Rice plays a critical role in human livelihoods and food security. However, its cultivation requires inputs that are not accessible to all farming communities and can have negative effects on ecosystems. simultaneously, ecological research demonstrates that biodiversity management within fields contributes to ecosystem functioning.This study aims to evaluate the mixture effect of four functionally distinct rice varieties in terms of characteristics and agronomic performance and their spatial arrangement on the upland rice performance in the highlands of Madagascar. The study was conducted during the 2021-2022 rainfall season at two close sites in Madagascar. Both site differ from each other’s in soil properties and soil fertility management. The experimental design at each site included three modalities: i) plot composition, i.e., pure stand or binary mixture; ii) the balance between the varieties within a mixture; iii) and for the balanced mixture (50% of each variety), the spatial arrangement, i.e., row or checkerboard patterns. Data were collected on yields (grain and biomass), and resistance to Striga asiatica infestation, Pyricularia oryzea and bacterial leaf blight (BLB) caused by Xanthomonas oryzae-pv from each plot.Varietal mixtures produced significantly higher grain and biomass yields, and significantly lower incidence of Pyricularia oryzea compared to pure stands. No significant differences were observed for BLB and striga infestation. These effects were influenced by site fertility, the less fertilized site showed stronger mixture effects with greater gains in grain yield (60%) and biomass yield (42%). The most unbalanced repartition (75% and 25% of each variety) showed the greatest mixture effect for grain yield at both sites, with a strong impact of the varietal identity within the plot. The mixture was most effective when EARLY_MUTANT_IAC_165 constituted 75% of the density associated with other varieties at 25% density. The assessment of the net effect ratio of disease, an index evaluating the mixture effect in disease reduction, indicated improved disease resistance in mixtures, regardless of site conditions. Our study in limited environments suggests that varietal mixtures can enhance rice productivity, especially in low-input situations. Further research is needed to understand the ecological mechanisms behind the positive mixture effect. In MadaGenotypic diversity has a positive effect on the functioning of ecosystems and enhance diverse ecological services . MoreoveOne way to increase the within field biodiversity is the use of varietal mixtures. The potential benefits of mixtures, including increased productivity , stabiliHowever, the effectiveness of varietal mixtures may depend on their growth conditions including both, spatial organization of the mixture and fertility of the field. Studies have shown that a crop’s mixture organisation can have a significant impact on crop performance and can influence, in different ways, vegetative and reproductive biomass production in favour of grain yield , as wellStriga asiatica, as well as the diseases Pyricularia oryzea, and Bacterial Leaf Blight (BLB).The main objective of the study was to assess the effect of varietal mixture of upland rice, their proportion and spatial arrangements on agronomical performance of yield and disease resistance. We then aimed at these three sub-objectives: i) assess the relevance in terms of yield and disease resistance of growing upland rice varieties in mixtures compared to monoculture, and whether the mixture effect is influenced by the environment; ii) test whether the spatial arrangement of the binary mixtures, i.e., both the proportion of each variety as well as their organisations, has an impact on the mixture effect; and iii) estimate if some genotypes are more efficient than others to build effective mixtures. To reach these objectives, we conducted field experiments in the highlands of Madagascar with four functionally contrasted upland rice varieties grown in mixture and pure stand. Two experimental sites were established with two different levels of fertilization. To assess the influence of environmental limitations on the performance of the varietal mixtures, we considered constraints such as the weed 22.1In order to compare the performance of upland rice under two contrasting growing conditions, the same experiment was conducted simultaneously at two relatively close sites both featuring “Ferralsol” soil type with differing properties Table S1During the growing season, which extended from October 2021 to May 2022, both sites experienced very similar meteorological conditions. The average monthly rainfall was 182mm at Site ANK and 183mm at Site IVO. The average temperature throughout the experiment was 25.4°C, with a minimum temperature of 18°C and a maximum temperature of 37°C at both locations.-1) compared to Site IVO (17.6g.kg-1), as was the case for the total nitrogen content . The assimilable phosphorus content was lower at Site ANK (mean =5.0mg.kg-1) compared to Site IVO (mean =6.9mg.kg-1) in all three horizons. Soil at the ANK site is rich in organic matter (20.9 g.kg-1) compared to soil at the IVO site (16.9 g.kg-1). Detailed information about soil characteristics and properties is available in Mucuna puriens, a leguminous cover crop, after being left fallow for two years. Given this history, we implemented a more intensive cropping management approach at the IVO site, involving higher fertilizer application.The soils at both sites are classified as Ferralsol , known f-1 of cattle manure resulted in an input of 31.2 kg.ha-1 of nitrogen (N) and 5.6 kg.ha-1 of phosphorus (P2O5). Subsequently, an application of 50kg.ha-1 urea further enriched nitrogen levels by 23kg.ha-1. In the case of IVO site, an initial application of 5t DM.ha-1 of cattle manure introduced 52.35 kg.ha-1 of N and 11.25 kg.ha-1 of P2O5. Dual applications of NPK (11:22:16) “Vano be” played a pivotal role in fertilisation. The first application at sowing time (50kg.ha-1), contributed 5.5kg.ha-1 of N, 11kg.ha-1 of P2O5, and 8 kg.ha-1 of potassium (K2O). The subsequent booting stage application (100kg.ha-1) added 11kg.ha-1 of N, 22 kg.ha-1 of P2O5, and 16kg.ha-1 of K2O. A 23kg.ha-1 of N by urea was also applied at this stage with the second application of NPK “Vano be”. As a result, the site ANK was fertilized with 54.2kg.ha-1 of N, 5.6kg.ha-1 of P2O5, and no amount of K2O. The site IVO received 91.85kg.ha-1 of N, 44.25kg.ha-1 of P2O5 and 24kg.ha-1 of K2O.We conducted this experiment on two sites with different levels of fertilisation. At ANK site, an initial application of 5t DM.ha2.2Pyricularia oryzea  alternating rows (referred to as L1/1) and ii) checkerboard pattern (CBD); iii) an unbalanced repartition, with two-thirds of the plants from one variety and one-third from the other one (67-33), obtained by alternating one in two rows (L1/2); and iv) a very unbalanced treatment, with three-quarters of one variety and one quarter of the other one (75-25), obtained by alternating one in three rows (L1/3). The mixtures were named using the codes of the varieties that composed them, considering first the most abundant one. For instance, the mixture composed of 67 percent of variety A and 33 percent of the variety B was named A2B1, with the first letter representing the most abundant variety in the mixture see Table 1.Both pure stands and mixtures were managed in the same way. At sowing,six to eight seeds (of a single variety) were placed in small holes about 3cm deep covered with a small amount of soil, this is what we call “hill”. The cattle manure, and the mineral fertiliser in IVO, were added to these holes, before covering the planting holes with small amounts of soil. These planted hills were considered as individual plants during our study. The crop depended entirely on rainwater as no irrigation was provided. Weeding was carried out manually three times in all plots to facilitate optimal growth, specifically two weeks after sowing, at the start and at the end of tillering.2.4Pyricularia oryzea (Xanthomonas oryzae pv. oryzae (Xoo) incidence  for each plot. This measure is obtained by adding up the relative yield of each variety in the mixture :mix(i) is the yield (grain or biomass) of variety (i) in the mixture and Ypure(i) is the yield (grain or biomass) of variety (i) in pure stand in the same main-plot. A RYT value greater than 1 indicates a global positive effect on yield in the mixture compared to what could be expected based on the performances of its components in pure stands. RYT was calculated per main plot within a block to account for the block effect. The pure stand yields are, therefore, the average of the yields per block. The calculation of Relative Yield Total (RYT) shares a common underlying principle with the Land Equivalent Ratio (LER). Both assess the advantages of intercropping by comparing combined yields or resource utilization in varietal mixtures with those achievable in separate pure stands  was calculated for the disease intensity (DI) (To assess resistance to ity (DI) . This inThe following formula was used to calculate the net effect ratio of disease:observed is the observed disease incidence or disease severity in the mixtures and DIexpected the expected disease incidence or disease severity based in the infections levels in pure stands. pi and pj are the proportion of the variety i and j in the mixture, respectively. DImix(i) is the disease incidence or the disease severity of the variety i in mixed plots and Dpure(i) the average disease infections in pure stands per block for variety i and likewise for the variety j. A NERDI value less than 1 indicates a higher resistance performance in the mixture than in the pure stand. Due to the low disease pressure during the experiment, only one variety (DOURADO PRECOCE) developed symptoms on leaves. As a result, the NERDI indicator was calculated for half of the mixtures, i.e. those that included this susceptible variety. Then, Pyricularia oryzea developed symptoms on panicles in two varieties: DOURADO PRECOCE and EARLY MUTANT IAC 165, but only at the IVO site. The NERDI on panicle was calculated in plots including these two varieties.Where NERDI (ij) is the net effect ratio of disease of the mixture of varieties i and j. DI2.7All the statistical analyses were done using R version 4.2.2 (2022-10-31 ucrt).We assessed the effects of the different modalities (pure stand and mixtures) and their interactions with the site on grain and biomass yields, diseases resistance and striga infestation using a mixed linear model (function lmer package lme4) . AssociaThe differences between RYT and NERDI and the value 1  were assessed using the one-sample Student’s t-test. We assessed a linear model considering association type ’, ‘spatial arrangement’, ‘main plot’) and site as fixed effects and the block and plot positions within each site were added as random factors. Adjusted means of each index were again calculated by lsmean. Then we compared the means between sites and the means for modalities of mixture repartition. Taking into account the comparison between modalities in each site and in general, the most efficient spatial arrangements was selected. Then we detailed the component mixtures of varieties in the identified spatial arrangement to highlight the one(s) inducing best performances. In this more efficient spatial arrangement (L1/3), the characteristics of each variety were compared across various proportions, both in pure stands and mixtures. Comparisons were performed using a Fisher’s multiple comparison test following an ANOVA, treating each trait as the dependent variable and the proportions as the independent variable for each variety.33.1-² and biomass yield by 236 g.m-². These positive effects of mixtures were observed in both sites, although there were significant differences in yields between the two sites. Specifically, site IVO, with high fertilizer inputs, had significantly higher yields of 631g.m-² for grain and 363g.m-² for biomass, compared to the ANK site. The interactions between crop conditions and sites also had significant effects (p<0.05) (Pyricularia oryzea and BLB have significantly