Given that steroidogenesis is achieved by coordinated functioning of multiple tissue specific enzymes, many steroids intermediates/metabolites are generated during this process. Both the steroid products as well as major lipoprotein cholesterol donor, high-density lipoprotein 3 , CREB transcriptional activity and mRNA expression of StAR, CPY11A1/P450scc and antioxidant enzymes, superoxide dismutases , catalase (CAT) and glutathione peroxidase 1 (GPX1). We also detected the steroid product in p38 MAPK inhibitor treated Y1 cells by HPLC-MS / MS.In the current study, we examined the effects of treatment of a mouse model of steroidogenesis, Y1-BS1 adrenocortical tumor cells with pregnenolone, 22(R)-Hydroxycholesterol [22(R)-diol] or hHDL2O2 greatly enhanced the phosphorylation of both p38 MAPK and CREB protein. Likewise, treatment of cells with pregnenolone, 22(R) diol or hHDL3 increased ROS production measured with the oxidation-sensitive fluorescent probe 2′,7′-Dichlorofluorescin diacetate (DCFH-DA). Under identical experimental conditions, treatment of cells with these agents also increased the phosphorylation of p38 MAPK and CREB. This increased CREB phosphorylation however, was associated with its decreased transcriptional activity. The stimulatory effects of pregnenolone, 22(R)-diol and hHDL3 on CREB phosphorylation was abolished by a specific p38 MAPK inhibitor, SB203580. Pregnenolone, and 22(R) diol but not hHDL3 upregulated the mRNA expression of SOD1, SOD2 and GPX1, while down-regulated the mRNA levels of StAR and CYP11A1. The p38 inhibitor SB203580 could increase the steroid production in HDL3, 22(R)-diol or pregnenolone treated cells.Treatment of Y1 cells with HOur data demonstrate induction of a ROS/p38 MAPK -mediated feedback inhibitory pathway by oxy-cholesterol and steroid intermediates and products attenuates steroidogenesis via inhibition of CREB transcriptional activity. Promega Dual-Luciferase Reporter Kit (E1980) was purchased from Promega Corporation . pCRE-Luc cis-reporter plasmid and pLuc-MCS vector were obtained from Agilent Technologies . 20α-hydroxyprogesterone was purchased from Chemsky International Co., Ltd. . Anti-phospho-CREB (Ser133), anti-CREB, anti-p38 MAPK, and anti-phospho-p38 MAPK (Thr180/Tyr182) antibodies were supplied by Cell Signaling Technology, Inc. . Anti-GAPDH was purchased from Santa Cruz Biotechnology . IRDye® 800CW Goat anti-Mouse IgG (H + L) and IRDye® 800CW Goat anti-rabbit IgG (H + L) were purchased from LI-COR Biosciences . SYBR Green Master Mix was supplied by TAKARA Biotechnology  Co. LTD . Lipofectamine® 2000 was obtained from Invitrogen . Tissue culture supplies were from Life Technologies through its Gibco Cell Culture Media Division .2O2 (100 nM) or Bt2cAMP (2.5 mM) for an appropriate time  and subjected to 12% SDS-PAGE. For each sample, a constant amount of protein (10–20 μg) was loaded on the gel. Protein markers were also loaded on the gel. After electrophoretic separation, the proteins were transferred to Immobilon PVDF membranes  using standard techniques. The protein blots were incubated with first antibody for 2 h at room temperature, then probed with IRDye infrared secondary mouse or rabbit anti-rabbit IgG and visualized using the odyssey® infrared imaging system .5 cells/well) and then cultured in F-12K medium supplemented with 10% LPDS ± p38 MAPK inhibitor SB203580 overnight. Subsequently, dishes were washed and incubated with hHDL3 (30 μg protein/ml), 22(R)-diol (10 μM) or pregnenolone (10 μM) for 5 h. The medium samples were collected for hormone analysis using isotope dilution high performance liquid chromatography-tandem mass spectrometry (ID-HPLC-MS / MS). HPLC-MS / MS was performed by Shanghai Dian Medical Testing Institute  as previously escribed [Y1-BS1 cells were pre-loaded into 12-well plate . A statistical difference of 2-, •OH and H2O2, promote the phosphorylation and activation of MAPK, among which p38 MAPK is highly responsive to oxidant stress which has important roles in cell signaling and homeostasis [2O2 or a lipid peroxidation product, 4-hydroxy-2-nonenal (HNE) reciprocally inhibited steroid production and increased the phosphorylation and activation of p38 MAPK [2O2 for 5 min caused a robust phosphorylation of p38 MAPK and a modest but significant stimulation of phosphorylated form of CREB for 20 min, thereafter the