Homogeneous aqueous phase reactions were conducted using soluble Fe(III), where particle growth/agglomeration were monitored using dynamic light scattering. Mass yield experiments of insoluble soot-like dark brown to black particles were as high as 40%. Hygroscopicity growth factors (κ) of these insoluble products under sub- and super-saturated conditions ranged from 0.4–0.6, higher than that of levoglucosan, a prominent proxy for biomass burning organic aerosol (BBOA). Soluble products analyzed using chromatography and mass spectrometry revealed the formation of ring coupling products of o- and p-aminophenols and their primary oxidation products. Heterogeneous reactions of aminophenol were also conducted using Arizona Test Dust (AZTD) under simulated aging conditions, and showed clear changes to optical properties, morphology, mixing state, and chemical composition. These results highlight the important role of iron redox chemistry in BrC formation under atmospherically relevant conditions.Nitrogen-containing organic carbon (NOC) in atmospheric particles is an important class of brown carbon (BrC). Redox active NOC like aminophenols received little attention in their ability to form BrC. Here we show that iron can catalyze dark oxidative oligomerization of  o- and p-aminophenols under simulated aerosol and cloud conditions.Iron is the most abundant redox active transition metal in mineral dust, but its role in nitrogen-containing organic carbon formation remains largely unexplored. Here, the authors show that Fe(III) catalyzes the dark oxidative oligomerization of  Atmospheric BrC refers to the class of organic compounds that efficiently absorb solar and terrestrial radiation. The majority of BrC originates from biomass burning events and their cloud condensation nuclei activity and hygroscopicity parameter, κ, ranges from 0.1 to 0.4, which slightly increases upon particle photochemical or oxidative aging5. Nitrogen-containing organic carbon (NOC) in atmospheric particles are increasing in importance as a class of BrC8, which originates from primary and secondary sources10. Primary emission sources of NOC particles were estimated to be 27.4 Tg yr−1 from biogenic, soil, ocean, and anthropogenic (that includes biomass burning) sources9. Molecular characterization and microscopy analysis of biomass burning organic aerosols (BBOA) including tar balls showed uniform distribution of nonvolatile NOC11, with only 14 ± 3% of the N-bearing compounds occur as ammonium sulfate12. Recent advances in analytical tools allowed for direct measurements of inorganic  and organic nitrogen in ambient aerosol samples as opposed to the difference method between total nitrogen and inorganic nitrogen13.Quantifying the contribution of atmospheric brown carbon (BrC) to climate forcing and aerosol-cloud interactions remains a large source of uncertainty in climate models due to their chemical complexity and variable sources14. In general, nitro-aromatic compounds are characterized as chemical tracers from biomass burning events along with levoglucosan, forming during pyrolysis of plant biopolymers17. Atmospheric amines include aliphatic and aromatic compounds19. Low molecular weight with 1–6 carbons aliphatic amines are the most abundant in the atmosphere with global emission fluxes dominated by trimethylamine (TMA) and ethanolamine from animal husbandry, marine and biomass burning sources18. Glycine was found to be the most abundant amino acid in atmospheric aerosols from the decay of biological matter21. The reactivity and hydration properties of some aliphatic amines were explored for their ability to form secondary organic aerosol (SOA)23 and act as cloud condensation nuclei (CCN)25. Aromatic amines in atmospheric particles on the other hand are dominated by phenylamine , p-aminophenol and phenylamine with alkyl substituents26. While the global fluxes of these molecules have not been reported, their concentrations in the gas phase and ultrafine particulate matter are source-dependent mostly from industrial emissions27. Multiphase redox chemistry could also lead to the formation of aromatic amines from the reduction of nitrobenzenes28 and nitrophenols30, which warrants further investigation under cloud and aerosol conditions. Supplementary Scheme a values of some of the abovementioned compounds with phenol and catechol as reference compounds. As summarized by Yu et al.13, the concentration of water-soluble organic nitrogen accounts for 10–30% of water-soluble total nitrogen and ranges from 1 to several hundred nmol N m−3 in remote oceanic to polluted urban areas, respectively.There are two major NOC classes in BrC that reflect the oxidation state of nitrogen: oxidized NOC that include nitrocatechols and nitrophenols, and reduced NOC that includes amines, imines and N-heterocycles 7. For example, particle phase formation pathways between ammonia and primary amines and carbonyl groups lead to BrC formation7. Efficient formation of NOC was observed from ammonia gas phase uptake on newly formed secondary organic aerosol (SOA) from the oxidation of α-pinene and m-xylene31, and evaporation of glyoxal-ammonium sulfate droplets32. The interaction of ammonium with oxidized organics in single particles during summer and fall seasons at an urban location  was found to explain 50% of NOC formation in processes facilitated by NOx and relative humidity33. The reactive uptake of glyoxal into ammonium-containing salts or methylaminium-containing salts was also found to produce BrC at different RHs35. Direct photosensitized oxidation of vanillin, a phenolic carbonyl, in the presence of ammonium nitrate was also found to produced aqueous SOA36. Reactions with nitrogen dioxide (NO2) and nitrate radical (NO3) led to nitration of polycyclic aromatic hydrocarbons yielding nitrophenols, nitrocatechols40, nitration of isoprene-derived epoxides during SOA formation41, and nitro-heterocyclic compounds such as nitropyrrole42.Secondary sources of NOC that contribute to BrC constitute an active area of research44. Iron-containing atmospheric particles are ubiquitous in the atmosphere45 from natural sources such as in mineral dust46 and anthropogenic combustion including coal burning48, biomass burning50, and brake wear51. As recently highlighted in references52, there are realistic scenarios in which phenolic compounds and NOC from biomass burning and industrial sources encounter iron-containing particles through gas-particle partitioning or particle mixing. This is because emissions from these sources containing semivolatile gases and particles are often spread by wind, which also lifts crustal particles off the ground. Long-range transport of mineral dust53 and BBOA54 by wind contribute to atmospheric aging and growth of particles partly due to partitioning of organic vapors into Fe-containing particles and reactions with acids. Other pathways that lead to dust aging and particle growth remain unexplored. One potentially important pathway is the one catalyzed by iron since aging processes increase the solubility of iron which can reach millimolar levels in aerosol liquid water as reported by Gen et al.55.The role of transition metals in NOC formation and contribution to BrC remains largely unexplored56. These studies suggested that the catalytic power of the catechol/o-quinone redox pair59 together with the complex equilibria for Fe(III) and Fe(II) ions with catechol and dicarboxylic acids should play a key role in the formation of secondary BrC. The compounds shown in Supplementary Scheme 2, –C(O)OH) capable of complexing iron. The arylamines are electron-rich and redox active and can undergo enzymatic and electrochemical oxidation leading to oligomerization and polymerization64. Supplementary Table o- and p-aminophenol (oAP and pAP). Metal-free oxidation of oAP and pAP results in the formation of polymeric products referred to as poly(o-aminophenol) and poly(p-aminophenol), which are desired for their corrosion inhibition properties63. The oxidation potential for glycine and other aliphatic amines is higher than 0.7 V at pH 1, hence, the redox reaction with Fe(III) would be non-spontaneous67.We recently reported the formation of soluble NOC from dark reactions between Fe(III) and catechol that formed insoluble black polycatechol particles and colored water-soluble oligomers under conditions characteristic of viscous multicomponent acidic aerosol systems containing ammonium68 with impacts on their morphology and optical properties.The objective of this investigation is to explore the role of iron-catalyzed reactions in forming BrC from aromatic amines under conditions that mimic dark processing in cloud water, in the absence and presence of ammonium sulfate. Our studies here show the efficient and rapid formation of dark and insoluble NOC oligomers in addition to soluble products. The cloud condensation nucleation (CCN) efficiency of these non-combustion particles was also investigated and shown to be higher than levoglucosan. The iron content in Arizona Test Dust (AZTD) was also found to catalyze reactions with aminophenols over atmospherically relevant timescales that simulate long-range dust transportoAP and pAP was investigated as a function of the organic:Fe molar ratio under acidic conditions and two different background solutions, 0.01 M KCl SO4  with pAP than oAP. According to Supplementary Table oAP involves dimerization, whereas that of pAP involves hydrolysis of quinone imine to form benzoquinone. Increasing the pH increases the dimerization reaction rate  and decreases the hydrolysis reaction rate69. Supplementary Table 70. Therefore, it is likely that redox reactions between the aminophenols/iron species pair become less thermodynamically favorable and the kinetics of the redox steps become slower with decreasing pH. Supplementary Scheme The mass yield of the insoluble products relative to the initial mass of the organic precursor ranged from 13 to 38% depending on pH and reactants molar ratio (Supplementary Table 3 (Supplementary Table oAP reacted with Fe(III) over a wide range of molar ratios, the colored solid products were further analyzed using thermogravimetric analysis (TGA) to examine their thermal properties relative to that of poly(o-aminophenol) and poly(p-aminophenol) reported in the literature63. Supplementary Figure oAP and the solid products from its dark aqueous phase reaction with Fe(III). oAP undergoes a one-step thermal degradation process that starts at 120 °C and ends around 200 °C during which the sample lost nearly 91% of its initial mass. However, the thermogram for the solid reaction product shows that the sample undergoes a three-stage decomposition pattern: ~267 °C with about 12% mass loss corresponding to the evaporation of water molecules and volatile compounds, ~321 °C with about 70% mass loss due to the loss of the oligomeric/polymeric part of the samples, and ~450 °C due to the thermal conversion of the iron content to α-Fe2O3, the most stable form of iron oxides at high temperature71, resulting in about 7% mass residue. The digital photos of the trays containing the samples show the dark red color of the residual material at the end of the TGA analysis. Supplementary Table α-Fe2O3 (x%) to be 4.8% and 1159 g mol−1, respectively. The thermogram in Supplementary Figure o-aminophenol) suggesting structural differences between the two materials analyzed, which is expected