negative association with grain yield (p<0.001) (The results show that the variety EARLY MUTANT IAC 165 was the most productive in terms of grain yield (p<0.001) and it also performed well in biomass yield even the p-value was not significant between the monocultures of the four variety Table 2.Pyricularia oryzea infection levels on leaves and panicles compared to pure stands  and a reduction of 7 lesions number in Pyricularia oryzea severity (p<0.001). Interestingly, the pathogen Pyricularia oryzea only affected panicles at the IVO site, with an incidence rate of 6% and an average of 9 lesions in severity. Additionally, Bacterial Leaf Blight (BLB) infection was observed only at the IVO site, while striga infestation occurred solely in the experiment at the ANK site. In summary, the highly fertilized IVO site demonstrated higher yields and lower Pyricularia oryzea infection levels compared to the ANK site.The results of the site-specific analysis reveal significant differences between the two experimental sites. The IVO site showed significantly lower values of 3.2The RYT analysis demonstrated an overall 45% increase in grain yield and a 33% increase in biomass yield in mixtures compared to monocultures Figure 2Pyricularia oryzea infection on leaves and panicles in mixtures. In comparison to pure-stands, mixtures showed an average 10% decrease in disease incidence on leaves .Results also demonstrated an overall reduction in Regarding the spatial arrangement, there were no significant differences between lines and checkerboard patterns within the L1/1 arrangement for all the considered, variables including yields and disease resistance Figure 3Pyricularia oryzea, and were not affected by this pathogen in our study. On the other hand, the association composed by 25% EARLY MUTANT IAC 165 and 75% of DOURADO PRECOCE had the lowest RYT for biomass  was present. Disease incidence and severity were higher in pure stands of this variety compared to mixtures. However, no significant differences between mixtures and pure stands were found for Bacterial Leaf Blight (BLB) and striga infestation.This study demonstrated that binary varietal upland rice mixtures had significantly higher grain and biomass yield compared to the varieties in pure stand, indicating the benefits of varietal mixtures. The Relative Yield Total (RYT) indicated an average of 45% gain in grain yield and 33% gain on biomass yield when applying varietal combination. These benefits of intercropping can be interpreted as improved land use efficiency .This finSignificant variations in yields were observed between the experimental sites according to the It was demonstrated that soil organic matter content, which is a major driver of soil fertility, could impact mixture performance, i.e. the more organic matter available, the lower the mixture effect and thus the relevance of using varietal mixtures . This stPyricularia oryzea incidence and severity. These findings are in line with previous research that has demonstrated the potential of mixtures to reduce pest and disease pressure through diversity-mediated pest suppression via specific ecological processes (Pyricularia oryzea infection could be attributed to reduced host-plant density and increased diversity, making mixtures less favourable to the spread of pathogens (In our study, varietal mixtures demonstrated efficiency to reduce rocesses . These rathogens . This meathogens . Howeverathogens . Studiesathogens . Overallathogens Table 3.Regarding the spatial arrangement, the most unbalanced repartition showed a higher RYT for grain yield in both sites, highlighting that the balance between the varieties is not a prerequisite to obtaining a positive effect of mixtures . These rPyricularia oryzea infection, no significant differences were found for BLB and striga infestation. This suggests that the impact of mixtures on different diseases may vary. Further research is needed to fully understand the relationship between pure stands and the spread of these specific infections. The study, furthermore, provided insights into the positive effects of varietal mixtures on crop performance, however, the underlying mechanisms driving these outcomes remain largely unexplored. Future studies should aim to investigate the specific ecological processes and interactions contributing to the observed benefits. Our results highlight the potential benefits of an unbalanced spatial arrangement regarding grain yield. However, the specific reasons behind these findings, such as the selection effect, driven by the dominance of a particular genotype, require further investigation. Additionally, the relationship between spatial arrangement, complementarity effects, and resource competition must be explored further. Lastly, the experiment duration of a single growing season limited our insights into the sustainability and stability of the observed benefits. Long-term experimentation is needed to confirm the above results.The results of this study indicated important benefits of varietal mixtures, albeit these results had limitations, as they were specific to the experimental conditions and locations. Extrapolating the results to different environmental conditions or rice varieties should be treated with caution, as local factors such as soil type, climatic conditions, and genotype selection can influence outcomes. While the study demonstrated the effectiveness of mixtures in reducing Our study highlighted the benefits of varietal mixtures in upland rice in the mid-west of Madagascar. The highland rice production systems of Madagascar often face challenges such as limited access to inputs such as fertilizers and pesticides. Intercropping can help to address some of these challenges by reducing the reliance on external inputs and enhancing ecosystem functioning. However, understanding ecological mechanisms that drive interaction among varieties is crucial for optimizing mixture composition and improving rice production sustainability. However, further research is needed to explore these mechanisms in more detail and to assess their long-term implications for rice production in the context of highland agroecosystems in Madagascar.https://figshare.com/articles/dataset/Assessing_the_impact_of_rice_varietal_mixtures_on_crop_performance_in_Madagascar/24488386.The data and the script for data analysis are available at the following link: KR: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Supervision, Writing – original draft, Writing – review & editing. BM: Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Validation, Writing – review & editing. CV: Conceptualization, Data curation, Formal analysis, Funding acquisition, Resources, Writing – review & editing. KB: Investigation, Writing – original draft, Writing – review & editing. RP: Supervision, Validation, Writing – review & editing. JM: Investigation, Methodology, Validation, Writing – review & editing. EB: Methodology, Software, Writing – review & editing. FF: Conceptualization, Data curation, Formal analysis, Methodology, Supervision, Validation, Writing – original draft, Writing – review & editing.


Arabidopsis SLAC1 WT and its phosphomimetic mutants in open and closed states. Comparison of the open structure with the closed ones reveals the structural basis for opening of the conductance pore. Multiple phosphorylation of an intracellular domain (ICD) causes dissociation of ICD from the transmembrane domain. A conserved, positively-charged sequence motif in the intracellular loop 2 (ICL2) seems to be capable of sensing of the negatively charged phosphorylated ICD. Interactions between ICL2 and ICD drive drastic conformational changes, thereby widening the pore. From our results we propose that SLAC1 operates by a mechanism combining the binding-activation and inhibition-release models.The anion channel SLAC1 functions as a crucial effector in the ABA signaling, leading to stomata closure. SLAC1 is activated by phosphorylation in its intracellular domains. Both a binding-activation model and an inhibition-release model for activation have been proposed based on only the closed structures of SLAC1, rendering the structure-based activation mechanism controversial. Here we report cryo-EM structures of  Phosphorylation-mediated activation of SLAC1 anion channel is crucial in plant responses for abiotic stresses including drought. Here, authors determine the open and closed structures of SLAC1, revealing how phosphorylation can activate SLAC1. However, the impermeability of the cuticle restricts gas exchange crucial for photosynthesis2. Consequently, stomata allow land plants to access aerial environment3. Stomata opening is regulated by a pair of guard cells by turgor pressure change. In the long evolutionary history, basic strategy of stomata regulation using osmotic pressure has not changed5. Stomata opening facilitates gas exchange and transpiration, but provokes water loss and pathogen entry8. Abiotic and biotic cues such as drought, light, humidity, CO2, ozone and pathogens are perceived by abscisic acid (ABA), salicylic acid (SA), Ca2+, ethylene, nitric oxide, hydrogen peroxide and hydrogen sulfide, activating pertinent signaling pathways to regulate the stomata opening17. Osmotic pressure change in the stomata opening is mediated by osmolyte movements through diverse ion channels and transporters2.Land plants have adapted to dry terrestrial environments by having cuticles on their surface to suppress water loss- and nitrate) efflux on the guard cell plasma membrane and acts as a key effector of stomatal closure19. Trimeric SLAC1 harbors N-terminal and C-terminal intracellular domains (ICDs) with the transmembrane domain (TMD) positioned in the middle. Signaling molecules such as ABA, CO2, Ca2+, SA, and hydrogen peroxide, participate in regulation of SLAC1 by means of phosphorylation and dephosphorylation via kinases and phosphatases26. Multiple serine/threonine phosphorylation in the N-terminal and C-terminal intracellular domains activates SLAC128. Kinases responsible for such multi-site phosphorylation include OPEN STOMATA 1 (OST1), calcium-dependent protein kinases (CPKs), calcineurin B-like/CBL interacting protein kinases (CBL-CIPKs), STRESS INDUCED FACTOR 2 (SIF2) and GUARD CELL HYDROGEN PEROXIDE-RESISTANT 1 (GHR1)30. Phosphatases responsible for the deactivation of SLAC1 by dephosphorylation include ABA-INSENSITIVE 1 (ABI1), ABI2 and protein phosphatase 2 C (PP2CA)22. Major phosphorylation sites  and several minor ones that are important for SLAC1 activation have been well defined31. These multiple phosphorylation of SLAC1 enables it to integrate signals from various signaling pathways. However, SLAC1 activation mechanism remains elusive due to limitation of our knowledge on how multiple phosphorylation events are coupled to conformational changes leading to the channel opening.Slow anion channel associated 1 (SLAC1) is a slow (S)-type anion channel that generates anion (ClHaemophilus influenzae TehA (HiTehA), a monocot plant Brachypodium distachyon (Bd) and a dicot model plant Arabidopsis thaliana (At)32. From structural analysis of the closed SLAC1 structures, two alternative models have been suggested for how phosphorylation leads to the activation of SLAC1: the binding-activation model28 and the inhibition-release model31. The binding-activation model claims that negatively charged phosphorylated serine/threonine residues of the intracellular domains (ICDs) bind to the positively charged surface of the transmembrane domain (TMD), leading to channel opening. The inhibition-release model states that ICDs work as self-inhibitory plugs that block the pore and phosphorylation of serine/threonine residues of ICDs relieves such inhibition, thereby activating SLAC1. Currently available SLAC1 structures in the closed state implicate controversy in the activation mechanism. The closed BdSLAC1 structure supports the binding-activation model because the closed BdSLAC1 does not interact with the ICD28. In contrast, a closed and phosphorylation-incapable mutant AtSLAC1-S59A structure reveals the ICD bound to TMD to act as a plug, supporting the inhibition-release model31.Despite extensive structural studies, only