phosphorylation of p38 and CREB decreased. Pretreatment of cells with a specific p38 MAPK inhibitor, SB203580 abolished the stimulatory effects of H2O2 on p38 MAPK and CREB phosphorylation. These results not only complement the previously published studies from our laboratory [2cAMP could stimulated both the phosphorylation of p38 MAPK and CREB -diol and pregnenolone to modulate ROS production in Y1-BS1 cells. For these studies, Y1-BS1 cells were cultured in regular F-12K medium containing 10% FBS for a brief period of time to allow the cells to attach to the plate and then culture medium was replaced with a fresh medium containing 10% LPDS and dishes incubated overnight. Subsequently, the cells were treated ± HDL3, ±22(R)-diol or ± pregnenolone in the presence of 10% LPDS for various time points. At the end of incubation, intracellular ROS generation was evaluated using DCFH-DA assay. The results depicted in Fig. 3, 22(R)-diol or and pregnenolone, all stimulated ROS generation with a biphasic response; an initial 10 min rapid response and a gradually declining slow phase during the next 60 min -diol and pregnenolone, which could potentially further enhance the intracellular ROS production. This possibility is in line with the earlier evidence showing that the steroid intermediates, testosterone, testosterone precursors or analogs e.g., epitestosterone and 17a-methyltestosterone, can cause oxygen tension sensitive decrement in P450 hydroxylase activity . In addiignaling –42 and i3, 22(R)-diol and pregnenolone to promote the phosphorylation of p38 MAPK. Treatment of Y1-BS1 with hHDL3 (30 μg protein/ml), 22(R)-diol (10 μM) or pregnenolone (10 μM) increased p38 MAPK phosphorylation in a time dependent manner -diol or pregnenolone treatment, like H2O2, increased the phosphorylation of CREB with maximal stimulation occurring around 20 min. These results led us to conclude that HDL3, 22(R)-diol and pregnenolone promote CREB phosphorylation via upstream p38 MAPK signaling pathway. To provide additional evidence for this conclusion, we treated cells with these three agents in the presence and absence of a specific p38 MAPK inhibitor, SB203580 and reassessed the phosphorylation status of both p38 MAPK and CREB proteins. The results presented in Fig. 3, 22(R)-diol and pregnenolone to stimulate the phosphorylation of both p38 MAP and CREB, further confirming the p38 MAPK catalyzed phosphorylation of CREB.We next tested the ability of HDLner Fig. . In eachner Fig. . We next factors –45. It a3, 22(R)-diol or pregnenolone compared to vehicle control -diol or pregnenolone) loading increased the steroid production, inclusion of SB203580 to inhibit the p38 activity could increase the steroid production in HDL3, 22(R)-diol or pregnenolone treated cells. These findings are consistent with previous results showing that inhibition of p38 MAPK by SB203580 could ameliorate the oxidative stress-induced repression of steroidogenesis [We have demonstrated that inhibition of p38 MAPK activity with SB203580 potentiated the Btal cells . Althougogenesis .Fig. 7In3, 22(R)-diol or pregnenolone. Contrary to our expectation, we were surprised to see that 22(R)-diol and pregnenolone but not hHDL3 treatment induced the mRNA expression of SOD1, SOD2 and GPX  and comprise molecules with varying oxidant properties. At low concentrations, ROS modulate many cellular processes through redox-dependent signaling including proliferation, differentiation, apoptosis, immune regulation, cellular adaptation [Steroidogenesis represents a highly complex and multistep metabolic process involving the participation of several catalytic enzymes, regulatory molecules and cellular organelles. More specifically, it involves the participation of various steroidogenic enzymes , cholesterol transport proteins , transcription factors  and availability of adequate amounts of substrate cholesterol , 53, 54.aptation  and now aptation , 55, 56.aptation , 58. Thu3 can promote ROS production and ensuing oxidative stress in Y1-BS1 adrenal cells. Our observations are in line with previous studies showing super oxide/ H2O2 produced during ACTH-stimulated adrenal steroid production is involved in the termination of the steroidogenic response [2O2 produced by P450 enzymes during steroidogenesis inactivates potent mitochondrial antioxidant enzyme, peroxiredoxin 3, which in turn