given the differences in the experimental conditions that led to the polymerization of the aminophenols63. The average molecular weight determined for both poly(o-aminophenol) and poly(p-aminophenol) using gel permeation chromatography was 297,000 g mol−163. Hence, the lower effective molar weight calculated above from our experiments suggests oligomers as the main products under our experimental conditions.Fig. oAP and pAP with the assignment of observed vibrational modes. There are similarities between the spectra in Supplementary Figure o-aminophenol)64 and poly(p-aminophenol)63 whose suggested structures are shown in Fig. − = 212.1 and 213.06 were observed and assigned to the dimer from the reaction of Fe(III) with oAP (Mwt = 109 g mol−1). These mass features were not observed using pAP as the organic precursor. The mass spectrum of the reactant FeCl3 is also shown as a reference. The most intense mass features were observed at the same locations for the thin films from the reaction of FeBr3 with oAP confirming their assignment to organic species. The mass ranges corresponding to the trimers and tetramers contained much less intense features or overlapping cluster features assigned to residual iron chloride and iron hydroxide species72. In summary, the characterization of the colored solid products suggests that the reaction conditions produced oligomers of oAP and pAP that are not as polymeric as those produced from metal-free polymerization reactions64.Moreover, Supplementary Figure oAP and pAP with Fe(III) was monitored in situ using DLS as shown in Fig. oAP as the organic reactant  molar ratios. Slower rates of particle growth/agglomeration were observed for the 1:2 and 1:3 molar ratios. Fig. oAP:Fe molar ratio, which decreased with either reactant present in excess. This observation can be explained in light of the mechanism shown in Supplementary Scheme oAP:Fe molar ratio greater than 1. For molar ratios less than 1, the initial concentration of the reactant oAP is rate-limiting.Particle growth/agglomeration rate of ant Fig. , average4)2SO4, as the background solution appeared to increase the rate of particle growth/agglomeration for 1:3 and the 1:2 oAP:Fe molar ratio  by a factor of 4 relative to rates observed at low ionic strength using 0.01 M KCl 2SO4 and sodium chloride solutions, Wang et al.75 reported that for the same neutral organic compound, (NH4)2SO4 has a higher ‘salting out’ effect than NaCl. As emphasized by the authors75, (NH4)2SO4 does not only affect the solvation of organic compounds in water, but could influence the reactive fate of some solvated organics. Since both Fe(III) and oAP are present at 1000x lower concentration than ammonium sulfate, it is likely that in our studies here, the ‘salting out’ effect of oAP in the 1:3 and 1:2 ratio by ammonium sulfate resulted in concentrating the organics with Fe(III) in a solvation cage leading to enhancements in the rate of particle growth/agglomeration. This process might be facilitated by the presence of four and five water molecules complexed to Fe(III) since the Fe(SO4)+ and Fe(SO4)2− are the dominant aqueous phase species56. This finding warrants further investigation. The next section describes in detail the optical properties of the soluble and insoluble products from the reactions of aminophenols with Fe(III).Higher ionic strength adjusted using 1 M ammonium sulfate, (NHoAP and (b) pAP with Fe(III) in the pH range 3–4.5. For reference, the absorbance spectra of standard oAP (1.6 × 10−4 M) and pAP (3 × 10−4 M) solutions are also shown, which were converted to mass-normalized absorption coefficient (MAC)7 spectra per Eq. l is the sample path length and Cmass is the mass concentration of the aminophenol. The spectra show π→π* transitions of the aromatic ring at 277 and 296 nm for oAP and pAP. The spectra for the filtrates are different than the standard solutions and from each other suggesting that soluble products have distinct structures and amounts depending on the aminophenol:Fe ratio. Chromatographic and mass spectrometric analyses of these products are presented in the next section. Here we emphasize that some of these products have absorbances in the near UV and visible (λ > 290 nm) wavelengths that overlap with the actinic flux, most notably the broad feature at 400 nm from the reactions of oAP with Fe(III) at the 3:1 and 1:1 oAP:Fe molar ratios. The spectrum of the filtrate from the reaction containing 2:1 oAP:Fe molar ratio is identical to those observed for the aforementioned ratios, and hence not shown here. This broad feature is absent from the 1:3 oAP:Fe molar ratio suggesting undetectable amounts of the soluble reaction products. This broad feature at 400 nm together with nitrogen from oAP suggests the presence of a quinone imine as we previously analyzed56. The MAC values in the 300–500 nm range between (1–2) × 104 cm2 g−1 are comparable to those from primary BBOA (103–104 cm2 g−1)76. The next section describes in detail the analysis of soluble products in the filtrate solutions using UHPLC-UV-MS and ion chromatography MS.Fig. Moreover, the insets in Fig. The spectra show that scattering from the suspended particles is dominant with wavelength-dependent features. The broad band between 300 and 500 nm in the insets of Fig. oAP (purple filled trace) with Fe(III) in a 3:1 ratio. The wavelength range was selected to illustrate the absorption of relevant products from the near UV to the center of the visible spectrum. Additionally, the use of an ESI/MS(+) detector allow us to extract the m/z+ values for the corresponding chromatographic peaks reported. Further, analysis of the previous sample was performed after spike addition with 4-nitrophenol, catechol, hydroquinone, and 4,4’-dihydroxybiphenyl. The green filled trace in Fig. tr) of oAP reactant labeled as peak 1 in Fig. tr = 3.08 min. The absorption maxima of oAP occur at maximum wavelengths of λmax = 201, 230 and 284 nm  with H+ (M + H+) at m/z+ 110.14 (M = C6H7ON), and also with acetonitrile (M + CH3CN + H+) at m/z+ 151.10. For product identification, multiple formulas could be assigned to the many isomers that exist for a given mass. The nitrogen rule was taken into consideration when assigning even and odd molecular formulas. No additional constrains were used to limit the number of atoms or double bond equivalents. More details are provided in the Figure  nm Fig. . The postr = 4.07 min, which forms an ion at m/z+ 227.15. This mass-to-charge ratio corresponds to an adduct of M + 2 NH4+ + 2 CH3CN–H+. The elemental formula of the cationic peak adduct is C10H19O2N5+, as confirmed for the spike addition of catechol to the same sample that increased the peak height without changing peak shape and position. The extracted UV–visible spectrum for peak 2 is displayed in Fig. 2 group of oAP and generate catechol43 Alternatively the substitution of an H atom by HO should  displays an extended weak absorption at λmax = 555 nm for an ion at m/z+ 336.27, which is 4 Da lighter than the previous trimer suggesting the formation of two new rings among two oAP and one 4-aminocatechol molecules. Such a structure for Peak 7 can be represented by the molecule of 2-amino-8-hydroxy-14,14a-dihydrobenzooxazinophenoxazin-3(12H)-one  in Fig. oAP and one catechol molecules. Peak 9 is shown at tr = 7.36 min -one  with λmax = 312 and 426 nm and m/z+ 319.17 for the ion C18H11O4N2+ is a proton adduct of similar origin to the previous peaks as described by the molecule of 2-hydroxybenzooxazinophenoxazin-3(5aH)-one  with Fe(III) followed by the interpretation of these peaks per the suggested mechanism in Supplementary Scheme o- and p-aminophenols and their primary oxidation products. Given their structure and optical properties, these products would be reactive under day- and night-time conditions in the bulk and at interfaces, which is worth exploring.Supplementary Figure oAP and pAP with Fe(III) were investigated under subsaturated and supersaturated conditions using a Humidified Tandem Differential Mobility Analyzer (H-TDMA) and a Cloud Condensation Nuclei Counter (CCNC), respectively. Fig. oAP + Fe(III) reaction were slightly more hygroscopic than from the pAP + Fe(III) reaction, and both types were more hygroscopic than levoglucosan. Organic particles exposed to 85% RH swelled and the measured growth factor, Gf , was 1.54 ± 0.06, 1.46 ± 0.03, and 1.20 ± 0.03 for particles from oAP + Fe(III), pAP + Fe(III), and levoglucosan, respectively , pAP + Fe(III). The high growth factors measured for oAP + Fe(III), pAP + Fe(III) can be attributed to the presence of high-density hydrophilic functional groups in their polymeric structures. Previous literature has ascribed the high hygroscopicity measured for insoluble polymers due to the presence of hydroxyl groups in their structures78. In addition, The high surface area of their polymeric structures is conductive for water condensation which can drive droplet growth as reported by Gohil et al.79. At supersaturated conditions, oligomers from the oAP + Fe(III) and pAP + Fe(III) continued to grow, and the dry particle critical diameters (Dd) were measured at four constant supersaturations (0.4–1.1%). At each supersaturation, the critical diameters increased from the oAP + Fe(III) to pAP + Fe(III) to levoglucosan  is also reported  were slightly more hygroscopic than those from the pAP + Fe(III) with 81. For comparison, κCCN values for the oligomers from the oAP + Fe(III), pAP + Fe(III), and levoglucosan were 0.57 ± 0.06, 0.48 ± 0.02, and 0.21 ± 0.03, respectively.The average single hygroscopicity parameter ted Fig. . Under boAP + Fe(III) and pAP + Fe(III), the κCCN values are greater than their κCCN values to surface tension depression of a pure water droplet83 and adsorption-driven droplet growth84. Surface tension pendant droplet measurements were conducted for both oligomer types in range of 0.3–2.0 g L−1  and the κ values reported here for oligomers under sub- and supersaturated conditions are within range of previously published literature values of direct biomass emissions88. Here, the measured oligomer κ-values are greater than 0.3 and less than 0.6. In addition, when comparing the CCN efficiencies of these oligomers relative to levoglucosan, we observe that these nitrogen-containing oligomers are more hygroscopic than levoglucosan. Köhler theory predicts water uptake to be inversely correlated with molecular weight and this relationship has been previously illustrated in literature with various organic aerosols89. Since the insoluble oligomers from the reaction of oAP + Fe(III) and pAP + Fe(III) are shown to have a higher molecular weight than levoglucosan (see above), other factors need to be considered in explaining their hygroscopic properties. Recent work78 showed that organic polymeric compounds found in BBOA may be more hygroscopic than previously thought. A study by Malek et al.78 investigated the hygroscopicity of two relevant and biomass burning based insoluble polymeric aerosols: polycatechol and polyguaiacol and found their к-values to range from 0.03 to 0.25. Their hygroscopicity was attributed to their chemical structure, specifically the presence of hydroxyl groups. Here, the к-values of the oligomers from the reaction of oAP + Fe(III) and pAP + Fe(III) are above 0.3. This can be attributed to the presence of a high density of hydriphilic carbonyl groups within their polymeric