closed state structures of SLAC1 have been determined from a bacterial orthologue 29 in both open and closed states, and 8D (S59D/T62D/S65D/S86D/S107D/S124D/S146D/S152D) in a closed state. Conformational changes around the ion pore allows us to uncover a pore opening mechanism and several putative anion binding sites. A conserved, positively charged sequence motif in the intracellular loop 2 (ICL2) shows conformational changes that are essential for phosphorylation-mediated activation. The conformational changes caused by the ICL2 are coupled to partial unwinding of TM5 and helical rearrangements of other TMs. These coupled conformational changes widen the intracellular side of the pore just as in a putative open mechanism from a previous study31. The discovered latch motif in a closed state supports that phosphorylation sites on ICD are not randomly spread in intracellular space, but that their spatial distributions are restricted by the latch. We propose a combined activation model compatible with both the binding-activation and the inhibition-release models. These findings give us a perspective on diverse phosphorylation sites and their roles in SLAC1 regulation.To reveal the activation mechanism of SLAC1 unequivocally, here we report cryo-EM structures of AtSLAC1 with different phosphomimetic states: wild-type (WT) in an open state, 6D (T62D/S65D/S107D/S124D/S146D/S152D)29, to determine the AtSLAC1 structures in different conformational states . To evaluate the functional activities of the recombinant AtSLAC1 channels, we performed whole-cell patch clamp recordings on HEK293T cells expressing AtSLAC1 WT or 6D mutant in the presence or absence of the C-terminal sfGFP-HA-H10 tag  aligned in one direction  and cholesteryl hemisuccinate (CHS) 28 and dicot AtSLAC1 S59A (PDB: 7WNQ)31, are well conserved in our AtSLAC1 structures: 10 transmembrane helices (TMs) comprising the pore formed by five odd-numbered inner TMs and outer helices formed by five even-numbered TMs32 s32 Fig. . We alsos32 Fig. . The clo31 MFRTK112, revealing features for unequivocal modeling: an apparent N-terminal direction due to a right-helical turn and side chain orientation; readily discernible densities of the aromatic amino acid pattern (F/Y)xx(F/Y)R; distinct density of the phosphomimetic mutation S107D compared to that of the corresponding S107 in the AtSLAC1 S59A31  nearby the plug region in the cryo-EM maps of the closed structures of AtSLAC1: the maps of 6D and 8D as observed in that of S59A 31 Fig. . HoweverThe open state structures of AtSLAC1 revealed distinct conformational changes from closed states. AtSLAC1 6D open state was modeled with the TMD region  except the faint density of intracellular loop 2 (ICL2) (residues 319–328), and closed state was modeled with the TMD and an intracellular plug (residues 150-516) Fig. . Key strKlebsormidium nitens (Kn) and a liverwort Marchantia polymorpha (Mp) based on a previous study34  that might interact with negatively charged, phosphorylated residues of ICDs: (K/R)RRL(C/S)K in dicot and monocot plants and x(K/R)RLSK where “x” indicates any amino acid residue including aspartate, glutamate, lysine, glutamine and valine in basal plants  and its ICL2 mutants were expressed in HEK293T cells, and whole-cell currents from these constructs were recorded by whole-cell patch clamp recordings. ICL2 mutants showed similar or higher expression level than that of WT AtSLAC1  in the middle of the pore helices may contribute to pore regulation by helix movements such as rotation and kink32. Cα r.m.s.d.s among our AtSLAC1 6D open, closed, and two known closed structures are negligible on the extracellular side of TMs based on these helix breakers  to pass through in both the closed and open structures along with negligible structural differences on the extracellular side . The density of SExt is weak and dispersed, reflecting long distance, weak electrostatic interactions between a chloride ion and K347 and K461: 5.6 Å/4.0 Å in the 6D closed; 4.4 Å/5.1 Å in the 8D closed; 4.3 Å/4.7 Å in the 6D open; 5.8 Å/5.8 Å in the WT open  in the open and closed structures: one site is located in the extracellular exit of pore, S32. In the closed structure of AtSLAC1 6D, F276 on TM3 adopts an unfavorable rotameric state  in a manner similar to those in the previously reported structures, F285 of BdSLAC1  and F276 of AtSLAC1 S59A  , and three valine residues  Fig. . These cχ2) Fig. . In the χ2) Fig. , 4c, d. χ2) Fig. . The rotχ2) Fig. . F450 inχ2) Fig. , 4c. TM9χ2) Fig. . Previou451 Fig. . Such a 451 Fig. .39. In the closed structure, F276 represents an unfavorable conformation due to the confined space. Compared to the closed structure, the expanded space adjacent to F276 could permit small oscillation of χ1 and χ2 angles of F276, thereby widening the central gate transiently.How are these movements linked to the central gate widening? In the closed structure, constriction of the central gate is caused by two sites: a triad of V272, V336 and F450 on the intracellular side , and a quad of F276, V336, V340 and F450 in the center  Figs. . Slight 31. Our structures uncover that distribution of phosphorylation sites in the intracellular space is restricted by spatial constraints from the ordered plug and latch  than other residues  is conserved despite small BSAs 40 spans the C-terminal half of the latch and the first serine (S113) of the patch 1  that are crowded with minor phosphorylation sitestch Fig. . To conftch Fig. . All platch Fig. . The lattch Fig. . In the ues Fig. . The motues Fig. . By contues Fig. . InteresSAs Fig. . We founh 1 Fig. .Fig. 5ReMultiple sequence alignment uncovered that the latch and the patch 1 motifs are juxtaposed and the OST1-mediated phosphorylation motif overlaps with both the latch and patch 1 motifs Fig. . Structu27. Two models have been proposed for how phosphorylation leads to the activation of SLAC1: the binding-activation model28 and the inhibition-release model31. In the binding-activation model, negatively charged phospho-serine/threonine residues of ICDs bind to the positively charged surface of TMD, causing conformational changes for channel opening. The closed BdSLAC1 structure supports the binding-activation model based on the observation that the closed BdSLAC1 does not interact with the ICD28. In the inhibition-release model, intramolecular binding of ICDs to the rest of SLAC1 locks SLAC1 in a self-inhibitory state and subsequently phosphorylation of ICDs relieves SLAC1 of such inhibition, leading to the activation31.Phosphorylation in the N- and C-terminal ICDs of SLAC1 is essential for activation31. Such phosphorylation by kinases from heterologous origins may have led to heterogeneity in phosphorylation states, which in turn would complicate the interpretation of phosphorylation effects by phosphomimetic mutants. We also observed that some aspartate or glutamate mutations were phospho-defective  also apparently features a closed TMD with a partially unfolded plug43. The ICL2 was predicted to maintain a bent state despite a negligible interaction toward the plug. These results suggest that phosphorylation events, presumably inducing destabilization and subsequent dissociation of the plug and latch regions, are necessary for SLAC1 to escape from inhibition, reflecting the inhibition-release model, but not sufficient to convert it into the open state. In our open structures, phosphorylation events are connected to the channel opening via interactions between the phosphorylated ICD and ICL2, a feature of the binding-activation model  and C- 506-517) terminal regions of ICD and the latch (residues 105-112) make contacts with TMD in similar manners despite different phosphorylation states. Phosphorylation sites are located in the disordered region of ICD except S107, S152 and T513 on the plug terminal48-182 anWe propose a speculative activation mechanism combining elements from the binding-activation and the inhibition-release models. The combined activation model consists of the initial dissociation phase (inhibition-release) and the subsequent activation phase (binding-activation) Fig. . In the 32. SLAC1 and its homologues possess a pore on each protomer, but reported high cooperative opening indicates interprotomer relationship upon activation as previously suggested37. In trimer, each three ICL2s and ICL3s are alternately arranged and face each other  that blocks wide poreges Fig. . F266A mges Fig. . F276A a34. Isoleucine substitution shows nitrate-dependent gating, but valine substitution shows nitrate-independent gating. In our structures, V272 induces change of the rotameric state of F276, a key residue of opening despite a slight movement by itself. In the open state of a monocot SLAC1, Cδ1 atom of I281 differing from valine may narrow the triad constriction in the central gate  and dicot ate Fig. . It is p28. Previously, these phosphorylation sites were regarded as activators with a similar role in single activation mechanism. Our proposed combined activation model challenges whether each phosphorylation site contributes to either or both of the inhibitory component dissociation or the ICL2-mediated activation. The discovery of the ordered plug and latch from the disordered ICD may give a hint about the role of phosphorylation sites.Our results raise questions about role of various phosphorylation sites. Multiple phosphorylation events on the two major sites and patch 1 are responsible for activation of SLAC1Arabidopsis thaliana SLAC1  was cloned into a modified version of pACEBac2 (Geneva Biotech) containing a C-terminal tobacco etch virus (TEV) protease cleavage site, a HA tag (YPYDVPDYA) and a decahistidine tag (H10). To improve solubilization efficiency and protein stability, superfolder GFP (sfGFP)44 was introduced in the downstream of the TEV protease cleavage site. We achieved a higher protein yield through the replacement of the hexahistidine tag with a decahistidine tag. A phosphomimetic mutant 6D was prepared by a megaprimer PCR method. Other mutations used in this study, were introduced by site-directed mutagenesis. The final constructs, pACEBac2-(AtSLAC1-WT or mutants)-sfGFP-HA-H10 plasmids, were used to produce the corresponding recombinant baculoviral genomes.The coding region of the full-length E. coli DH10EmBacY (Geneva Biotech) strain. After 2 days, recombination clones were selected from blue-white screening followed by colony PCR. Bacmids were gently extracted by midi-prep due to their fragility. Subsequently, freshly prepared Sf9 cells   in 30 mL Sf-900 II SFM (Gibco) were transfected with 12.5 μg of recombinant bacmid using ExpiFectamine Sf transfection reagent (Gibco). Transfected cells were grown at 27 °C for 4 days and centrifuged at 1000 g for 10 min. The supernatant was filtered through 0.2 μm cellulose acetate filter, followed by the addition of 0.1% (w/v) bovine serum albumin to prevent proteolysis of recombinant P0 baculovirus and stored at 4 °C until use. Viral titer was measured by qPCR. To achieve high protein expression level, we produced P1 virus by infection of 1 × 106 Sf9 cells mL−1 with P0 virus at a multiplicity of infection (MOI) of 0.1.To produce recombinant baculoviral genomes, expression vectors were transformed into chemically competent 10 WT and phosphomimetic mutants were used to produce recombinant proteins. Sf9 cells at 2 × 106 cells mL−1 in 1.4~2.1 L Sf-900 II SFM were infected with recombinant P1 virus at a MOI of 5.0 and grown at 27 °C for 66 h. After expression, fluorescence of YFP and GFP was used to confirm infection level. Expressed cells were harvested at 3300 g for 10 min and kept at −80 °C until use. For lysis of cells, frozen cell pellets were resuspended in a hypotonic buffer , incubated for 30 min on ice and centrifuged at 22,000 g, 4 °C for 1 h. Pellets were resuspended in a hypertonic buffer , and incubated for 1 h on ice and centrifuged at 22,000 g, 4 °C for 1 h. To solubilize the proteins, a solubilization buffer  was added to pellets, followed by gentle bounce homogenization. The lysate was solubilized by stirring at 4 °C for 3 h and centrifuged at 22,000 g, 4 °C for 1 h to remove insoluble materials. For capturing the solubilized his-tagged proteins, pre-equilibrated TALON Superflow  resin was poured into the cleared lysate and the resulting solution containing the TALON resin and the solubilized protein was stirred at 4 °C for 3 h. The lysate was applied into a gravity-flow column to discard unbound lysate, and washed with 5 column volume (CV) of wash I buffer , followed by 15 CV of wash II buffer . Elution buffer  was applied into the column, followed by addition of 0.1 mM EDTA into the eluate. Eluted proteins were enriched to a final volume of 600 μL with an Amicon Ultra-15 100 kDa centrifugal filter unit (Merck Millipore). To remove aggregates, the enriched proteins were centrifuged at 16,000 g, 4 °C for 1 h. After centrifugation, soluble supernatant was loaded on a Superdex 200 increase 10/300 GL  column with final buffer . For cryo-EM, fractions containing homogeneous protein from the Superdex 200 column were enriched to a final concentration of 2–4 mg mL−1 using an Amicon Ultra-0.5 100 kDa centrifugal filter unit (Merck Millipore).Recombinant baculoviruses encoding sfGFP-fused AtSLAC1-sfGFP-HA-H10 proteins were diluted in the final buffer to a final concentration of 1.9, 2.4 and 3.3 mg mL−1. No particle was observed in the hole at 1.6 mg mL−1. Prior to sample application, Quantifoil R 1.2/1.3 Au 200 or 300 (Electron Microscopy Sciences) grids were negatively glow-discharged for 60 s in a PELCO easiGlow glow-discharge cleaning system . 