triggers a sequence of events including increased accumulation of H2O2, activation of p38 MAPK, suppression of StAR mRNA/protein expression and inhibition of steroidogenesis [2O2 produced during Mn-SOD catalyzed dismutation of superoxide anions and ensuing inactivation of peroxiredoxin 3 that inhibits steroidogenesis, but also steroid metabolites such as 22(R)-diol and pregnenolone possess similar capabilities in promoting ROS production and interfering with normal steroidogenesis. However, we were surprised to find that hHDL3 a cholesterol donor for steroidogenesis also promotes ROS production. This is even more surprising given that HDL is considered an antioxidant with relevance to cardiovascular disease [3 being a cholesterol donor, delivers excessive amounts of cholesterol to cells for its catabolism and this leads to exaggerated production of ROS and excessive oxidative stress. Obviously, more experimental studies are needed to sort out among these various possibilities.In this study we made a novel observation that endogenous steroid intermediates such as 22(R)-diol and pregnenolone as well as a donor of cholesterol substrate, hHDLresponse . They de disease . At pres3, 22(R)-diol or pregnenolone treatment of Y1-BS1 results in robust phosphorylation of p38 MAPK as a consequence of enhanced ROS production. Under identical experimental conditions, the phosphorylation of one of the important steroidogenic transcription factors, CREB was also induced and based on the use of inhibitors, the observed phosphorylation of CREB is mediated by p38 MAPK. This observation is in agreement with previous reports showing that ROS upregulates CREB phosphorylation via p38 MAKP signaling cascade [Previous data suggest that hydrogen peroxide is a potent activator for p38 MAPK , 60. Oth cascade . Althoug cascade , 64. Naq cascade , 62, 65. cascade , 62. It 2, very-low-density lipoprotein (VLDL) and glyco-oxidized VLDL can induce Cyp11B2 expression and stimulate steroid production in a human adrenocortical carcinoma cell line, NCI H295R [3 repress the gene expression of StAR and CYP11A1. We further provided the evidence that 22(R)-diol and pregnenolone-mediated repression of StAR and CYP11A1 gene expression is achieved through excessive oxidative stress and associated p38 MAPK signaling cascade. Our steroid hormone production data provide additional support to the notion that SB203580 inhibition of p38 MAPK augments the steroids production in Y1-BS1 cells treated with steroid metabolites/intermediates. A number of studies have implicated p38 MAPK signaling cascade in the regulation of steroidogenesis [3, 22(R)-diol or pregnenolone. In addition, we will examine whether ROS/p38 MAPK modulate the expression of some of the critical SNARE proteins. In a recent publication, we identified several SNAREs, whose expression is essential for cholesterol transport to outer mitochondrial membrane for optimal steroid production [Currently, we are exploring the potential mechanism by which ROS/p38 MAPK inhibition of CREB activity may lead to impaired steroidogenesis. One possibility we are considering is the inhibition of StAR gene transcription as a result of oxidant-mediated impaired CREB activity. This is in line with the observation that StAR expression is sensitive to both physiological and pathophysiological levels of ROS. Moreover, earlier studies have shown that CREB protein in cooperation with SF1 is a major regulator of StAR protein gene transcription , 67. FinCI H295R –70. Sahaogenesis , althougogenesis . Other sogenesis , 73. Likogenesis , 74. Intogenesis . In thisoduction .3 leads to increased mRNA expression of three antioxidant enzymes, SOD1, SOD2 and CAT. In contrast, studies by Kil et al [Another surprising finding from our studies was that treatment of Y1-BS1 with 22(R)-diol and pregnenolone but not HDLil et al  observedil et al . Obvious3 led to increased ROS production and associated enhanced phosphorylation of p38 MAPK and CREB. This oxidant-mediated up-regulation of CREB phosphorylation is mediated by p38 MAPK. The increased CREB phosphorylation however, was accompanied by a significant loss of CREB’s transcriptional activity. Furthermore, treatment of cells with 22(R)-diol and pregnenolone and ensuing oxidative stress resulted in decreased mRNA levels of StAR and CYP11A1 and increased mRNA levels of antioxidant enzymes SOD1, SOD2 and CAT. From these studies we conclude that ROS/p38 MAPK inhibition of CREB transcriptional activity is likely responsible for ROS-induced feedback inhibition of steroidogenesis.In conclusion, our studies provide evidence that exposure of adrenal cells to steroid intermediates/metabolites, 22(R)-diol and pregnenolone, and hHDL