structure. Additionally, the high water uptake can be also explained by the polymers’ propensity for organic aerosol swelling analogous to water uptake of hydrophilic polymer substances91. Furthermore, previous literature93 also showed organic compounds containing sulfates or nitrates have enhanced hygroscopicity compared to organic carbohydrates. This enhancement in hygroscopicity is comparable to what we observe with oAP + Fe(III) and pAP + Fe(III). Peng et al.94 investigated the hygroscopicity of organosulfate species  and found κ values to range between 0.459–0.206. In addition, Estillore et al.95 examined the water uptake of more than 10 organosulfates using H-TDMA, and reported the growth factor range to be between 1.30–1.50 The results of these two reports are comparable to the water uptake results obtained in this study. Hence, nitrogen-containing oligomers formed here offer a fascinating insight on the structural composition and their effect on water uptake. Thus, our results indicate that nitrogen-containing organic oligomers in BBOA may be as efficient as inorganic and/or organosulfate aerosol-cloud seeds from biomass sources.For both oligomer types from the reaction of ues Fig. . PreviouoAP and pAP as a function of pH over two weeks. AZTD has abundant Lewis acid sites97 that act as electron acceptors including Fe(III). Figure 98. At pH 1, the average (±1σ < 8%) of triplicate measurements of DFe in all filtrate vials equals 7.7, 10, and 7.6 ppm for the control, reaction with oAP, and pAP, respectively. This result suggests that oAP slightly enhanced the dissolution of iron by 1.3x than pAP. At pH 1, below the first pKa1 for oAP, the protonated -NH3+ substituent is present with the -OH group on the benzene ring. Also, most surface sites on the minerals in AZTD are positively charged due to points of zero charge below 7 (Table 99). oAP forms a complex with iron(III)(hydr)oxides in AZTD in a monodentate fashion via the -OH group with -NH3+ stabilized by hydrogen bonding with neighboring sites  to solution and the formation of a phenoxide radical. pAP was reported to adsorb poorly on hematite100 due to the location of the -NH3+ substituent in the para position  to the solution .At pH 7, DFe values are below the limit of detection of the instrument due to the significant decrease in acid-promoted dissolution kinetics. Reaction slurries containing the aminophenols appear much darker than the control slurry. Both aminophenols are neutral and have their maximum adsorption capacity at pH 7pAP reaction. Since the filtrates are transparent, the dark brownish to black color observed in the AZTD slurries was due to the formation of insoluble products associated with reacted AZTD particles. Supplementary Figure Upon filtration following the 15 d of simulated dust aging, photographs of the dry filters in Fig. oAP and R-AZTD-pAP). The MAC spectra were calculated using the initial mass concentration of oAP and pAP of 1 mM (1.1 × 10−4 g mL−1). The MAC spectrum of R-AZTD-oAP filtrate shows similar features to the one shown in Fig. oAP and Fe(III) at the (1:1) and (3:1) oAP:Fe molar ratios. Similarly, the MAC spectrum of R-AZTD-pAP filtrate in Supplementary Figure pAP and Fe(III) at the (3:1) pAP:Fe molar ratio. These results suggest that our simulated AZTD aging experiments reflect excess organics relative to dissolved iron reaction conditions. Importantly, the proposed reactions of soluble oAP and pAP with Fe(III) result in absorption features of oligomers formed between the reactants and their primary oxidation products that explain the registered changes of MAC and MEC . The dissolved Fe(III) and organic concentrations are atmospherically relevant for air masses containing aged dust from long range transport over urban areas and aim to simulate reactions in deliquesced aerosol liquid water. In real aerosol systems, aminophenol concentrations are source-dependent and might be lower than the ones used here, especially in remote areas. Also, there are other organic and inorganic components that can complex Fe(III) and affect its reactivity. For the experiments using 1 M ammonium sulfate, which is about 1000x higher concentration than Fe(III) and the aminophenols, oligomerization reactions took place despite sulfate being a strong chelating agent for Fe(III). This result is similar to what we observed earlier for catechol104. Here, we show that dust aging chemistry due to reaction with redox active nitrogen-containing compounds may change the radiative forcing of dust aerosol to that of black carbon. It also shows that reaction products may enhance the hygroscopic properties of dust in cloud seeding due to changes to surface functional groups. Changes to dust morphology, mixing state, and chemical composition due to the chemistry we report here opens avenues for exploring changes to dust photochemical reactivity influenced by the nitrogen-containing oligomeric content. Given the wide variety of NOC in ambient aerosol particles and the analytical challenges of directly measuring their concentration and speciation, more field research is needed to confirm and evaluate the relevance and importance of these lab results in the atmosphere. In particular, collecting ambient particles in the fine and ultrafine size modes from urban/industrial geographical regions on the path of long-transported dust plumes would be most influenced by this type of chemical reactions.Our results provide insights into our understanding of mineral dust redox reactivity and its impact on climate forcing and hygroscopic properties. The key variables that affect the radiative forcing (heating or cooling effect) of dust include height of dust layer, particle size and aerosol optical depthAll chemicals—listed in the SI—were used as received without further purification.4)2SO4 background solution. Additional details are listed in the SI.DLS experiments were performed to monitor growth of insoluble polymeric materials forming in solution within the first hour of reaction time. Each reaction took place in a 20 mL vial containing 5 mL of a known concentration of the organic precursor prepared in either 0.01 M KCl or 1 M (NHThe mass yields of insoluble products were determined from 2 h reactions using 3:1 and 1:1 molar ratio organic reactant:Fe by weighing the filters before and after filtration followed by overnight drying followed by characterization. More details are in the SI.Ultraviolet-visible (UV–vis) spectra of diluted control and reacted solutions and AZTD slurries were collected using a UV–vis spectrophotometer (Ocean Optics USB 4000) in a 1-cm quartz cuvette. Selected filtrate samples and the corresponding controls were analyzed after dilution in water (18.2 MΩ cm) with 5% acetonitrile (LC/MS grade) by 10 and 16.67 times, respectively, by UHPLC-UV-(ESI)MS  equipped with a C18 column . More details are in the SI.106 and Cloud Condensation Nuclei Counter (CCNC) measurements108 with more details in the SI.Supplementary Figure 99. More details are in the SI.These experiments were performed using AZTD and following a modified procedure from that reported by Link et al.Supplementary Information


Phytophthora infestans populations from five locations along a mountainous hill in China and sequenced the effector gene Pi02860 from >300 isolates. To minimize the influence of other ecological factors, isolates were sampled from the same potato cultivar on the same day. We also expressed the gene to visualise its cellular location, assayed its pathogenicity and evaluated its response to experimental temperatures. We found that Pi02860 exhibited moderate genetic variation at the nucleotide level which was mainly generated by point mutation. The mutations did not change the cellular location of the effector gene but significantly modified the fitness of P. infestans. Genetic variation and pathogenicity of the effector gene were positively associated with the altitude of sample sites, possibly due to increased mutation rate induced by the vertical distribution of environmental factors such as UV radiation and temperature. We further found that Pi02860 expression was regulated by experimental temperature with reduced expression as experimental temperature increased. Together, these results indicate that UV radiation and temperature are important environmental factors regulating the evolution of effector genes and provide us with considerable insight as to their future sustainable action under climate and other environmental change.Effector genes, together with climatic and other environmental factors, play multifaceted roles in the development of plant diseases. Understanding the role of environmental factors, particularly climate conditions affecting the evolution of effector genes, is important for predicting the long-term value of the genes in controlling agricultural diseases. Here, we collected  Plant diseases have deleterious effects on ecological integrity and agricultural productivity, greatly threatening natural sustainability and global food security. For example, damage caused by plant diseases such as chestnut blight and Dutch elm disease to primary and secondary forests not only reduces species richness and the associated ecological services  but has Effector proteins, either directly secreted into the apoplast or translocated into host cells, play critical roles in the antagonistic interaction between hosts and pathogens . They arPhytophthora infestans Avr1 and Avr4 were removed from the effector repertoire by pseudogenization to escape the corresponding R1 and R4 detection in host (Phytophthora infestans Avr2 escapes host immunity by protein disordering through which shifts the effector from an avirulent type to virulent type (P. infestans Avr3a overcomes R3a recognition by potato crop through point mutations in two residues (K80I103 to E80M103) where protein (effector)-protein (receptor) interactions take place  How is the genetic diversity generated? (iii) What are the pathogenicity and cellular consequences of the observed mutations? and (iv) how may climatic or climate-related factors such as UV radiation and air temperature affect the performance and spatial distribution of genetic variation in the effector gene? Altitude change provides a strong reflection of climatic gradients across short spatial distances, offering a great opportunity to study the influence of climate conditions particularly UV light and temperature on the biology and ecology of species  what is the genetic variation of  species . For exaPhytophthora infestans isolates were collected in a single day during the 2016 late blight epidemic season from a single potato cultivar (Hui-2) grown in five fields along along a mountainous hill located at Huize, Yunnan. The altitudes of the five fields ranged from 1,976 to 2,677 m and were designated as A–E from lower to higher altitude, respectively  and rifampicin (50 μg/ml). Detailed information on the sample collection and P. infestans isolation can be found in our previous publications (ectively . One hunications .Phytophthora infestans isolates were cultured on rye B agar at 18°C in the dark. Mycelia (~100 mg) were harvested after 15 days cultivation, transferred into sterile, 2 ml centrifuge tubes and lyophilized with a vacuum freeze dryer . The lyophilized mycelia were ground to powder with a mixer mill . Total DNA was extracted using a Plant gDNA Miniprep Kit  according to the manufacturer’s