3 μL of sample was applied, and grids were blotted , followed by plunge freezing in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). The 1.9 and 2.4 mg mL−1 datasets were collected on a Talos Arctica G2 (Thermo Fisher Scientific) operated at an acceleration voltage at 200 kV with a K3 detector (Gatan) and a BioQuantum energy filter (Gatan) at the Korea Basic Science Institute (KBSI). The 3.3 mg mL−1 dataset was collected on a Titan Krios G4 (Thermo Fisher Scientific) operated at an acceleration voltage at 300 kV with a K3 detector (Gatan) and a BioQuantum energy filter (Gatan) at the Institute of Membrane Proteins (IMP). Movie data were collected using automated process of EPU software in electron counting mode. Total 3562 movies (2.4 mg mL−1), 4792 movies (1.9 mg mL−1) and 12,149 movies (3.3 mg mL−1) were recorded and fractionated into 50 frames with total dose of 50 e-/Å2, a pixel size of 0.84 Å per pixel (1.9 and 2.4 mg mL−1) and a pixel size of 0.85 Å per pixel (3.3 mg mL−1), and defocus range of −1.0 to −1.9 μm (2.4 mg mL−1), −0.8 to −1.7 μm (1.9 mg mL−1) and −0.7 to −1.9 μm (3.3 mg mL−1), respectively.The purified AtSLAC1-6D-sfGFP-HA-H10 were diluted in the final buffer to a final concentration of 1 mg mL−1. Prior to sample application, Quantifoil R 1.2/1.3 Au 300 (Electron Microscopy Sciences) grids were negatively glow-discharged for 60 s in a PELCO easiGlow glow-discharge cleaning system . 3 μL of sample was applied, and grids were blotted with similar condition , followed by plunge freezing in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). The datasets were collected on a Talos Arctica G2 (Thermo Fisher Scientific) operated at an acceleration voltage at 200 kV with a K3 detector (Gatan) and a BioQuantum energy filter (Gatan) at the KBSI. Total 2662 movies were recorded and fractionated into 50 frames with total dose of 50 e-/Å2, a pixel size of 0.84 Å per pixel and defocus range of −0.7 to −1.9 μm.The purified AtSLAC1-WT-sfGFP-HA-H10 were diluted in final buffer to a final concentration of 2.1 mg mL−1. Prior to sample application, Au-Flat 1.2/1.3 Au 300 (Protochips) grids were negatively glow-discharged for 60 s in a PELCO easiGlow glow-discharge cleaning system . 3 μL of sample was applied, and grids were blotted with similar condition , followed by plunge freezing in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). The datasets were collected on a Talos Arctica G2 (Thermo Fisher Scientific) operated at an acceleration voltage at 200 kV with a K3 detector (Gatan) and a BioQuantum energy filter (Gatan) at KBSI. Total 11,523 movies were recorded and fractionated into 50 frames with total dose of 50 e-/Å2, a pixel size of 0.84 Å per pixel and defocus range of −0.8 to −1.8 μm.The purified AtSLAC1-7D-sfGFP-HA-H10 were diluted in the final buffer to a final concentration of 2.3 mg mL−1. Prior to sample application, Au-Flat 1.2/1.3 Au 300 (Protochips) grids were negatively glow-discharged for 60 s in a PELCO easiGlow glow-discharge cleaning system . 3 μL of sample was applied, and grids were blotted with similar condition , followed by plunge freezing in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). The datasets were collected on a Titan Krios G4 (Thermo Fisher Scientific) operated at an acceleration voltage at 300 kV with a K3 detector (Gatan) and a BioQuantum energy filter (Gatan) at IMP. Total 11,112 movies were recorded and fractionated into 50 frames with total dose of 50 e-/Å2, a pixel size of 0.858 Å per pixel and defocus range of −0.7 to −1.9 μm.The purified AtSLAC1-8D-sfGFP-HA-H45. Each dataset was evaluated by data processing with a randomly sampled subset from the dataset. From initial screening and processing, we observed that particles predominantly represent open conformation in two datasets of low concentration samples (1.9 and 2.4 mg mL−1). Therefore, we combined these datasets and used to determine the open structure. By contrast, particles representing closed conformation were observed in the datasets of relatively high concentration samples. The dataset of 3.3 mg mL−1 sample was used to determine the closed structure. All movies were motion-corrected by patch motion correction and processed by patch CTF estimation. Next, randomly selected small subsets were used to train particle picking models. In detail, initial blob picking and following 2D classification were performed for template picking. The particles from template-based picking were 2D classified, and then selected particles were used to train a Topaz picking model. After particle extraction by the trained Topaz model, these particles were sorted by 2D classification, ab initio reconstruction, heterogeneous refinement and used to improve the Topaz model. Particles were extracted from all micrographs using the improved Topaz model. These particles were classified by 2D classification and heterogeneous refinement with an ab initio model, decoys and junks. Selected particles were refined with C3 symmetry by non-uniform refinement and subsequent local refinement was performed with a mask excluding micelles and disordered regions. Further 3D classification and local refinement were used to refine final map and local filtered map were used to build models.All image processing was performed using CryoSPARC46 based on known HiTehA structures. The homology model was fitted into the open AtSLAC1 6D map using Phenix47. Coot48 was used to manually rebuild models and real space refinement of Phenix was performed to deduce a final model. For model building of AtSLAC1 WT open, 6D closed and 8D closed, similar procedures were conducted, but the open AtSLAC1 6D and closed AtSLAC1 S59A model was used as an initial model. The final models contained residues 150-516 for AtSLAC1 6D closed structure, residues 182-318 and 329-506 for AtSLAC1 6D open structure, residues 182-317 and 327-508 for AtSLAC1 WT open structure and residues 105-112 and 148-516 for AtSLAC1 8D closed structure. The cryo-EM data collection, refinement and validation statistics of all structures are summarized in Supplementary Table 49, UCSF Chimera50 and UCSF ChimeraX51, and were analyzed using MOLE38 and PISA52.For high concentration dataset, we extracted particles by a trained Topaz model and a consensus map was obtained by similar data processing procedure. The Topaz model was further trained from these particles to yield a refined consensus map. This consensus map represented weak density of TMD-bound density of ICD and weird density around TM5. Without any prior information, these particles were divided into closed and open states by ab initio reconstruction and heterogeneous refinement. Entire particles from 2D classification were classified by a series of heterogeneous refinement with these open and closed models, decoys and junks. Closed particles and closed-like particles (absence or weak ICD density) were further classified by heterogeneous refinement and refined by similar process to yield a closed state map. The rest open state particles were used to yield an open state map, but we obtained a low-resolution map due to heterogeneity of open state particles. Other constructs were also processed by similar data processing procedure. Detailed information of data processing is summarized in Supplementary Figs. -1 (per 35 mL) EZ-Link Sulfo-NHS-SS-Biotin for 15 min on ice. Remaining reactive biotin was washed out with PBS and additionally quenched with 50 mM glycine pH 7.5. Cells were harvested and lysed in a lysis buffer . Biotinylated plasma membrane (surface) proteins were purified from whole cell lysate  using NeutrAvidin Plus UltraLink resin. After washing the resin with the lysis buffer, proteins were eluted from resin by 2X LDS sample buffer. Total and surface proteins were separated on a Bolt™ 4–12% Bis-Tris Plus Gel (Invitrogen) and transferred to a PVDF membrane using iBlot 2 Transfer Stack. 250–500 ng antibodies /mL iBind Flex Solution were used for immunoblotting. Protein bands were visualized by Clarity Western ECL Substrate and images were acquired by the ChemiDOC imaging system. Three independent experiments were conducted. Densitometric analysis was performed by ImageJ53 software.To test cell surface expression level of AtSLAC1, surface biotinylation was performed. HEK293T cells  were transfected with wild-type and mutant constructs using the Transporter 5™ Transfection Reagent and incubated for 40 h. Cells were washed with ice-cold PBS and biotinylated using 2 mL of 0.25 mg mL2 incubator. Cells at 50–60% confluence in a 6-well plate were transiently transfected with 0.5 ~ 1.0 μg of AtSLAC1 plasmid DNA in complexed with polyethylenimine transfection reagent according to the manufacturer’s instruction. For identifying transfected cells in whole-cell patch clamping, GFP-expressing plasmid DNA was co-transfected at 1:5 ratio. A day after transfection, the cells were moved to the poly-L-lysine (PLL) coated coverslips and allowed to be attached at least 6 h. Typically, electrophysiological recordings were conducted 30 ~ 48 h after transfection. Borosilicated micropipettes (WPI) were pulled by using P-1000 puller (Sutter) and fire-polished by using MF-900 microforge (Narishige). The patch pipettes had resistance of 2 ~ 3.5 MΩ were backfilled with the internal solution . The external solution contained 140 mM NaCl, 20 mM HEPES, pH 7.4 with NaOH. Whole-cell patch clamp recordings were performed with a Multiclamp 700B amplifier (Molecular Devices) controlled by pCLAMP 11 software via Digidata 1550B data acquisition system (Molecular Devices). After formation of whole-cell patch clamp mode, currents were evoked by 500-ms step stimulations of −100 mV to +100 mV with 20-mV increments from a holding potential of 0 mV. Data were sampled at 5 kHz and filtered at 2 kHz by low-pass vessel filter. Experiments were performed at room temperature (20~25 °C).HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Thermo) at 37 °C in a 5% COFurther information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


