubilization in agriculture waste-modified soils

Application of agricultural waste such as rapeseed meal(RM)is regarded as a sustainable way to improve soil phosphorus(P)availability by direct nutrient supply and stimulation of native phosphate‐solubilizing microorganisms(PSMs)in soils.However,exploration of the in situ microbial P solubilizing function in soils remains a challenge.Here,by applying both phenotype‐based single‐cell Raman with D_(2)O labeling(Raman‐D_(2)O)and genotype‐based high‐throughput chips targeting carbon,nitrogen and P(CNP)functional genes,the effect of RM application on microbial P solubilization in three typical farmland soils was investigated.The abundances of PSMs increased in two alkaline soils after RM application identified by single‐cell Raman D_(2)O.RM application reduced the diversity of bacterial communities and increased the abundance of a few bacteria with reported P solubilization function.Genotypic analysis indicated that RM addition generally increased the relative abundance of CNP functional genes.A correlation analysis of the abundance of active PSMs with the abundance of soil microbes or functional genes was carried out to decipher the linkage between the phenotype and genotype of PSMs.Myxococcota and C degradation genes were found to potentially contribute to the enhanced microbial P release following RM application.This work provides important new insights into the in situ function of soil PSMs.It will lead to better harnessing of agricultural waste to mobilize soil legacy P and mitigate the P crisis.

Exogenous nitrogen input skews estimates of microbial nitrogen use efficiency by ecoenzymatic stoichiometry

Background Ecoenzymatic stoichiometry models(EEST)are often used to evaluate microbial nutrient use efficiency,but the validity of these models under exogenous nitrogen(N)input has never been clarified.Here,we investigated the effects of long-term N addition(as urea)on microbial N use efficiency(NUE),compared EEST and^(18)O-labeling methods for determining NUE,and evaluated EEST’s theoretical assumption that the ratios of standard ecoenzymatic activities balance resource availability with microbial demand.Results We found that NUE estimated by EEST ranged from 0.94 to 0.98.In contrast,estimates of NUE by the^(18)O-labeling method ranged from 0.07 to 0.30.The large differences in NUE values estimated by the two methods may be because the sum ofβ-N-acetylglucosaminidase and leucine aminopeptidase activities in the EEST model was not limited to microbial N acquisition under exogenous N inputs,resulting in an overestimation of microbial NUE by EEST.In addition,the acquisition of carbon by N-acquiring enzymes also likely interferes with the evaluation of NUE by EEST.Conclusions Our results demonstrate that caution must be exercised when using EEST to evaluate NUE under exogenous N inputs that may skew standard enzyme assays.

Salicylic acid biosynthesis is not from phenylalanine in Arabidopsis

The phytohormone salicylic acid(SA)regulates biotic and abiotic stress responses in plants.Two distinct biosynthetic pathways for SA have been well documented in plants:the isochorismate(IC)pathway in the chloroplast and the phenylalanine ammonia-lyase(PAL)pathway in the cytosol.However,there has been no solid evidence that the PAL pathway contributes to SA biosynthesis.Here,we report that feeding Arabidopsis thaliana with Ring-13C-labeled phenylalanine(13C6-Phe)resulted in incorporation of the13C label not into SA,but into its isomer 4-hydroxybenzoic acid(4-HBA)instead.We obtained similar results when feeding13C6-Phe to the SA-deficient ics1 ics2 mutant and the SA-hyperaccumulating mutant s3h s5h.Notably,we detected13C6-SA when13C6-benzoic acid(BA)was provided,suggesting that SA can be synthesized from BA.Furthermore,despite the substantial accumulation of SA upon pathogen infection,we did not observe incorporation of13C label from Phe into SA.We also did not detect13C6-SA in PAL-overexpressing lines in the kfb01 kfb02 kfb39 kfb50 background after being fed13C6-Phe,although endogenous PAL levels were dramatically increased.Based on these combined results,we propose that SA biosynthesis is not from Phe in Arabidopsis.These results have important implications for our understanding of the SA biosynthetic pathway in land plants.

Quantitative and Functional Phosphoproteomic Analysis Reveals that Ethylene Regulates Water Transport via the C-Terminal Phosphorylation of Aquaporin PIP2;1 in Arabidopsis

Ethylene participates in the regulation of numerous cellular events and biological processes, including wa- ter loss, during leaf and flower petal wilting. The diverse ethylene responses may be regulated via dynamic interplays between protein phosphorylation/dephosphorylation and ubiquitin/26S proteasome-mediated protein degradation and protease cleavage. To address how ethylene alters protein phosphorylation through multi-furcated signaling pathways, we performed a lSN stable isotope labelling-based, differential, and quantitative phosphoproteomics study on air- and ethylene-treated ethylene-insensitive Arabidopsis double loss-of-function mutant ein3-1/eill-1. Among 535 non-redundant phosphopeptides identified, two and four phosphopeptides were up- and downregulated by ethylene, respectively. Ethylene- regulated phosphorylation of aquaporin PIP2;1 is positively correlated with the water flux rate and water loss in leaf. Genetic studies in combination with quantitative proteomics, immunoblot analysis, protoplast swelling/shrinking experiments, and leaf water loss assays on the transgenic plants expressing both the wild-type and S280A/S283A-mutated PIP2;1 in the both Col-O and ein3eill genetic backgrounds suggest that ethylene increases water transport rate in Arabidopsis cells by enhancing S280/S283 phosphorylation at the C terminus of PIP2;1. Unknown kinase and/or phosphatase activities may participate in the initial up- regulation independent of the cellular functions of EIN3/EIL1. This finding contributes to our understanding of ethylene-regulated leaf wilting that is commonly observed during post-harvest storage of plant organs.