instructions. The genomic DNA was suspended in 50 μl of ultrapure water and stored at −20°C until use.Pi02860 specific primers (For: 5′-ACTCACCGTCACCCTCATTC-3′ and Rev: 5′-AACTTTGACTCCGACCGTTG-3′) were designed according to the conserved upstream and downstream of the reference sequence (PITG_02860) downloaded from NCBI and used to amplify the Pi02860 effector gene. PCR reactions were carried out in a 25 μl reaction volume using a thermal cycler (Applied Biosystems 2720). The reaction cocktail contained 1 × PCR buffer, 0.2 mM dNTPs, 1 unit of TransStart KD Plus DNA Polymerase, 0.2 μM of primers and 20 ng of template DNA. PCR amplification was started with an initial denaturation step of 94°C for 2 min, followed by 30 cycles of amplification at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 50 s, and ended with a further extension cycle at 72°C for 10 min. The PCR products were separated by 1% agarose gel electrophoresis and sent to Sangon Biotech company for sequencing using an ABI3730 automated DNA sequencer .A pair of Nicotiana benthamiana and a universal susceptible potato variety Desiree were grown in a greenhouse at 20°C in 60% humidity. The greenhouse was supplemented with 16 h-light at the intensity of 120–150 μmol m−2 s−1, nutrient and water were supplied at necessary. Nicotiana benthamiana plants were used for agroinfiltration at 4–5 weeks old and potato leaves at 5–6 weeks old were used for P. infestans infection analysis.Pi02860 haplotypes without a signal peptide were amplified from genomic DNAs of corresponding P. infestans isolates. PCR products were ligated into pEarlyGate 104  using a Vazyme ClonExpressII One Step Cloning Kit, and then transformed into Agrobacterium tumefaciens strain AGL1. The Avr3a and INF1 constructs with N-terminal GFP tag were kindly provided by Dr. Qin He from Huazhong Agricultural University, Wuhan, China.After removing those with mutations in the start codon or translating to the same isoforms, 16 Agrobacterium tumefaciens strain AGL1 transformed with the vector constructed above was grown overnight in LB medium containing selective antibiotics at 28°C, pelleted, resuspended in infiltration buffer , and adjusted to the required optical density measured at 600 nm (OD600) before being infiltrated into N. benthamiana leaves . For co-expression, agrobacterial cultures carrying the appropriate vector constructs were mixed prior to infiltration.Phytophthora infestans strain 88,069 .n 88,069  was usedPi02860 effectors was investigated on N. benthamiana plants by confocal imaging. In this analysis, N. benthamiana cells were imaged at 2 DAIs using a Nikon Ti-E inverted microscope with Plan Apo 40X/0.95 dry objective. GFP was excited by 488-nm light shed from an argon laser, and 500–530 nm emissions were captured. To minimize potential artifacts of ectopic protein expression, only single optical images from leaf cells expressing low levels of the protein fusions were collected and analysed.Cellular localization of P. infestans isolates with different Pi02860 haplotypes were inoculated onto the detached leaves of the universal susceptible potato cultivar at low (10°C), near optimum (18°C) (near optimum) and high (25°C) temperature of the pathogen infection (EasyPure plant RNA Kits (TransGen Biotech) according to the manufacturer’s instructions. RNA was quantified using a Nanodrop 1000 (Thermo Scientific) and cDNA was synthesized using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech) according to the manufacturer’s instructions. QRT-PCR was performed using TransStart Top Green qPCR SuperMix (TransGen Biotech) and run on a QuantStudio 5 Real-Time PCR Instrument (Applied Biosystems) using QuantStudio Design & Analysis software v1.4.2 with specific primers . The expression of the effector gene was analyzed using the ΔΔCt method  of haplotype i and Y is the mean lesion size (INF1 cell death suppression) in the population. Associations of sequence diversity with biological characteristics of the effector gene and altitude were evaluated by Pearson’s correlation  translate into one identical isoform and two haplotypes (Hap_2 and Hap_4) translate into another identical isoform , produced lesions larger than Hap_8, Avr3a and the negative control (EV). Hap_1, Hap_3 and Hap_6 which have one SNP in 48K-R compared to the wild type Pi02860 effector, as well as other haplotypes except Hap_8, produced larger lesions than Hap_2 and Hap _4 did. Hap_8, which has a terminal truncation starting at the 36th amino acid position, produced significantly smaller lesions than other haplotypes including Avr3a  compared to the near optimum infection temperature of the pathogen (~19°C), and the expression level was further reduced as the experimental temperature increased to 25°C .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer RV declared a shared affiliation with one of the authors JZ to the handling editor at the time of review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A total of 40 of the 70 genes were present in > 90% of the samples, and significant differential gene expression identified for 29 genes. We identified 4 distinct regulation patterns for synthesizing enzymes, as well as a distinct regulation pattern for degradations and transporting enzymes. This study charts the expression of endometrial endocannabinoid system genes across the menstrual cycle. Altered expression of genes that control endocannabinoid may allow fine control over endocannabinoid concentrations and their influence on cellular function, maturation and differentiation as the endometrium matures through the menstrual cycle.Endocannabinoids mediate cellular functions and their activity is controlled by a complex system of enzymes, membrane receptors and transport molecules. Endocannabinoids are present in endometrium, a cyclical regenerative tissue requiring tightly regulated cellular mechanisms for maturation. The objective of this study was to investigate the gene expression of key elements involved in the endocannabinoid system across the menstrual cycle. RNA was isolated from endometrial tissue and genome-wide gene expression datasets were generated using RNA-sequencing. An a priori set of 70 genes associated with endocannabinoid system were selected from published literature. Gene expression across the menstrual cycle was analyzed using a moderated  Collectively endocannabinoids and these proteins are termed the endocannabinoid system (ECS)2. Altered expression of any ECS component can lead to variation in endocannabinoid activity and alter their impact of cellular function.Endocannabinoids are small lipid-based molecules that play critical roles in regulating cellular functions. The most well characterised endocannabinoids are anandamide (AEA) and 2-arachdonoylglycerol (2-AG). Their production, concentration and cellular influence is mediated by a complex interaction of proteins that regulate their synthesis, transport, membrane binding, trafficking and degradation6. The remaining three pathways are two-step processes involving phospholipase C (PLC) and the protein tyrosine phosphatase N22 (PTPN22)8, phospholipase A2 (PLA2) and 2-lyso-phospholipase D (LysoPLD)10, and α/β hydrolase 4 (ABHD4) and glycerophosphodiester phosphodiesterase 1 (GDE1)12. Fatty acid amide hydrolase (FAAH) is the main enzyme responsible for degradation of AEA14. In addition AEA is subjected to oxygenation by a number of enzymes including cyclooxygenase-2 (COX-2)16, 5-, 12- and 15-lipoxygenase (5-/12-/15-LOX)18, and several cytochrome P450 monooxygenases (P450s) including CYP3A4, CYP4F2, CYP4X1, and CYP2D620. Two two-step synthetic pathways have been identified for the synthesis of 2-AG, and involve PLC21 followed by sn-1-diacylglycerol lipase (DAGL), or phospholipase A1 (PLA1) followed by lyso phospholipase C (lyso-PLC)23. The metabolism of 2-AG involves several enzymes including the main degrading enzyme monoacylglycerol lipase (MAGL)24, FAAH, ABHD6 and ABHD1225, COX-2 and LOXs26.Synthesis and degradation of AEA involves a number of enzymes. Four routes for AEA synthesis from NAPE have been reported and the most widely accepted pathway is the single-step, direct synthesis by the enzyme NAPE-PLD28, proliferation29, survival30 inflammation32 and cellular differentiation33. The endometrium lines the uterus and is comprised of epithelial glandular structures, vascularised stroma and infiltrating immune cells. It is unique in that it undergoes cyclical growth, regeneration and shedding each month. Endometrial maturation during the menstrual cycle is controlled by female sex hormones, but also requires tightly regulated cellular responses to facilitate the cellular and structural changes. The proliferative phase is dominated by estrogen-driven cellular proliferation, vascularisation and immune stimulation while the secretory phase is dominated by progesterone mediated stromal cell decidualisation, vascular remodelling and immune cell modulation34. Regeneration of the endometrium following menses is mediated through adult stem and progenitor cells located in the basalis layer of the endometrium that proliferate, mature and differentiate across the menstrual cycle36. The consistent cyclical shedding and regrowth creates the monthly potential for errors in cell replication that may underlie both permanent, or transient endometrial abnormalities.Endocannabinoids are found in many organs and tissues including the endometrium and influence cell migration27, and AEA impairs both cellular proliferation and differentiation of an immortalised endometrial stromal cell line (St-T1b) and human decidual fibroblast from placenta, suggesting a crucial role in decidualisation39. AEA was also shown to influence migration in the immortalized human endometrial epithelial cell line HEC-1B28. Variations in concentrations of endocannabinoids may influence cellular proliferation and differentiation as the menstrual cycle progresses and the endometrium matures. Genes within the ECS are increasingly being recognised as targets to modulate endocannabinoid activity for multiple reproductive diseases including endometriosis, miscarriage, ectopic pregnancy, pre-eclampsia and endometrial cancer40. How these are altered across the menstrual cycle is not yet clear.Endocannabinoids are abundantly expressed in the endometrium and influence endometrial cellular function. Synthetic endocannabinoid, methandamide induces endometrial stromal cell migrationWe hypothesised that the ECS plays a role in the cellular function of the endometrium, that they are regulated across the menstrual cycle and that this may have implications for how the endometrium matures. The goal of this study was to define an a priori set of ECS genes from the literature, analyse their mRNA expression in human endometrial tissue and determine variations in their expression across the menstrual cycle.PLA2G2E), 64 were expressed in at least 1 sample with > 10 counts, 61 were expressed in 3 or more samples, 40 were expressed in greater than 90% of all samples  or MS stage (PLCB4) followed by a decline in expression in the LS  and the LP to ES stage (p = 8.28 × 10−15), but was subsequently down regulated in the MS stage (p = 0.007). There was a significant increase in PLCB4 from the LP to ES stage (p = 0.007) with a further increase from the ES to MS stage (p = 0.003). A significant difference was recorded in the transition from the ES to MS stage