This article discusses the results of the excavations conducted in the Iron II site near the En-Gedi Spring in 1961–1962 and 2019. The site, consisting of a prominent stone platform documented as early as the 19th century and other recently discovered structural remains, is interpreted as a Judahite outpost built in a strategic location within the oasis of En-Gedi. On the basis of the ceramic assemblage, it is suggested that this site was founded during the early 7th century BCE and was abandoned before the end of that century—making it the earliest Iron Age occupation in the oasis. Combined with historical considerations and a regional analysis, the En-Gedi Spring site enhances our understanding of the Judahite expansion into the Judaean Desert during the late Iron Age. The 7th century BCE is widely regarded as a transformative era in the history of the Southern Levant in the Iron Age Koch . Shelteraman 1991; Finkelstein One region in which the transformations of the 7th century BCE are most evident is the eastern fringe of Judah—the Judaean Desert and the western coast of the Dead Sea. Here, the region that was desolate and barren until the end of the 8th century BCE experienced an unprecedented wave of activity, including the establishment of agricultural estates in the oases of the northwestern Dead Sea shore and the Hyrcania Valley  and numerous forts and towers in the desert uplands Stager : 249–271The key site for reconstructing the Judahite expansion into the Judaean Desert is, evidently, En-Gedi—the most prominent oasis along the western shore of the Dead Sea . Here, on a low hill in the southern part of the oasis plain (Tel Goren), a thriving Judahite centre was founded during the late Iron Age (Stratum V), on what would become the main mound in the oasis in subsequent centuries. Since the first publications of the Hebrew University excavations at Tel Goren in the early 1960s, it has been suggested that the late Iron Age activity in the oasis of En-Gedi began no earlier than the last third of the 7th century BCE . This operation took place at the same time as the excavations at Tel Goren and other sites within the oasis  headed by Nimrod Marom, a project that explores Holocene human–environment interactions in the Judaean Desert , followed by excavation of a circular probe (L301), ca. 2 m in diameter, near its northeastern corner b.2 In thFurther excavations along the outer walls of the platform were conducted by the Mazar expedition in three probes near its northern, western, and eastern walls, labelled L302, L303 and L304, respectively, in which the platform’s outer foundations were exposed. These excavations demonstrated that the platform was erected directly on bedrock, atop a natural rocky outcrop protruding from the terrace surface. According to assessments in the field journal, this outcrop was hewn and flattened by the platform’s builders prior to its construction, but no evidence for this was found in the renewed investigation of the site.L302 was a narrow trench excavated by the Mazar expedition perpendicular to the northern outer wall of the platform, to a distance of 3 m. A description provided in the excavation log recounts an exceptional amount of ‘stone collapses’ in this locus and mentions a ‘course of stones’ that runs ‘parallel to the outer wall of the tower’.The architectural purpose of this stone revetment cannot be determined with certainty due to the limited exposure and meagre architectural information collected thus far. It should be emphasised, however, that similar constructions do not exist along other faces of the platform and that topographically, the most convenient approach to the platform is from the north. It may thus be suggested that this feature was intended to serve as a base for a ramp or staircase leading up to the platform , exposed new architectural remains associated with in situ Iron Age deposits and confirmed our initial hypothesis regarding the extension of Iron Age remains in this area. These remains were covered by a thick (Ꜥ 0.5 m) geological layer consisting of compacted silts, sands, gravel and cobbles, interpreted as debris flow, i.e., colluvial deposits induced by heavy torrential rainstorms over the nearby steep western Dead Sea Escarpment . This accumulation ‘sealed’ the ancient remains, and, if not for the 1960s excavations that removed part of this debris and exposed the edges of the features to be described below (without noticing them), it is unlikely that we would have suspected the presence of these remains.The 2019 excavation focused on the area to the west and northwest of the platform, following the detection of hitherto unknown ancient architectural remains in this area.The most prominent of the newly exposed remains is W320—a 1 m wide wall built of two rows of medium-sized and large fieldstones and cobbles in a north–south orientation, parallel to and ca. 6.5 m to the west of the western face of the stone platform . In the A small segment of an additional north–south wall, W309, was exposed ca. 3 m east of W320 and at a similar distance west of the platform. This wall was built as a single line of medium-sized fieldstones and was poorly preserved to a maximal height of 0.5 m and a length of 2 m b; its soin situ deposits dated to the Iron Age clearly suggests that the square platform was not an isolated feature, as previously assumed. As for the relations between the platform and the newly discovered architecture, the removal of the accumulation west of the platform by the Mazar expedition (L303) and the insufficient documentation related to this operation make it impossible to establish a stratigraphic connection between them. Nonetheless, the lack of evidence for chrono-stratigraphic phasing at the site coupled with the homogeneous character of the pottery assemblage found by both expeditions (see below) suggest that all the architectural elements at the site belong to a single, probably short, phase of activity and possibly to a single architectural complex.The limited excavations conducted in 2019 do not permit a comprehensive reconstruction of the nature of the Iron Age activity in the En-Gedi Spring site. Except for limited ceramic assemblages, no other artefacts were retrieved, and the preservation of organic materials  was limited to tiny unidentified fragments, possibly due to moist and saline conditions in the sub-surface associated with the nearby spring. Nevertheless, the exposure of hitherto unknown architectural remains associated with The ceramic assemblages from the En-Gedi Spring site collected by the two expeditions  contain et al.et al.aman 2004; Davidovich The ceramic typology of the site, coupled with the relatively short duration of activity indicated by the limited anthropogenic accumulation and refuse, seems to suggest that the En-Gedi Spring site was active most probably during the first half of the 7th century BCE. The assemblage is comparable to other Judahite sites with strata from this period, particularly in Jerusalem  Iron IIC assemblage. Interestingly, the notion that the site near the En-Gedi Spring is earlier than Tel Goren Stratum V was already implied in the field journals of the Mazar expedition. In the March 14, 1962 entry, in reference to the finds from the Spring site, the excavators state that: ‘The few sherds [from the excavations in the platform] give the impression that they are earlier than the vessels of Stratum V at Tel Goren and ended together with it’.The data presented in this article suggests that the area of the En-Gedi Spring housed, during the early 7th century BCE, the earliest Judahite occupation in the En-Gedi oasis. The location of the site must have been carefully selected, as it combines three important qualities: 1) proximity to the main spring irrigating the central part of the oasis; 2) a commanding view of the lion’s share of the oasis, especially to the south and southeast towards the outlet of Naḥal ꜤArugot  and apparently comprised a single architectural complex. The small size of the site suggests that it did not house a large sedentary community and probably served other functions. This is also indicated by the very thin anthropogenic accumulations and the strikingly restricted quantity and diversity of refuse deposited at the site, which may testify to a short occupation, possibly lasting no longer than several decades.The solid raised platform, which drew the attention of travellers and archaeologists since the early days of exploration at En-Gedi, is the most notable feature at the site. Its construction method, involving the use of a wide dense stone mass supported by strong single-faced walls, is widely attested in public constructions in Judah and the Southern Levant in the Iron II. It was employed primarily in urbanized contexts, featuring prominently in fortification walls and towers . Following its excavation in the early 1960s by the Mazar expedition, this stratum was dated to the last third of the 7th century BCE on the basis of historical considerations coupled with the ceramic evidence, which accords well with the date of strata attributed to the Babylonian conquest (Lachish Level II horizon) elsewhere in Judah  preceded that of Stratum V in Tel Goren. This implies that Judahite control over the oasis of En-Gedi began earlier than previously hypothesised, with the foundation of a relatively small outpost near the spring. This site, with its massive platform erected for either military/administrative or ritual purposes, reflects an incipient Judahite foothold in the major oasis of its eastern wilderness. Its strategic location granted its occupants good control over the oasis and its environmental resources, allowing Judah to establish its presence in this desolate region.ibid.; Parker The new data attesting to an early 7th-century BCE date for the beginning of the Judahite occupation of En-Gedi also allows for a better understanding of the enigmatic Hebrew inscription found almost 50 years ago south of Naḥal Yishai, ca. 1.2 km northeast of the En-Gedi Spring Bar-Adon . This infossiles directeurs of the Iron IIB (contra Vaughn The early occupation of En-Gedi, epitomised in the foundation of the En-Gedi Spring site, was evidently one component—albeit a significant one—in the much broader process of Judahite territorialisation under Assyrian hegemony during the early 7th century BCE. A recent reevaluation of all Iron Age sites in the Judaean Desert, which included a detailed inspection of both published and unpublished pottery assemblages and new surveys in multiple sites (Davidovich et al. Toward the mid-7th century BCE, possibly still within the Assyrian era, the centre of gravity in En-Gedi shifted to Tel Goren in the oasis plain. This process may perhaps be attributed to the consolidation of Judahite rule in the Judaean Desert, resulting in a general shift in the nature of the Judahite presence at En-Gedi from isolated outpost to regional economic hub. This reconstruction accords well with the historical processes associated with the period of Assyrian hegemony in the Levant. The initial establishment of En-Gedi in the form of a small outpost near the spring should be attributed to the years that followed the punitive Assyrian campaigns of the late 8th century BCE, during the later part of Hezekiah’s reign or the early days of Manasseh, whose reign is perceived as a prosperous era of demographic and economic growth (e.g., Finkelstein