Although activation of the ErbB family of receptor tyrosine kinases (ErbB1-4) is driven by oligomerization mediated by intermolecular interactions between the extracellular, the kinase and the transmembrane domains, the transmembrane domain has been largely neglected in this regard. The largest contributor to the intramembrane electric field, the dipole potential, alters the conformation of transmembrane peptides, but its effect on ErbB proteins is unknown. Here, we show by Förster resonance energy transfer (FRET) and number and brightness (N&B) experiments that the epidermal growth factor (EGF)-induced increase in the homoassociation of ErbB1 and ErbB2 and their heteroassociation are augmented by increasing the dipole potential. These effects were even more pronounced for ErbB2 harboring an activating Val → Glu mutation in the transmembrane domain (NeuT). The signaling capacity of ErbB1 and ErbB2 was also correlated with the dipole potential. Since the dipole potential decreased the affinity of EGF to ErbB1, the augmented growth factor-induced effects at an elevated dipole potential were actually induced at lower receptor occupancy. We conclude that the dipole potential plays a permissive role in the clustering of ErbB receptors and that the effects of lipid rafts on ligand binding and receptor signaling can be partially attributed to the dipole potential. ErbB proteins are known to a wide array of scientists ranging from cell biologists to clinical researchers1Clustering plays a key role in the activation of ErbB proteins. The dogma, formulated for ErbB1  receptor), involves the ligand-induced dimerization of monomeric receptors as a result of a transition of the extracellular domain from a closed to an extended conformation in which the dimerization arm is exposed6811In addition to the extracellular domain the transmembrane and the intracellular domains also play a role in the clustering of ErbB receptors. The structure of the autoinhibited conformation of the ErbB1 kinase domain resembles that of inactive cyclin-dependent kinases. Upon activation two kinase domains form an asymmetric dimer in which the “receiver” kinase is turned on by the “activator” kinaseWe have a much less clear view about the role of the transmembrane domain (TMD) in dimerization. Although the linkage between the extra- and intracellular domains provided by the transmembrane and juxtamembrane domains seems to be very flexible1517232425268–109 V/m as opposed to the much weaker (~2.5·107 V/m) electric field associated with the transmembrane potential29The electric field in the membrane alters the conformation of the TMDAlthough the dipole potential is expected to influence the conformation of the TMD of membrane proteins, its effect has not been tested on ErbB proteins. Here, we show that alterations in the dipole potential modify the clustering and signaling properties of ErbB1 and ErbB2, and conclude that the dipole potential plays a permissive role in the activation of these receptors.Phloretin and 6-ketocholestanol were used to decrease and increase the dipole potential, respectively see , Fig. S2Since transmembrane signaling induced by EGF is preceded by clustering of ErbB receptors, we investigated whether alterations in the dipole potential have any effect on receptor oligomerization in quiescent or stimulated cells. According to flow cytometric FRET  and S3 aIn order to reveal the effect of the dipole potential on EGF-induced receptor clustering both the FRET and the molecular brightness values had to be normalized in order to eliminate variability in the values in unstimulated cells. Contrary to resting cells, an elevated dipole potential significantly and systematically promoted the EGF-induced homoassociation of ErbB1 and ErbB2 according to FRET and N&B measurements A,B and 3Since ErbB2 is the preferred heterodimerization partner of EGF-activated ErbB1Ligand-induced clustering of ErbB receptors leads to phosphorylation of tyrosine residues in the receptors and spreading of