for PLCB1 (p = 1.31 × 10−11). A significant decrease was observed for PLA2G4A in the transition from the MS stage to the LS stage (p = 1.31 × 10−11).The first group contained five genes with a high median gene expression that increased consistently across the menstrual cycle from the EP stage and peaked in either the ES  and PLA2G12A (p = 0.001) that also showed a significant decrease from the LP to ES (p = 0.007), as well as the ES to MS stage (p = 1.44 × 10−8). Significant decreases were also observed for GDE1 from the LP to ES stage (p = 7.81 × 10−8) and for PLCB3 in transition from the MS to LS stage (p = 0.003).The second group of genes was characterised by a moderate mean expression with a gradual decrease across the cycle after initially peaking during the EP stage. An exception was observed with age Fig. B. SignifPLD1 were observed in the transition from the M to EP stage (p = 0.0005) and again from the LP to ES transition (p = 0.002). A significant increase was observed for PLCG2 (p = 0.005) and PLCD3 (p = 8.01 × 10−5) during the LP to ES transition. PLD1 began to increase in the MS stage (p = 0.0003) continuing into the LS stage (p = 2.8 × 10−8). Both PLCG2 (p = 0.005) and PLCD3 (p = 1.44 × 10−8) continued their increase during the early to mid-secretory stage.The third group was characterised by medium mean expression and that decreased directly after the menstrual stage and then subsequently increased again after the ES stage Fig. C. SignifDAGL that was increased (p = 0.003) and PTPN22 that was decreased (p = 0.003). There was minimal regulation across the proliferative stage, however in the transition from the LP to ES stage there was a decrease in GDE4 (p = 5.48 × 10−5), PLCL2 (p = 0.007) and an increase in GDE7 (p = 2.93 × 10−11), ABHD4 (p = 0.002), PLCH2 (p = 0.0001) and PLA2G6 (p = 0.007). Significant changes also occurred in the ES to MS stage with a reduction in PLCH2 (p = 2.19 × 10−16), DAGL (p = 0.0004), GDE7 (p = 0.0009) and an increase in PLCB2 (p = 0.002) and PTPN22 (p = 0.001). NAPE-PLD decreased from the MS to LS (p = 0.003). Finally, PLCH2 (p = 0.003), PTPN22 (p = 0.0002) and GDE4 (p = 0.003) were all significantly increased in LS compared to the MS stage.The final group was characterised by low mean expression that showed multiple and varied expression changes across the EP to ES stage Fig. D. SignifFABP5 which was significantly upregulated from the LP to ES stage (p = 0.002) (Supplementary Table p = 0.01)  (Supplementary Table We identified 7 genes known to encode fatty acid-binding proteins (FABPs) that transport endocannabinoids intracellularly. The largest changes were observed for 01) Fig. A (Supple01) Fig. . FABP3 wALOX5 (p = 0.0005) and COX-2 (p = 0.008) were significantly down regulated and NAAA significantly upregulated in the transition from the M to the EP stage (p = 0.008)  and ABHD12 was increased (p = 0.001)  and ABHD12 downregulated (p = 0.004) (Supplementary Table NAAA (p = 5.04 × 10−7) and COX-2 (p = 1.15 × 10−6) that were downregulated and ALOX5 (p = 0.0002) that was upregulated.We identified expression of 8 of the 12 genes related to endocannabinoid degradation in endometrial tissue. Of these 08) Fig. B (Supple08) Fig. . In the 01) Fig. B (Supple01) Fig. . In contTRPV1 was expressed in > 90% of the samples. It had a low but consistent expression and was not significantly regulated across the menstrual cycle or influenced by endometriosis status. The expression of all other membrane receptors occurred in < 90% of the samples. This suggests either a consistently low expression below read depth, or a binary on/off regulation. Based on the potential for binary regulation we determined the percentage of samples expressing membranes receptor genes in each menstrual stage . 35 of the 40 genes were detected in at least 50% of the microarray samples and included in the analysis to ensure sufficient pairwise comparisons to estimate an accurate correlation. Using the ‘cor.test' function in R we estimated the Pearson correlation between normalized and batch corrected counts from RNA-Seq (log2-CPM) and microarray  for each gene across the 161 samples. All 35 genes detected were positively correlated with a mean correlation value of 0.62 confirming a robust measurement of gene expression using the RNA-seq technique, despite the differences between microarray and RNA-seq including the inability of microarray to measure all transcript variations and different detection thresholds . PLCs selectively catalyze the degradation of phosphatidylinositol 4,5 bisphosphate resulting in soluble inositol-1,4,5-triphosphate (IP3) and membrane delimited 1,2 diacylglycerol (DAG)55. FAAH has previously been implicated in the survival of endometrial stromal cells from ectopic endometriosis lesions56 and has received significant interest as a pharmacological target, modulating pain through both peripheral and central mechanisms57. Physiological concentrations of progesterone stimulated FAAH activity in human lymphocytes, decreasing AEA levels58, while in a mouse model progesterone was reported to decrease uterine FAAH activity59. Progesterone levels and FAAH expression have been correlated during the menstrual cycle60, in agreement with the finding that progesterone up-regulates FAAH gene expression58. Combined with the regulation of the PLCs during this period, this suggests a regulation of endocannabinoid turnover during the transition between menstrual cycle stages that is tightly regulated.Endocannabinoids continue to exert their activity until degraded. A number of enzymes responsible for the degradation of endocannabinoids, including FAAH, MGLL and ABHD12 were also significantly downregulated during the transition from the late proliferative to early secretory stage. A down regulation of these enzymes would potentiate the ability of endocannabinoids to continue signaling. Of these enzymes FAAH is of particular interest. FAAH is a principal enzyme in the degradation of AEA and previous evidence suggests a relationship to progesteroneFABP5 across the menstrual cycle. FABP5 is a fatty acid binding protein that transports endocannabinoids through the aqueous cellular environment, preferentially directing it towards FAAH61. FABP5 was also significantly downregulated during the transition from the late proliferative to the early secretory stage. A reduction in the transport towards the degrading enzyme could potentiate the presence of the endocannabinoids during this period and may contribute to cellular and inflammatory changes62 that enables the regeneration of the endometrium in the early and mid-secretory phase in non-conceptive cycles.Additional factors, other than their production and inhibition, can influence endocannabinoids concentrations and their activity. Intracellular degradation relies on lipophilic endocannabinoid reaching the intracellular enzymes. One of the most significant changes we observed in this gene set was the regulation of CNR1, CNR2, TRPV1, TRPA1, GPR55, and GPR119 we found that only one of these (TRPV1) was consistently expressed in > 90% of the samples. Both CNR1 and CNR2 were previously shown to be expressed in the endometrium with variation in expression between women with and without endometriosis31. We found expression in the majority of samples, but not sufficient to reach the > 90% cut off. Analysis of gene expression for the remaining receptors as a binary variable found changes across the menstrual cycle. Although we did not find any that reached significance after multiple testing, this may be related to the power of the sample size, or the complex milieu of individual cell types and maturing cell states that exist within the endometrium at any point in time. The results showed non-significant changes in number of samples expressing CNR1, CNR2 and GPR55 during the late proliferative to early secretory period. While CB1 receptor (the protein encoded by the CNR 1 gene) was found to inhibit human decidualization and stimulates apoptosis38, studies on CB1 expression in the epithelial glands found no significant regulation across the menstrual cycle51, and CB2 expression in stromal cells was similar between the proliferative and the secretory phase30. Previous studies have also reported that progesterone exerted minimal effects on CB1 expression in lymphocytes58. It is therefore likely that the lack of significant changes in the expression of these receptors across samples and across the menstrual cycle indicates they may not be significantly regulated by reproductive hormones in endometrial tissue. The ability of these genes to be switched on or off may reflect the selective use in patients, depended on local requirements and provide the ability to fine tune the activity of the ECS.Although we included an extensive list of genes encoding endocannabinoid receptors, including NAPE-PLD and FAAH expression in the endometrium of patients compared to women without endometriosis throughout the menstrual cycle63. Another study reported no difference in CB1 protein expression during the proliferative phase between patients with and without endometriosis63, whereas lower levels of CNR1 and the protein CB1 have been reported in endometrial tissue from women with endometriosis compared to controls regardless of the cycle phase31. No difference in TRPV1 expression between women with and without endometriosis throughout the menstrual cycle has also been reported63. In this current study, when accounting for menstrual stage and multiple testing, we found no significant difference in any of the ECS genes investigated between women with and without endometriosis. Our data therefore correlates with previously published data that shows the ECS is not significantly dysregulated in eutopic endometrial tissue from patients with endometriosis.From our study cohort, 143 (69.4%) were diagnosed with endometriosis. Previous studies on the ECS in women with endometriosis have shown conflicting results. One reported no significant difference in This is the largest number of endometrial samples utilized to investigate gene expression of the ECS through menstrual cycle, and samples were assigned to detailed 7 menstrual stages following the Noyes criteria. However, the distribution of samples across the menstrual stages varied and the results should be interpreted with caution when the sample sizes for menstrual stages were small. While we were able to analyze the majority of the ECS at the gene level with RNA-seq data, we are yet to translate this to protein. Difference in gene and protein expression are commonly identified, which is likely to replicated in the ECS system. We aim to generate a truly representative picture of the ECS at the protein level once sufficient sample and resources have been collected.FAAH and FABP5 that work in concert to deactivate endocannabinoid activity and are already receiving considerable attention as drug targets for pain, inflammation and cancer65. The important ability of endocannabinoid receptors to be regulated may also hold particular interest for the potential of selective targeting of patients and the personalisation of medication.In summary, we have utilized genome wide gene expression to investigate a specific predefined gene set based on their previously identified involvement in the ECS. Targeted analysis of a defined gene set will reduce the burden of multiple testing in gene expression data of modest