Ice formation on solid surfaces is a ubiquitous process in our daily life, and ice orientation plays a critical role in anti-icing/deicing, organ cryo-preservation, and material fabrication. Although previous studies have shown that surface grooves can regulate the orientation of ice crystals, whether the parallel or perpendicular alignment to the grooves is still under debate. Here, we systematically investigate ice formation and its oriented growth on grooved surfaces through both in situ observation and theoretical simulation, and discover a remarkable size effect of the grooves. With the designability of surface groove patterns, the preferential growth of ice crystals is programmed for the fabrication of a crisscross-aligned graphene aerogel with large negative Poisson’s ratio. In addition, the size effect provides guidance for the design and fabrication of solid surfaces where the effective control of ice orientation is highly desired, such as efficient deicing, long time organ cryo-preservation, and ice-templated materials. Ice formation on grooved surfaces is ubiquitous, but controlling orientation is difficult due to lack of mechanistic insight. Here, the authors observed oriented growth using graphene oxide nanosheets as probes, revealing the effect of groove size, and programmed ice growth to fabricate freeze-cast metamaterials. In recent years, ice orientation controlled by selective ice nucleation and preferential ice growth has been the focus of research in various aspects, including anti-icing/deicing4, cell preservation5, and fabrication of multifunctional materials7, which are related to ice adhesion, cell survival, and formation of porous structure, respectively. Many works reported that oriented ice crystals have been successfully achieved through regulating the intrinsic properties of solid surfaces8, such as surface microstructure13, surface charge15, and surface wettability18. Specifically, surface grooves have been proven to be capable of inducing both parallel and perpendicular orientation of ice crystals23. Lo. et al. reported that water vapor nucleate on the V-grooved surface, preferentially grows along the direction perpendicular to the grooves, and aligns parallelly to the grooves at the macroscopic level19. Munch. et al. reported that ice crystals tend to grow parallelly or perpendicularly to the grooves during the freeze-casting process depending on the cold surface23. Therefore, ice orientation on grooved surfaces is still under debate, which can be attributed to the limited approach and instruments to directly observe the ice orientation in pure water without any probes. Additionally, the effect mechanism of surface grooves on ice orientation through controlling ice nucleation and growth is still unknown. Therefore, systematic research of ice formation on grooved surfaces is highly demanded.Ice formation on cold surfaces is a ubiquitous phenomenon in nature with wide implications for our daily livesHere, we investigated ice orientation on various grooved surfaces through both in situ observation and theoretical simulation and proposed a size effect of the grooves. A small amount of dye-labeled graphene oxide nanosheets (0.5 wt.%) were dispersed in water as non-invasive probes to visualize the boundaries of ice crystals. It was found that both parallel and perpendicular preferential ice growth can be achieved on grooved surfaces depending on the groove size. With the designability and versatility of groove patterning, the preferential growth of ice crystals can be facilely achieved and applied to fabricate materials with complex porous architectures and therefore unprecedented properties. As a proof of concept, we fabricated an ultralight graphene aerogel with a unique crisscross-aligned architecture and ultralow negative Poisson’s ratio. Moreover, the groove size effect also provides guidance for engineering solid surfaces to control ice formation, which is highly desired in various aspects, such as efficient deicing, high-percent-survival cell preservation, and ice-templated materials with controllable pore structures.W) and distances (D) were observed using a fluorescent microscope  platelets, and boron nitride (BN) nanosheets were obtained and shown by the scanning electron microscopy (SEM) images observed in the cross-section parallel to the grooved surface . As shown in Supplementary Fig. λ-W/D map distribute in a concentrated region, which varies among the suspensions with different ksuspension. Therefore, we infer that ice orientation is controlled by λ, ksuspension, and W/D. As shown in Fig. λksuspension < 30 (×10−6 W K−1)  or λksuspension > 120 (×10−6 W K−1) (large λ), ice crystals grow into small-area alignment. When 30 <λksuspension < 120 (×10−6 W K−1) (medium λ), the growth mode of ice crystals is dependent on the ratio of W and D. This phase diagram indicates that the desired ice orientation in the suspension with a certain thermal conductivity can be obtained under the induce of surface grooves with a certain W and D , which holds great promise for the design and fabrication of functional grooved surface. It should be noted that with higher thermal conductivity, smaller λ is required to effectively generate preferential orientation. When λ is at nanometer scale, other mechanisms like nucleation barrier difference may play a more important role and new mechanism should be developed.A series of substrates with a wide combination of grooves ves Fig. . As showJ = J0exp(−ΔG kB−1T−1)8, ice crystals preferentially nucleate at the bottom of the grooves with a lower temperature. Additionally, as our grooves are around several to tens of micrometer scale, three orders of magnitude than the critical radius of ice crystals24, we consider the grooves size have a negligible effect on nucleation energy barrier. According to the experimental results and theoretical analysis, typical freezing processes on grooved surfaces with different sizes are illustrated in Fig. λ, ice crystals nucleate at the bottom of the grooves and grow randomly towards different directions, resulting in a multidomain structure with small-area alignment  determines the ultimate orientation, the temperature distribution at this place was quantitatively evaluated. This temperature variation is plotted with respect to the corresponding y position for λ = 20 μm at different t. As illustrated in Fig. T1 and T2 represent the temperature of the midpoint at the top of the concave and convex parts, respectively. Figure T1 and T2 on λ when ice crystals grow up to the top position of the concave parts (t = t1) /(λ/2), representing the localized temperature gradient along the y axis  to promote the preferential growth of ice crystals perpendicular to the grooves. It is noteworthy that the specific periodicity is closely relevant to the freezing conditions, such as the cooling rate, the thermal conductivity of the cold substrate, and suspension properties. Figure In order to explain the perpendicular ice growth, we carried out theoretical simulations to describe the temperature field on various grooved surfaces, as it is challenging to experimentally detect this localized temperature gradient within grooves. Since perpendicular orientation occurs on the grooved surfaces with ces Fig. . Becauset1) Fig. . The corxis Fig. . It showλ (30 μm) but different W/D was performed. As shown in the Supplementary Fig. W = 25 μm and D = 5 μm, there is no significant temperature gradient to induce the preferential growth of ice crystals, thus ice crystals nucleate at the bottom of the grooves and grow towards random directions until ice crystals in a single groove merge together due to the geometric constraints. For the grooves with W = 5 μm and D = 25 μm, there is also no significant temperature gradient to induce the preferential growth of ice crystals, thus ice crystals nucleate at the bottom of the grooves and grow until they randomly merge together into a small-area alignment without geometric constraints. For the grooves with W = D = 15 μm, an obvious temperature gradient was generated to induce the perpendicular growth of ice crystals. Therefore, we propose that the preferential growth of ice crystals on the grooved surface can be programmed through either the geometric constraint or a localized temperature gradient.Additionally, in order to further prove the formation of three growth modes, the simulation of the temperature field on the grooved surfaces with a constant 26, underflow freezing27, bidirectional freezing7, and surface wettability induced freezing18. In this work, surface grooves induced freezing was investigated based on the localized temperature gradient between two microgrooves, holding great potentials for the construction of complex porous architectures.It is well-known that the structure of freeze-casted materials plays the critical role in the final property, thus many researches have been focusing on the tailoring of freeze-casted architecture and developed various techniques, such as external field assisted freezingIn order to demonstrate the potential application of our discovery, porous material with a unique architecture was fabricated by freeze-casting on a grooved surface with a composite pattern. In particular, a cross-aligned structure  behavior would endow aerogels with a substantial enhancement of mechanical robustness to resist damage through volume shrinkage when under inevitable deformation,38 which requires precise design for the porous architecture of the aerogel. As shown in Fig. θ) between the lamellae orientation and vertical compression direction on the NPR behavior was firstly investigated through a finite element simulation . As depicted in Fig. θ and θ = 45° shows that pattern-45 generates the lowest NPR value.In recent years, aerogels have been extensively explored due to their excellent thermalθ = 45°) has been successfully produced through the controllable ice growth induced by the surface grooves with corresponding pattern , outperforming those of previously reported graphene oxide-based (GO-based) aerogels41  comparable to the commercial silica aerogel, and maintain excellent thermal insulating property after compression and recovery from a 90% strain, while the commercial aerogel is broken after compression. Notably, such programmable ice growth on the grooved surface provides an efficient route for the fabrication of multifunctional graphene aerogels with tunable NPR behaviors, such as soft actuators with positive Poisson’s ratio, sensors with zero Poisson’s ratio, and body armors with negative Poisson’s ratio.As a proof of concept, graphene aerogel with the typical architecture  were purchased from Merck, Germany. Boron nitride (BN) powders with average size of 10 µm were purchased from Dandong Chemical Engineering Institute Co., Ltd., China. Poly , 5-fluorescein hydrochloride (5-DTAF), and dimethyl sulfoxide (DMSO) were purchased from Aladdin Chemistry Co., Ltd., China. Waterborne polyurethane solution  was purchased from ShunDe SanSheng Trade Co., Ltd., China. Poly(ethylene glycol) (PEG-300) was purchased from Sigma-Aldrich Chemistry Co. Ltd., USA. Darvan 811 was purchased from R.T. Vanderbilt Co., Norwalk, CT, USA.The grooved silicon wafer was fabricated by photolithography by commissioning Suzhou Research Materials Microtech Co., Ltd., China. Polydimethylsiloxane  was purchased from Dow Corning Co., Ltd., USA. Ethanol (AR) and sodium bicarbonate (AR) were purchased from Sinopharm Chemistry Co., Ltd., China. Octadecyltrichlorosilane (OTS) was purchased from Macklin Biochemical Co., Ltd., China. Graphene oxide sheets  were purchased from Hangzhou Gaoxi Tech Co., Ltd., China. The alumina platelets  under the vacuum atmosphere and 70 °C for 2 hours. The modified surface turned out to be hydrophobic. Then the silicon wafer was put in an aluminum plate and PDMS was poured to cover the silicon wafer. After curing at 70 °C for 2 hours, the grooved PDMS substrate that duplicated the pattern on the silicon wafer could be obtained. The PDMS substrate is 1 mm in thickness and the grooves are 5 μm in height, respectively.−1) was added to 100 mL sodium bicarbonate  solution. 5 mg of 5-DTAF dissolved in 1 mL of anhydrous DMSO was further added into 100 mL of the sodium bicarbonate solution to form a reactive dye solution. And the solution was stored for 12 h under 4 °C in a dark environment to form conjugated fluorochromes. Finally, dye-labeled graphene oxide dispersions were obtained by centrifuging multiple times with deionized water to remove excess dye molecules.In order to observe ice orientation, we added 0.5 wt% graphene oxide (GO) nanosheets into the water, and the GO nanosheets made the ice boundaries clear. At the same time, in order to clearly observe GO nanosheets, we labeled them with fluorescent dyes and observed them using fluorescence microscopy. Firstly, 1 mL GO dispersion (10 mg mL−1) and PVA (3 mg mL−1). The suspension was sonicated for 1 hour at 60% power and degassed before freezing. Alumina suspension was prepared by mixing distilled water with alumina platelets (20 vol.%), Darvan 811 , poly (ethyl glycol) , which were regarded as the dispersant and the lubricant respectively. The suspension was ball-milled for at least 24 hours and degassed before freezing. The BN/PU suspension was prepared by mixing distilled water with exfoliated BN nanosheets (10 vol.%) and PU solutions (25 wt.% to BN). The suspension was sonicated for 1 hour at 60% power and degassed before freezing. Square Teflon tubes were placed on the grooved PDMS substrate with various groove sizes. The suspension was poured into the mold and frozen on the grooved PDMS substrates at a cooling rate of 5 °C min−1 from −30 °C. After being entirely frozen, the sample was freeze-dried for more than 24 hours with freeze dryer under 0.05 mbar pressure . The commercial aerogel is silica aerogel fabricated by sol-gel method, having a thermal conductivity of around 16 mW m−1 K−1.The GO/PVA suspension was prepared with GO  images were collected by Hitachi S-3500N at an acceleration voltage of 5 kV. The thermal conductivity Supplementary informationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Source Data