the tyrosine phosphorylation signal. In order to test whether the dipole potential-related changes observed in the clustering of ErbB1 and ErbB2 are mirrored in their signaling the level of tyrosine phosphorylation was determined in quiescent and EGF-stimulated cells using antibodies against phosphotyrosine, ErbB1 phosphorylated at Tyr1068 and ErbB2 phosphorylated at Tyr1248. Similar to the FRET and N&B experiments normalization of the tyrosine phosphorylation to unstimulated control cells eliminated the variability in the baseline level of activation. Alteration of the dipole potential did not modify the low level of tyrosine phosphorylation in unstimulated cells, while the EGF-induced response was correlated with the dipole potential . SimilarSince the dipole potential is assumed to be different in lipid rafts than in the rest of the membrane, we expected some sort of correlation between the activation state of ErbB2, its raft localization and the dipole potential. Cells treated with 6-ketocholestanol to increase their dipole potential were compared to control cells. The tyrosine phosphorylation of ErbB2 was normalized to its expression level and separately evaluated in raft and non-raft regions of the cell membrane corresponding to areas with high and low CTX labeling, respectively. In cells with an unmodified dipole potential there was no difference between the normalized tyrosine phosphorylation of ErbB2 inside and outside lipid rafts . IncreasIn the current manuscript we showed that the dipole potential is significantly correlated with the clustering of ErbB1 and ErbB2. The effect was systematically observed in EGF-stimulated cells, whereas changes in the dipole potential did not exert substantial effects on ErbB1 and ErbB2 in unstimulated cells. The increased growth factor-induced association of ErbB proteins at an elevated dipole potential was correlated with enhanced signaling which is in line with receptor dimers or clusters being the active signaling units. However, the fact that increased signaling at an elevated dipole potential was correlated with reduction of ligand binding was unexpected. If this suppressed ligand binding at an elevated dipole potential is taken into account, the dipole potential-induced augmentation of the EGF effect is even larger since a lower amount of bound ligand brings about the larger changes in the clustering and phosphorylation of ErbB1 and ErbB2. This apparent contradiction can be resolved by assuming that the increase of the dipole potential favors the active conformation of the transmembrane domain dimers which eventually induces conformational transitions in the extra- and intracellular domains as well resembling or identical to those induced by growth factor binding. In light of the above reasoning a lower amount of bound ligand at an elevated dipole potential can induce stronger signaling since both growth factor binding and the dipole potential induce the conformational changes leading to receptor activation.The reduced affinity of EGF binding is also in line with the dipole potential-induced conformational change in the extracellular domain. It has been shown that the ligand binding site is structurally restrained in ErbB1 dimers6Lipid rafts regulate EGF-mediated signaling since both ErbB13638393536The dipole potential may only exert a permissive effect on clustering which is not strong enough for altering the weak and transient constitutive dimers and oligomers.It has been suggested that constitutive dimers or oligomers are generated by corralling or confinement by the cytoskeleton41Parallel alpha helices, present in dimers of single-pass transmembrane proteins, exert a repulsive electrostatic force on each other1724Interactions of the juxtamembrane segment with negatively charged lipids in the inner leaflet of the plasma membrane have been shown to prevent the formation of active receptor dimers, while dimerization of the juxtamembrane domains orient the kinase domain to adopt their active conformation2324Exposure of the TMD helix termini to water decreases their dipole moment and repulsion46Since the interaction with lipids affects the conformation of ErbB proteins, changes in the dipole potential and consequent alterations in lipid-protein interactions may change the distribution of ErbB receptors between raft and non-raft domains. The majority of