size providing the potential to uncover subtle variations. We detected differences in the gene expression across the menstrual cycle that are most pronounced during the transition from the proliferative to secretory stage and reflect a potential to dynamically modulate endocannabinoid concentrations during this period. As the activity of endocannabinoids are rapid and short-lived, we speculate that the altered regulation of expression for many components of the ECS across the menstrual cycle provides a quick acting, fine tuning potential to modulate endocannabinoid expression as needed, particularly during the transition period in which the endometrium undergoes major structural rearrangement. Of particular interest, we noted the large changes in a number of PLC enzymes, and the significant variation in 43 and inclusion criteria were European ancestry and within reproductive age . Exclusion criteria were the use of hormonal medication within 3 months prior to surgery, abnormalities in histopathological examination, ambiguous disease status or menstrual cycle stage. Informed consent was obtained from all subjects. A histological assessment was performed on formalin fixed paraffin embedded tissue with each sample assigned to 1 of 7 possible menstrual stages following the Noyes criteria66: menstrual (M) (n = 14), early proliferative (EP) (n = 5), mid proliferative (MP) (n = 72), late proliferative (LP) (n = 22), early secretory (ES) (n = 31), mid secretory (MS) (n = 41) and late secretory (LS) (n = 21) later using the Allprep DNA/RNA Mini Kit  as per the manufacturer’s instructions and isolated RNA samples were treated with the Turbo DNA-free kit . RNA quality was confirmed using the Agilent Bioanalyzer 2100  with RNA integrity number (RIN) cut off values set at above 8 for inclusion and final RNA concentrations determined by the Nanodrop ND -6000 . Stranded RNA-sequencing (RNA-seq) libraries were prepared with the Illumina TruSeq Stranded Total RNA Gold protocol incorporating ribosomal depletion . The resulting libraries were pooled and sequenced with 75 bp paired-end reads on the Illumina HiSeq 4000 to a mean depth of 37,490,673 for 178 samples and with 120 bp paired-end reads on an Illumina Hi Seq 2000  for a mean depth of 40,818,062 reads for 28 samples.Total RNA was isolated from endometrial biopsies stored in RNA67 and MultiQC v1.668. Low quality reads, or reads containing HiSeq Illumina adapter sequences were trimmed using Trimmomatic v0.3669. The resulting trimmed reads were aligned to a reference assembly (Ensembl Homo sapiens GRCh38 release 91) with HISAT2 v2.0.5 and the transcript assembly performed with StringTie v1.3.171 and each read mapping to a known transcript counted. Transcript, exon and expression matrices in Fragments Per Kilobase of transcript per million mapped reads (FPKM) were determined with StringTie counts for each individual. Prior to normalisation of RNA-seq counts lowly expressed genes (counts per million (CPM) < 0.22) and expressed in < 90% of samples were removed. Gene counts were normalised for composition bias and total raw reads using TMM74 in edgeR R package v 3.22.375. The normalized counts were converted to CPM and log2 transformed (log2-CPM).The quality of raw RNA-seq reads were confirmed with FastQC v0.11.7A priori gene selection was performed through a literature search using the search term ‘endocannabinoid’, and combinations of ‘pathway’, ‘synthesis’, ‘metabolism’, ‘transport’ or ‘endometrium’. All genes relevant to the control of the ECS were catalogued by ENSEMBL gene expression ID and gene expression extracted from the curated database. ECS genes of interest were split into four categories based on their main function in the ECS including (i) synthesizing enzymes, (ii) receptors, (iii) transporters, and (iv) degradation enzymes. In total 70 potential genes known to play a role in the ECS were assessed  between M and EP, (ii) between EP and MP, (iii) between MP and LP, (iv) between LP and ES, (v) between ES and MS, (vi) between MS and LS and (vii) between MP and MS. Seven differential gene expressions comparisons were performed between women with and without endometriosis: (i) including stage of menstrual cycle as a covariate, (ii) within M, (iii) within MP, (iv) within LP, (v) within ES, (vi) within MS and (vii) within LS. Batch effects (flow-cell and lane) were fitted as covariates in all models. The voom function in limma43 to determine if there was as significant variation in the number of samples that show a positive expression of these genes across the menstrual cycle.In the case of membrane receptors where positive gene expression was consistently < 90% of the samples, we explored gene expression as a binary function of expressed/not expressed and performed a logistic regression analysis as described previouslySupplementary Figure 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.


The current study, using a pretest-intervention-posttest design, investigated the dynamic relationships among translation learning anxiety, foreign language learning proficiency, and English–Chinese translation strategies under an iterative translation continuation task (ITCT) that lasted 13 turns. The results yielded from 134 student translators showed a significant increase in their translation strategies comprehension and production, with those with a medium level of translation learning anxiety and foreign language learning proficiency achieving the most. It also showed that the significant partial mediating effect of translation learning anxiety between foreign language learning proficiency and the production of translation strategies in the pre-test became insignificant in the post-test, and the insignificant correlation between the comprehension and production of translation strategies in the pre-test became significant in the post-test. The dynamic relationships among translation learning anxiety, foreign language learning proficiency, and translation strategies confirmed the mediating role of the ITCT in attenuating the impact of higher level of translation learning anxiety and lower level of foreign language learning proficiency on the comprehension and use of translation strategies, though its effects could be different for student translators with different levels of translation anxiety and proficiency as measured by different assessments.Growing interest has been shown in the effects of the  CET-4 is a Chinese national test for non-English majors which has been proved to have high reliability . Nine stThe translation learning anxiety questionnaire was adapted from the Foreign Language Classroom Anxiety Scale invented by The pre-test and the post-test of the translation strategies both contain two parts: the comprehension and production of the translation strategies, each with a total score of 20 points in line with In light of the various orientations for translation quality assessment (TQA) provided by translation studies, following a critical analysis of some TQA models see , (ii) trr is 0.950, 0.991, 0.993, and 0.945, respectively. Totally, 10 kinds of translation strategies were categorized: negative-positive shifting, word class shifting, concrete translation, abstract translation, addition or deletion, word splitting or combination, sentence splitting or combination, trans-voice translation, transform translation, and degradation or eulogization translation (see r = 0.940).The translation strategies used were classified and judged according to Example (1) (a) We should by no means neglect the evaporation of water.我们不能忽视水的蒸发作用。(concrete translation) (pre-test).(b) We were all deeply touched by her modesty and loftiness.我们都被她的谦虚态度和高贵品质所深深打动。(concrete translation) (post-test).Example (2) (a) 他统治那个地区长达20年之久。He had been the ruler of that region for as long as 20 years (word class shifting) (pre-test).(b) 老师教导我们珍惜每一个机会。The teacher taught us the value of an opportunity (word class shifting) (post-test).Example (3) (a) I had expected to see a much worse house.我本来也没指望看到房子好到哪去。[negative-positive shifting] (pre-test).(b) Osborne had been one of the family anytime these 23 years.奥巴23年来对这个家庭来说一直就不是外人。[negative-positive shifting] (post-test).Example (4) (a) 过去的半个世纪见证了中国戏剧的巨大发展。 (pre-test).The past 50 years [transform translation] witnessed great development in Chinese drama.(b) 过去的一百年见证了中国的巨大变化。 (post-test).The past century [transform translation] has witnessed great changes in China.T-test, the correlation analysis, and the mediating effect analysis in order to see whether the student translators had made any progress in their translation strategies comprehension and production, whether there were any correlations between foreign language learning proficiency, and translation learning anxiety and translation strategies, and whether there were any changes in the relationships among the three after the ITCT treatment, respectively.SPSS23.0 was used to do the analysis, including the paired sample Book Worm, co-edited by Oxford Publishing House and Foreign Language Teaching and Research Publishing House. The topics relate to daily life, science and technology, scientific research, physics, movie comments, literature, education, and political reports, aiming to make the student translators familiar with different themes and styles to meet the requirement of translation featured by super-subjects , and in the post-test . Therefore, those with scores ≤ 3.02 – 0.53, 3.02 + 0.53 > scores > 3.02 – 0.53, or scores ≥ 3.02 + 0.53 were classified into the lower translation learning anxiety group (n = 21), medium translation learning anxiety group (n = 94), and higher translation learning anxiety group (n = 19), respectively. And those with scores ≤ 490.36 – 49.58, 490.36 + 49.58 > scores > 490.36 – 49.58, or scores ≥ 490.36 + 49.58 were classified into the lower foreign language learning proficiency group (n = 21), medium foreign language learning proficiency group (n = 94), and higher foreign language learning proficiency group (n = 19), respectively.T-test was used to analysze whether there were some differences between the pre-test and the post-test for student translators with different levels of translation learning anxiety , followed by those with a lower level of translation learning anxiety  with the higher translation learning anxiety learners facilitated the least . The findings indicated that although the ITCT could increase the student translators’ comprehension and production of translation strategies, its efficacy could be greatest for those with medium level of translation learning anxiety.Then, a paired sample iety see . Table 2T-test was also used to analyze whether there were some differences between the pre-test and the post-test for student translators with different levels of foreign language learning proficiency , followed by those with a higher level of foreign language learning proficiency  with the lower foreign language learning proficiency learners facilitated the least . The findings indicated that although the ITCT could increase student translators’ comprehension and production of translation strategies, its efficacy could be greatest for those with medium level of foreign language learning proficiency.A paired sample ency see . Table 3The findings in r = 0.20, p = 0.022), while no significant correlation was found between foreign language learning proficiency and the post-test translation strategies