How conformational dynamics affect the catalytic activity of enzymes remains a topic of active debate. We focus here on the domain closure dynamics of adenylate kinase (AK) and how they are affected by substrate inhibition. By screening an extensive mutant library, we show that this feature of the enzyme is well conserved in evolution. Importantly, domain closure is required in order to bring AK’s substrates to close together for their chemical reaction; single-molecule FRET studies directly measure the populations of the open and closed states. We find that overpopulation of the closed state can be detrimental to activity. The results allow us to develop a kinetic model that properly accounts for AK kinetics by combining conformational dynamics and biochemical steps. Enzymes play a vital role in life processes; they control chemical reactions and allow functional cycles to be synchronized. Many enzymes harness large-scale motions of their domains to achieve tremendous catalytic prowess and high selectivity for specific substrates. One outstanding example is provided by the three-domain enzyme adenylate kinase (AK), which catalyzes phosphotransfer between ATP to AMP. Here we study the phenomenon of substrate inhibition by AMP and its correlation with domain motions. Using single-molecule FRET spectroscopy, we show that AMP does not block access to the ATP binding site, neither by competitive binding to the ATP cognate site nor by directly closing the LID domain. Instead, inhibitory concentrations of AMP lead to a faster and more cooperative domain closure by ATP, leading in turn to an increased population of the closed state. The effect of AMP binding can be modulated through mutations throughout the structure of the enzyme, as shown by the screening of an extensive AK mutant library. The mutation of multiple conserved residues reduces substrate inhibition, suggesting that substrate inhibition is an evolutionary well conserved feature in AK. Combining these insights, we developed a model that explains the complex activity of AK, particularly substrate inhibition, based on the experimentally observed opening and closing rates. Notably, the model indicates that the catalytic power is affected by the microsecond balance between the open and closed states of the enzyme. Our findings highlight the crucial role of protein motions in enzymatic activity. Enzymes have been designed by nature to accelerate vital chemical reactions by many orders of magnitude. Quite a few of them have evolved to harness large-scale motions of domains and subunits to promote their activity. The study of the structural dynamics of these proteins is essential to decipher how they function and are regulated, as has been demonstrated in multiple experimental and theoretical reports –4. The e511616Interestingly, AK also incurs a pronounced AMP-mediated substrate inhibition (SI) , 27. DesIn this work, we carried out experimental and computational studies on several inhibition-prone as well as noninhibited mutants of AK  to deter5A . We then generated a library of 923 mutants wherein 13 different amino acids were introduced at each of these 71 locations identified above (Methods). Next, we developed a screening method  to measure the extent of AMP-mediated SI directly from the cell lysate of 1248 individual colonies by measuring the ratio of the initial reaction velocity at two different concentrations of AMP  and inhibition constant (IK) were obtained by a fit to a model of uncompetitive inhibition (Methods), and the values are given in SI Appendix, Table S1. We mapped the positions of the inhibition-prone mutants (IK ≤ 500) and the noninhibited mutants  on the structure of AK  higher conservation than those that led to an increase of inhibition.  strains . Paalme n source . For mitn source ], thus rn source . The curo the WT ; E.coli her side . Even sikcat), the explicit interconversion rates between the open and closed states do not determine enzyme activity. Instead, the relative population of the two states is what determines activity and inhibition.It is important to mention that previous work on AK dynamics suggested that the LID opening rate is the rate-determining step in catalysis , 15. In A matter of intense debate in enzyme catalysis is whether conformational fluctuations play a role in catalysis , 46, 47.This study goes beyond previous attempts to connect conformational dynamics and chemical steps in enzymes by developing a kinetic model that explicitly takes conformational changes into account. The model builds on the recent in silico observation that fast opening and closing cycles might help to find the correct mutual substrate orientation efficiently and to guarantee the high catalytic activity of the enzyme . It is fSI Appendix for a detailed description of the methodology for sample preparation, data collection, and analysis. Briefly, a large library of AK mutants was screened for the strength of substrate inhibition. A total of 86 unique mutants were purified and tested for their activity. The resulting inhibition constants were correlated with the conservation indices of mutated residues. Proteins for smFRET experiments were expressed and labeled with two fluorescent dyes. Single-molecule data were collected on freely diffusing molecules and analyzed using the statistical analysis algorithm H2MM, to obtain domain opening and closing rates. These rates served as input for simulation of enzyme kinetic curves using a model that included conformational dynamics.See SI Appendix, Figs. S1–S11 and Tables S1–S10, supporting references.Methods section containing a detailed description of the methodology, Appendix 01 (PDF)Click here for additional data file.


The end of the Middle Pleistocene Transition  was characterised by the emergence of large glacial ice-sheets associated with anomalously warm North Atlantic sea surface temperatures enhancing moisture production. Still, the direction and intensity of moisture transport across Eurasia towards potential ice-sheets is poorly constrained. To reconstruct late MPT moisture production and dispersal, we combine records of upper ocean temperature and pollen-based Mediterranean forest cover, a tracer of westerlies and precipitation, from a subtropical drill-core collected off South-West Iberia, with records of East Asia summer monsoon (EASM) strength and West Pacific surface temperatures, and model simulations. Here we show that south-western European winter precipitation and EASM strength reached high levels during the Marine Isotope Stage 18 glacial. This anomalous situation was caused by nearly-continuous moisture supply from both oceans and its transport to higher latitudes through the westerlies, likely fuelling the accelerated expansion of northern hemisphere ice-sheets during the late MPT. Iberian margin and Chinese Plateau climatic records combined with climate modeling reveal a warm ice age centered at ~730,000 years ago, which would have contributed to the accumulation of ice triggering the transition from the 40,000-year to the current 100,000-year climate cycles. Imbrie et al.’s conceptual model2 shows that the growth and decay of ice-sheets amplify, through albedo, winds and ocean mass properties, the system’s initial modest responses to orbital forcing by changing the transport of heat to boreal latitudes and atmospheric CO2 concentration. At the end of the MPT , which is marked by the manifestation of large continental ice-sheets, paradoxical responses are observed with warm sea surface temperatures (SST) in the North Atlantic mid-latitudes generating excess moisture that fuelled ice-sheet growth in Europe and further north5. This climatic evolution culminated in the maximum southern extent of the Eurasian inland ice-sheets during MIS 16 at ~650 ka6 contemporaneously with a shift to greater ice accumulation in North America7. Within the end of the MPT, MIS 18 (centred at ~730 ka) is a warm glacial in the North Atlantic Ocean8 and represents an anomaly during this period. However, the cause of the mild expression of MIS 18 remains unclear yet. Its glacial limits are unknown and geomorphological data suggests that the glaciation was limited to the high latitudes, especially in the Southern Hemisphere9. Furthermore, little is known about the Eurasian environments and atmospheric climate during the final stages of the MPT. We hypothesise that the combination of unusual warming in the mid/high-latitude North Atlantic, strong SST gradient in the tropical West North Pacific Ocean and intermediate glacial background conditions should have enhanced long-term moisture production leading to interglacial climates in the subtropical latitudes of Eurasia during MIS 18, and thus ice accumulation in the southern sectors of Eurasia.The strong non-linear behaviour of the climate system in response to the Milankovitch astronomical forcing is particularly well exemplified during the Middle Pleistocene Transition  when 100,000-year ice age cycles emerged without any significant change in orbital cyclicities dominated by the combination of 23,000 and 41,000-year cycles3  combined with published sea subsurface temperature records from the same site15, is supported by a wide array of studies on the modern relationship between pollen assemblages and vegetation  and, specifically, by the recent work of the pollen representation of the vegetation in the Tagus basin (South-West Iberia)18, the most important source of pollen preserved at IODP Site U1385. We infer the strong development of the Mediterranean forest cover, mainly composed of deciduous Quercus (~40–60 pollen %) and sclerophyllous trees and shrubs , during the MIS 18d-b interval, 740 to 725 ka, with a brief forest setback at around 733 ka (MIS 18c) , the last interglacial  and the Holocene21. Moreover, our data record all the phases characterising the succession of vegetation during interglacial periods in the Mediterranean region (Juniperus and Betula pioneer woodlands/deciduous and evergreen Quercus-Olea-Phillyrea-Pistacia forest/deciduous and evergreen Quercus woodlands)  Fig. . This in2 concentrations21, the strong Mediterranean forest cover development reflects high amount of winter precipitation, and thus relatively zonal and weak westerlies directed towards southern Europe. This interpretation is supported by the high statistical correlation between the NAO index, controlling the intensity and direction of the westerlies, i.e., winter precipitation, and the present-day Mediterranean forest cover changes24. The moderate values (~10%) of sclerophyllous plants  reflect furthermore a weak seasonal climate characterised by relatively cold and wet summers, compared to other interglacials and, particularly, to MIS 5, marked by sclerophyllous plant values peaking at 20%. Furthermore, the record of a few pollen grains of the summer-drought intolerant tree Castanea25 at c. 725 ka highlights the temperate and humid climate during MIS 18, with the westerlies affecting southern Iberia all over the year. SST at the South-West Iberian margin also reached the highest values, ~18 °C,  at Site U1385 and SST at mid-latitude Site U1313 (41°N) (thereafter “Ttherm SST gradient”), with a low gradient indicating enhanced subduction of warm mid-latitude waters in contrast to a high gradient indicative of a subpolar source3. Gradients of ~6 °C during warm phases of MIS 19-17 are among the lowest for the entire interval MIS 44-14 (ref. 3) and provide evidence for the high production of warm thermocline waters off Iberia. Atmospheric moisture provided by the accumulation of warm subtropical waters in the mid-latitude eastern North Atlantic could have contributed through the westerlies to the recorded extended mild-humid and forested conditions in central and South-East Europe from