production . Additionally, a significant correlation was found between the pre-test and the post-test in translation skill production , and between the comprehension and the production of translation strategies in the post-test , whereas no significant correlation was found between the comprehension and the production of translation strategies in the pre-test . r = −0.17, p = 0.05) which was significantly positive related to the pre-test production of translation strategies .A complex correlation among foreign language learning proficiency, translation learning anxiety, and translation strategies comprehension and production before and after the ITCT treatment can be seen from the correlation analyses see . First, The complex correlations among translation learning anxiety, foreign language learning proficiency, and translation strategies comprehension and production make it necessary to further analyze the relationships among them. Therefore, using Multiple-Linear Regression with pre-test translation strategies production as a dependent variable, the CET-4 score as an independent variable, and translation learning anxiety as the mediating variable, the present study made a path analysis.t = −2.762, p = 0.007), and (ii) CET-4 had a significant effect on the production of translation strategies in the pre-test both with translation learning anxiety  and without translation learning anxiety  with the former more significant. The findings demonstrated that translation learning anxiety played a partial mediating effect between foreign language learning proficiency and the production of translation strategies in the pre-test, indicating an indirect effect of translation learning anxiety on the use of translation strategies in the pre-test. However, in the post-test, no significant correlation was found between the student translators’ foreign language learning proficiency and their use of translation strategies , or between their translation learning anxiety and production of translation strategies . Namely, the significant partial mediating effect of translation learning anxiety between foreign language learning proficiency and the production of translation strategies in the pre-test became insignificant in the post-test.r = 0.01, p = 0.932) and the post-test , significant changes were found in the correlation between the comprehension and production of translation strategies from the pre-test  to the post-test . Therefore, generally speaking, dynamic relationships were found between translation learning anxiety, and foreign language learning proficiency, and the comprehension and production of translation strategies. The findings answered research question 2, indicating that the ITCT could activate the student translators’ comprehension and conscious use of translation strategies, though its effects could be different to some extent.Although path analysis hasn’t been done due to the insignificant correlation between the student translators’ CET-4 score and their comprehension of translation strategies in both the pre-test (xu-argument based ITCT through exploring the interaction between affective factors such as translation learning anxiety and cognitive factors such as foreign language learning proficiency. The student translators’ increased comprehension and production of the translation strategies, irrespective of their level of translation learning anxiety and foreign language learning proficiency, confirmed the efficacy of the ITCT as an effective treatment to increase the student translators’ comprehension and conscious use of translation strategies in line with xu which is characterized by a close combination of input and output in a rich interactive context. The present findings also extended the xu-argument, verifying that the ITCT could decrease the student translators’ affective and cognitive load (The present study provides a new evidence for the efficacy of the ive load , boostinive load , 2020.In addition, in contrast to the former static anxiety and foreign language learning proficiency research , the preThe increased agency of the student translators after the treatment might partly explain the efficacy of the ITCT and its mediating role in the relationships between translation learning anxiety and foreign language learning proficiency and translation strategies. Foreign language learners are dynamic individuals under the interaction of society, psychology, and affection, with a change in one possibly resulting in changes in another . During The optimal interactive learning context created by the ITCT also attributes to the student translators’ creative imitation in translation. Creative learning needs two conditions: sustainability and the resources for sustainable development . In the The unique feature of translation can partly explain the fact that the student translators with medium foreign language learning proficiency and translation learning anxiety could benefit the most from the ITCT. Different from listening, speaking, reading, and writing, translation requires a shift in the way of thinking because of the different cultures and the culture loaded concepts and conceptualization . Therefoxu-based continuation task can also increase the foreign language learners’ foreign language learning proficiency (The student translators with medium foreign language learning proficiency and translation learning anxiety benefited the most from the ITCT might also be partly due to the different interactions between the student translators and the ITCT treatment. Low learning efficiency may be due to the inefficient interaction among the main components in a system . As for ficiency , and thaxu-argument based ITCT, extending the xu-argument based research from an affective perspective.The present study investigated the dynamic relationships among translation learning anxiety, foreign language learning proficiency, and the comprehension and production of translation strategies. It was found that the ITCT could increase the student translators’ TS comprehension and production, though its efficacy could be shown to be greatest for those with a medium level of translation learning anxiety and foreign language learning proficiency. In addition, the present study also found that the ITCT could inhibit the negative effect of higher translation learning anxiety and lower foreign language learning proficiency on the comprehension and production of translation strategies. The findings of the present study provide a new evidence for the The present findings also have several pedagogical implications and applications. First, in order to maximize the effects of the ITCT, the student translators’ affective factors, such as translation anxiety should be paid attention to by the instructors. Just as Despite its findings and implications, this study has some limitations. First, it only indirectly investigated the effect of the ITCT on the student translators’ translation learning anxiety through its mediating role between foreign language learning proficiency and translation strategies production. Therefore, further studies should be done to explore the direct effect of the ITCT on translation learning anxiety. Additionally, the present study only investigated the student translators’ comprehension and production of translation strategies, albeit comprehensively, without differentiating Chinese–English translation from English–Chinese translation. Given the differences in translation strategies comprehension and production between Chinese–English translation and English–Chinese translation , future The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.SZ conceived and designed the study, collected the data, drafted, revised the manuscript, and got it ready for submission. YR collected and analyzed the data under the instruction of SZ. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The sensitivity of CGR to tropical temperature 2 records from Mauna Loa and the South Pole to compute CGR, and show that The atmospheric CO The authors show a recent decoupling of the tropical temperature variations and the carbon cycle that is driven by wetter conditions in the tropics. Increased atmospheric CO2 concentrations also have a positive effect on photosynthesis6, promoting vegetation growth7. Consequently, an increase in the net terrestrial carbon sink has been documented for the last 50 years8, which in turn has contributed to slowing down the ongoing global warming3. These processes constitute the interactions of climate and carbon cycle  when predicting future climate change5.Increased atmospheric concentrations of greenhouse gases have caused global warming that profoundly influences terrestrial ecosystems and the global carbon uptake, which in turn reinforces climate change2 growth rate (CGR) are tightly coupled to the variations in tropical air temperature10, which are themselves strongly associated with the El Niño Southern Oscillation (ENSO)10. The sensitivity of CGR to tropical temperature 9. A recent study documented that an increase in tropical extreme droughts could also amplify the CGR variability11. Moreover, a continuous increase in temperature that may approach or exceed an optimal temperature of plant photosynthesis12 may have negative effects on the net ecosystem carbon sink13, even though tropical forests may acclimate to a warmer climate16. In this case, a very warm year at present time could be more detrimental for photosynthesis than the same anomaly happening decades ago. Multiple lines of evidence indicate that the net terrestrial carbon uptake has saturated or decreased over tropical areas in recent decades19. This is caused by increased frequency of droughts, land degradation, deforestation, cropland expansion and changes in forest wildfires21. Collectively, these changes are expected to influence the established impact of temperature on carbon fluxes in the tropics, yet the primary factor controlling the trends of this effect remains unclear.Interannual variations of the atmospheric CO2 records from Mauna Loa (1960–2020) and the South Pole (1980–2020) atmospheric stations, and climatic data from CRU-4 and ERA5. We subsequently analyze the variations in 2 concentrations, nitrogen deposition and land cover change on the changes in Here, we investigate the changes in 22. Significant positive partial correlations between variations in CGR and temperature are identified in each moving window with an average r of 0.58 (P < 0.05)  in a partial regression analysis. The results indicate that the correlation of CGR with tropical temperature is stronger than with precipitation and solar radiation (with an average correlation coefficient of 0.31) in the tropical regions  to 1979–2000 (4.93 ± 1.66 PgC y−1 K−1), corresponding to a trend of 0.19 ± 0.03 PgC y−1 K−1 per year. However, for the second period we observe a negative trend of 0.13 ± 0.02 PgC y−1 K−1 per year and an overall decrease of 117% (2001–2020: 2.27 ± 1.19 PgC y−1 K−1), which brings the values of 2 data from the South Pole station  increasing and decreasing trends are identified with a specific Mann-Kendall trend test23 accounting for serial autocorrelations in To further study the changes in 60s Fig. . The decGRT Fig. . Changes11, so we use the standardized deviation (σ) of the long-term detrended precipitation, the self-calibrating Palmer Drought Severity Index (scPDSI) and the Standardized Precipitation and Evaporation Index (SPEI) to divide the time series at a high frequency  into very wet (σ ≥ 1), wet (0 ≤ σ < 1), dry (−1 ≤ σ < 0) and very dry (σ < −1) periods  correlated with changes in precipitation at a bi-decadal scale  with an average correlation coefficient r of −0.81  in Supplementary Table P < 0.05) correlated with changes in 25 and the simulated NBP can thus be used to explore the interannual changes in the We subsequently use the dynamic ecosystem model LPJ-GUESS to separate, by factorial simulations, the relative importance of changes in each climatic variables (precipitation and solar radiation), atmospheric COGRT Fig. . However2, nitrogen deposition, land cover or solar radiation being kept constant and the others varying over time, the simulated NBP shows similar variations as the results from SCE1  correlations of We further study the sensitivity of changes in RET Fig. . We analRET Fig.  where alCE2 Fig. , which sP < 0.05) between tropical NBP and temperature under very wet and very dry conditions determined by the standard deviation (σ) of the detrended precipitation. The ensemble mean of σ < −1) is higher than that during very wet years (σ ≥ 1)  Fig.  showing SMs Fig. . Of the ion Fig. . These r9, whereas the decrease since 1980 documented here needs to be understood in the context of continued warming and increased extreme climate events. The observed decreased 26. Recent studies have reported that a feedback between soil moisture and the atmosphere primarily controlled the variability and long-term trend of the global terrestrial carbon sink28 and that the role of terrestrial water storage in tropical regions may increase29. Moreover, existing studies show that semi-arid ecosystems dominate the interannual variability of global carbon cycle25 and that tropical extreme droughts are the cause for an increase in CGR variability11. These studies collectively suggest that water availability plays an important role in regulating the global carbon cycle. Our results show that water availability largely controls the bi-decadal variations in 2. These findings suggest that it is the interactions between water and temperature that control the carbon cycle in the tropical regions. Lastly, the simulation results show that water dryness controlling changes in We document an increase and decrease in the interannual 16. Changes in 30. Moreover, an increase in the frequency of climatic extremes and disturbances such as changes in wildfires and insect outbreaks, and decreasing availability of a necessary nutrient supply  could influence plant productivity that might indirectly alter the response of the carbon cycle to temperature. We finally show that the majority of ESMs could to only some extent capture the differences in 5. Improved knowledge on the complex interplay of processes regulating the response of the terrestrial carbon cycle to drought will require further research.Several other factors, however, may also contribute to changes in Changes in climate have altered terrestrial ecosystems in recent decades. In particular, the changes in temperature and precipitation seem to a large extend to regulate the exchange of carbon between land and the atmosphere, sustaining the important role of terrestrial ecosystems in the climate system. Our results provide clear evidence that water availability exerts an important control on the observed bi-decadal variations in CGR sensitivity to tropical temperature. This finding highlights the importance of the water-temperature interactions in regulating the CGR over the tropics and provides insights to our understanding of climate-carbon interactions.2) at the Mauna Loa Observatory for 1959–2020 and the South Pole station for 1980–2020 were accessed from the US National Oceanic and Atmospheric Administration (http://www.esrl.noaa.gov/gmd/ccgg/trends/). The Mauna Loa Observatory CO2 dataset represents the longest record of direct measurements of CO2 in the atmosphere. There are many CO2 observations missing in the South Pole station dataset before 1980, and we have thus chosen the time series of CO2 observations from 1980 to 2020, which allowed us to detect the changes CGR sensitivity for the recent period.The monthly mean atmospheric carbon dioxide concentration  Reanalysis (ERA5)32, which are the fifth generation ECMWF reanalysis data for the predictions of global climate and weather replacing the ERA-Interim reanalysis. The data are provided at a spatial resolution of 0.1° and a temporal coverage from 1950 to the present. We also extracted monthly gauges-based precipitation data for 1960–2019 from the Global Precipitation Climatology Centre (GPCC)33. We further used the self-calibrating Palmer Drought Severity Index (scPDSI) for the detection of changes in water stress conditions34, where negative values of scPDSI indicate drier conditions and vice versa. scPDSI is estimated based on precipitation, potential evapotranspiration (PET) and temperature, and the Penman-Monteith method is used to calculate PET. Likewise, the Standardized Precipitation and Evaporation Index (SPEI) data (using a three-month integration period) for 1960–2018 were extracted to indicate water availability conditions35. We used monthly root-zone soil-moisture data from the dataset of the Global Land Evaporation Amsterdam Model version 3 (GLEAM-v3) produced at a spatial resolution of 0.25° and with a temporal coverage of 1980–202036. We also extracted reconstructed data for terrestrial water storage driven by ERA5 climatic data37, which included GSFC-ERA5 and JPL-ERA5 with a temporal coverage of 1979–2018. It should be noted that spurious trends might occur in these reanalysis data due to the discontinuity of the assimilated observations9.We extracted monthly temperature, precipitation and solar radiation (cloud cover in CRU-4) for 1960–2020 from both the CRU-4 and ERA5 datasets. Monthly climatic data at a spatial resolution of 0.5° from the Climate Research Unit (CRU TS 4.05), University of East Anglia, are generated by scaling up rain-gauge observations38 is a dynamic vegetation model that simulates the cycling of carbon and nitrogen within vegetation and soil. Vegetation is represented with 12 plant functional types39, coexisting in patches of vegetation that undergo stochastically varying disturbances. Net biome productivity (NBP) was obtained from a simulation forced with monthly gridded data at a spatial resolution of 0.5 × 0.5° from CRU TS 4.0531, monthly model-derived estimates of nitrogen deposition and annual atmospheric CO2 concentration based on ice-core data and atmospheric observations in a simulation for 1901–2020. Land use was represented applying historical reconstructions of land use from previously published data39. In addition to this simulation, a set of factorial simulations  were kept constant at the mean conditions for 1960–2020, while all other drivers were time-variant as in the main simulation. We subsequently extracted time series of carbon flux components namely net primary production (NPP), heterotrophic respiration (Rh), and fire emission (FIRE) from these simulations.LPJ-GUESS40. Solar radiation was calculated as the difference between surface down- and upwelling shortwave radiation simulated in CMIP6. All CMIP6 outputs were resampled to a spatial resolution of 1°using bilinear interpolation.We collected monthly simulated NBP, temperature, precipitation and solar radiation for 1960–2014 from the historical simulations from 33 ESMs  to indicate El Niño and La Niña during 1979–2021.We extracted monthly NBP data under scenario 3 for 1960–2019 from 15 dynamic global vegetation models (DGVMs) from TRENDY version-92 between two successive months to calculate the monthly CO2 growth rate (CGR). We calculated annual sum of CGR for 1960–2020 (n = 59 after removing years of 1992–1993 due to the eruption of Mt Pinatubo) to identify the interannual variability and trends in the sensitivity of CGR to tropical temperature , which facilitates identification of the anomaly of changes in −1 into PgC y−1 by multiplying with the conversion factor of 2.124 PgC ppm−18. Climate variables of tropical temperature, precipitation and solar radiation over the surface area of vegetated land between 24° N and 24° S based on a land cover map of ESA CCI 2015 were derived at annual and high frequency scales to be consistent with CGR.We used the first-order difference of COmentclass2pt{minim3. The sensitivities were calculated in a 500-member bootstrap to estimate the uncertainties for each window. Similarity, the sensitivities of NBP  of data based on the standard deviation (σ) of detrended precipitation from CRU and GPCC into four bins. We chose the data at a high frequency in order to increase the degrees of freedom to test for statistical significance of the sensitivity analysis in each bin. These bins were defined as the periods of very wet (σ ≥ 1), wet (0 ≤ σ < 1), dry (−1 ≤ σ < 0) and very dry (σ < −1) following the ref. 9. We thus calculated σ ≥ 1) and very dry (σ < −1) conditions with the outputs  from ESMs. The very wet and dry conditions were determined by the anomalies of detrended precipitation for 1960–2014 derived from ESMs.We explored the variations of σ) of mean annual precipitation, soil moisture, scPDSI, SPEI and terrestrial water storage at the pixel level. These groups were defined as the regions of very wet (σ ≥ 1), wet (0 ≤ σ < 1), dry (−1 ≤ σ < 0) and very dry (σ < −1) to indicate four distinct gradients of water conditions. These variables at the pixel level in each group were aggregated to derive time series of climatic variables for 1978–2020 that were used to calculate 9.We then calculated mentclass2pt{minim2 (SCE4), land use (SCE5) and nitrogen deposition (SCE6), with the remaining driver variables time-varying. The driver variables kept constant were assigned the value of their long-term annual mean over 1960–2020 in each scenario. NBP derived from each simulation was used to calculate 2 but considers the dependence on ordering of explanatory variables by averaging over the total number of combinations of ordering in a multiple regression.We next designed several simulations to explore the underlying mechanism of changes in P < 0.05) in 23, to conduct the trend test. The slope of a linear regression between trends in 45. Moreover, we used the Cochrane-Ocrutt procedure to remove the impacts of serial autocorrelation on the relationships established between these time series variables (Supplementary Table ρ (the first-order autocorrelation parameter) from the original values and these adjusted values were used in a multiple linear regression, with i – ρPRi-1. The Durbin-Watson indicator (DWI) was used to detect the autocorrelation at lag 1 in the residuals (ei) from the multiple linear regression of a target variable against time46 using a significance level of P < 0.001. DWI was calculated through the Eq. (n is the number of observations, ei is the ith residual of the linear regression of the investigated variable against time. The DWI values range between 0 and 4. A value of 2 indicates no autocorrelation, whereas values from 0 to 2 indicate positive autocorrelation and values from 2 to 4 denote negative autocorrelation in the time series of observations.The Theil–Sen slope and Mann–Kendall trend test were used to identify trends and their significance (entclass1pt{minimaSupplementary InformationPeer Review File