MIS 21 to 175. Surprisingly, the subduction of warm waters to thermocline level during MIS 18 was similar and even stronger compared to both interglacials MIS 17 and 19, respectively, despite that insolation26, summer energy27, atmospheric CO2 concentrations28 and relative sea level30 were lower during MIS 18 , under the influence of both similar ice volume and higher atmospheric CO2 concentration, is marked by limited forest expansion32 indicating lower winter precipitation compared to MIS 17. More interestingly, the MIS 18 glacial was more forested, reflecting stronger winter rainfall, compared to the preceding MIS 19 despite that the latter interglacial was characterised by higher insolation, sea level, atmospheric CO2 concentrations and similar warm SST compared to MIS 18  the position of the North Atlantic moisture source using the Ttherm SST gradient3, and (d) the growth and decay of the northern hemisphere ice-sheets using the benthic foraminifera δ18O record from Site U1385 (ref. 30) that strongly parallels the LR04 δ18O stack curve35. Since large-scale reconstructed climate time series are unevenly spaced, we used the binned correlation technique37 to convert the unevenly spaced paleoclimate time series to evenly spaced time series  between Mediterranean forest cover and winter latitudinal insolation gradient. The progressive increase in warmer and wetter conditions in South-West Iberia, induced by the northward latitudinal shift of the oceanic moisture source to the Iberian margin, appears to be a distinctive feature of South-West Europe at the end of the MPT. Simulated relative humidity, i.e., the moisture content in the lowest atmospheric layer (at 800 hPa), between Sites U1313 and U1385 shows the northward migration of the moisture source from MIS 19 to MIS 18 although it does not reproduce the northern position of the moisture source during MIS 17  the10, both regions being located at similar subtropical latitudes (~35–37°N). The δ13C of the loess carbonate from the Jingyuan sequence  also shows summer precipitation during MIS 18 as high as during MIS 1941, also indicating the interglacial character of MIS 18 glacial. The EASM was as strong or stronger during the weaker summer insolation and more glaciated MIS 18 period than during the MIS 19 interglacial, and weaker compared to MIS 17 12 and grain size loess (GSL) stack records from the Chinese Loess Plateau11 18 Fig. S. During 33.During MIS 17, interglacial conditions associated with higher insolation would produce the intensification of the SST gradient in the West North Pacific and Indian tropical sectors and, therefore the increase of the EASM amplified by the contemporaneous increase of cold outbursts Fig. , compare4. Therefore, the nearly continuous moisture production in the North Atlantic and West North Pacific subtropical sectors and its progressive northward transport by the westerlies during the end of the MPT may have substantially contributed to the strongest glaciation of the last millions of years in Eurasia and North America during MIS 16, leading to the strong dominant 100,000-year ice age cycles. This process could also be at work in previous moderate glacial/interglacial periods of the MPT. The long-term decreasing Ttherm SST gradient trend in the North Atlantic from MIS 25 to 22 is similar to that observed from MIS 19 to 173 and could have led to the remarkable expansion of the Northern Hemisphere ice during MIS 22.These results reveal for the first time the paradoxical interglacial character of MIS 18 glacial in the Eurasian subtropical latitudes, and the role of the oceanic moisture production in explaining the strong precipitation during a glacial period. Not only the direction and the intensity of the westerlies but the amount of oceanic moisture production should be taken into account to explain the recorded evolution of South-West European precipitation and EASM. Furthermore, sensitive model experiments show that during the intervals marked by northern ice-sheets of intermediate size, such as our studied interval, the ice growth rate will potentially increase with the strengthening of the North Atlantic Current as summer temperature remains at 0 °C at the south of the major ice-sheets while precipitation increase over this area at least in the Eurasian sector30. The water depth of Site U1385 places it under the influence of Northeast Atlantic Deep Water today, although it was influenced by southern sourced waters during glacial periods47.Site U1385 was recovered during IODP Expedition 339 “Mediterranean Outflow”. The site is located on a spur, the Promontorio dos Principes de Avis, along the continental slope of the South-West Iberian margin, which is elevated above the abyssal plain avoiding the influence of turbidites48. The general distribution of water masses is influenced by the seasonal migration of the Azores anticyclonic cell and its associated large-scale wind pattern.The South-West Iberian margin is located in the north-eastern edge of the subtropical gyre, under the influence of Eastern North Atlantic central water (ENACW). The surface water column is affected by the Portugal current (PC), which brings cold nutrient-rich water from the northern latitudes and forms the ENACW of subpolar origin (ENACWsp), and by the Azores current (AC) which brings warm water from the Azores front generating the ENACW of subtropical origin (ENACWst)50. During winter the North Atlantic westerlies bring moisture to South-West Iberia, while a high-pressure cell develops in the North Atlantic during summer, which generates strong northerly trade winds inducing coastal upwelling51. This climate seasonality is characterised by wet and mild winters  and hot and dry summers, annual precipitation <600 mm52, and leads to the development of a Mediterranean vegetation in the adjacent landmasses dominated by deciduous oak at middle elevation, and evergreen oak, olive tree, Pistacia, Phillyrea and rockroses (Cistus) at lower elevations18. Ericaceae (heathlands) are restricted to rainy mountains , strong oceanic conditions and locations with wet soils53.Climate in South-West Iberia is directly affected by the intensity and direction of the westerlies that are, in turn, controlled by the North Atlantic Oscillation (NAO)54. The stratigraphy of Site U1385 was built upon a combination of chemo-stratigraphic proxies30. Ca/Ti ratio measured every cm in all holes by core scanning X-ray fluorescence (XRF) was used to construct a composite section, and the low resolution (20 cm) benthic foraminifera oxygen isotopic record (δ18Obenth) was correlated to the marine δ18Obenth stack of LR0435 to provide the age model that we present here30.The sedimentary section recovered at Site U1385 (150 m-long composite record) shows hemipelagic continental margin sediments deposited under normal marine conditions with a fully oxygenated water column and average sedimentation rates of 10 cm/ky3 volume were prepared for pollen analysis using the standard protocol for marine samples , employing coarse-sieving at 150 μm, successive treatments with cold HCl, cold HF at increasing strength and microsieving (10 μm mesh). Known quantities of Lycopodium spores in tablet form were added to permit the calculation of pollen concentrations. Slides were prepared using a mobile mounting medium to allow rotation of the grains and counted using a Primo Star light microscope at 400 and 1000-fold magnifications for routine identification of pollen and spores, respectively. One hundred twenty-four samples were analysed every 4 cm . Pollen counts oscillate between 100 and 152 terrestrial pollen grains excluding Pinus (main pollen sum), aquatics and spores. The main pollen sum plus Pinus oscillates between 205 and 1438. The number of pollen morphotypes in most of the samples, 98 samples out from 124, ranges from 20 to 27, and from 16 to 19 morphotypes in the remaining samples. Pollen percentages for terrestrial taxa were calculated against the main sum of terrestrial grains, while percentages for Pinus were calculated against the main sum plus Pinus. Aquatic pollen and spore percentages are based on the total sum (Pollen + spores + indeterminables + unknowns) , can also be used to track episodes of massive cold freshwater input (from iceberg melting or river discharges), which are responsible for decreasing salinities in the surface water masses58. Alkenones are part of the total lipid extracted (TLE) fraction which can be extracted from 2 g of sediment by sonication with dichloromethane and hydrolysed with 6% potassium hydroxide in methanol. After derivatization with bis(trimethylsilyl)trifluoroacetamide, the TLE was identified using a Bruker mass spectrometer detector and quantified with a Varian gas chromatograph Model 3800 equipped with septum programmable injector and a flame ionisation detector with a CPSIL-5 CB column. As a gas carrier was used hydrogen at 2.5 ml.min−1. Alkenone concentrations were determined using n-hexatriacontane as an internal standard. Reproducibility tests showed that uncertainty in the alkenone index Uk’37 determinations were lower than 0.015 (ref. 59). The Uk’37 was calculated based on the di- and tri- unsaturated concentrations60, and converted into temperature values using the global core top calibration of annual SST61. The average temporal resolution between samples is 528 years and the uncertainty of SST reconstruction is ±0.5 °C (ref. 62).Sea Surface Temperature (SST) was reconstructed using di- and tri-unsaturated alkenones of 37 carbons atoms, which are organic compounds synthesised by marine coccolithophore algae in a temperature-related proportion. Alkenones, in particular the tetra-unsaturated compounds , we used a robust correlation approach that takes into account the serially dependence of the time series under study. We have followed the method developed by38 to estimate the Pearson’s correlation coefficients that takes into account the serially dependence. This method also includes an estimation of a confidence interval (95%) obtained through a nonparametric stationary bootstrap technique with an average block length proportional to the maximum estimated persistence time of the data under analysis. This statistical technique is implemented in the software PearsonT3, which is freely available from http://www.climate-risk-analysis.com.To correlate the paleoclimate records, we used first the binned correlation technique64), which is a development branch of the LOVECLIM model in version 1.2 described in Goosse et al.65. iLOVECLIM does not present major differences in the physics of the atmosphere and ocean compared to LOVECLIM. The based components of this version are the atmosphere ECBilt66, the ocean CLIO67 and the vegetation and land surface VECODE68. The modelled tree fraction in VECODE includes shrub plants such as Ericaceae68. Oxygen isotopes (18O and 16O) have been implemented in the coupled model and evaluated against observed data in water samples and carbonates69. An equilibrium run was first performed for 5000 years under climatic conditions at 800 ka. We then ran a transient run between 800 and 670 ka. The insolation70, greenhouse gas concentrations71 and prescribed ice-sheets reconstruction72 have been updated with an acceleration factor of 10 for the transient simulation. The model has been run at T21 spatial resolution (5.6° in longitude and latitude) and the output are computed with an annual timestep and with a monthly timestep for the precipitation only. iLOVECLIM was previously successfully applied in the Asian monsoon region to investigate the oxygen isotopic composition of precipitation and calcite in association with monsoonal precipitation changes over the last 150 ka73. The δ18O in precipitation computed in the model is extracted over the East Asia region (~25°N–41.5°N and ~90°E–118°E). The mean δ18Ocalcite is then computed from δ18Oprecipitation, corrected from ice-sheet contribution to the global seawater δ18O (ref. 74) and from atmospheric temperature at 2 m height using the following Eq. , and smoothed using a 10-year running mean:18.We used the intermediate complexity climate model iLOVECLIM in version 1.1.5 (ref. Supplementary informationPeer Review File