studies found that lipid rafts inhibit ligand binding and signaling mediated by ErbB1 and that ErbB1 activation takes place upon concomitant migration of ErbB1 out of lipid rafts38We considered six possible models for explaining the phenomenon that the dipole potential only affects ligand-induced clusters without influencing constitutive ones.By one of these mechanisms monomers of ErbB proteins are more prone to dimerization after 6-ketocholestanol treatment. In this way, more monomers are available to form homo- and heterodimers after growth factor stimulation. Therefore, EGF-induced formation of ErbB1-2 heterodimers does not lead to decreased homoassociation of ErbB1 or ErbB2 because the dimers are formed from the dimerization-prone pool of monomers.The dipole potential may also have implications for the evolution and treatment of cancer. Tumor cells are characterized by an increased density of cholesterol-rich lipid raftsIn summary, we have shown that the dipole potential exerts significant effects on the ligand binding, clustering and signaling of ErbB proteins. We suggest that the dipole potential primarily affects receptor dimerization mediated by TMDs. This dimerization induced by an elevated dipole potential will bring about TMD-driven receptor activation leading to reduced affinity of growth factor binding and increased signaling mediated by dimers stabilized by interactions among the extracellular domains and the TMD dimerization motifs stabilizing active dimersSKBR-3, HeLa and A431 cells were obtained from the American Type Culture Collection  and grown according to their specifications. F1-4, a CHO subline stably expressing ErbB1-EGFP has been described previouslyFor microscopic experiments, cells were cultured on Lab-Tek II chambered coverglass . For flow cytometry, cells were harvested by trypsinization. Trastuzumab (against ErbB2) and Ab11 (against ErbB1) were purchased from Roche  and Thermo Fisher Scientific , respectively. These antibodies were labeled with AlexaFluor546 and AlexaFluor647 dyes according to the manufacturer’s specifications . Antibodies against phosphotyrosine , ErbB2 phosphorylated at Tyr1248  and ErbB1 phosphorylated at Tyr1068  were visualized by secondary staining with AlexaFluor546-goat-anti-mouse antibodies (Life Technologies/Thermo Fisher Scientific). Tetramethylrhodamine-labeled EGF was purchased from LifeTechnologies. Transient transfection of HeLa cells was carried out with an Amaxa Nucleofector device . The transfection solution and the program were selected according to the “Cell & Transfection Database” of the manufacturer.The dipole potential was measured using a ratiometric assay. Cells were incubated with 2 μM di-8-ANEPPS (4-(2-[6-(dioctylamino)-2-naphthalenyl]ethenyl)-1-(3-sulfopropyl)pyridinium inner salt, Invitrogen, Carlsbad, CA) for 20 min at 4 °C, and their fluorescence was measured using a ratiometric assay shown to be responsive to changes in the dipole potentialControl cells and those with an altered dipole potential were treated with EGF followed by staining with subunit B of cholera toxin (CTX) and antibodies against ErbB2 and tyrosine phosphorylated ErbB2. Details of the labeling protocol and image analysis are described in The dissociation constant characterizing the binding affinity of EGF to ErbB1 was determined by competitive and non-competitive binding assays using fluorescently labeled EGF. Tetramethylrhodamine-congjutated EGF  was reconstituted at a concentration of 10 μM. A dilution series was prepared with each vial containing 250 μl EGF diluted in Tyrode’s buffer supplemented with 0.1% (w/v) BSA. The solutions were kept on ice before adding 20 μl of cold cell suspension containing 100000 cells. Cells were incubated in the presence of TAMRA-EGF for 30 min on ice with shaking. The fluorescence intensity of the samples was measured on a FACS Aria III flow cytometer without washing to prevent the dissociation of fluorescent EGF from ErbB1. TAMRA was excited at 561 nm, and its emission was detected through a 595 nm band-pass filter. Analysis of flow cytometric data was carried out with FCS Express . The binding curves were fitted to the Hill equation on a logarithmic scale:I is the intensity of the sample labeled with fluorescent EGF at a concentration of c, Imin and Imax are the minimal and maximal intensities, respectively. Kd and n are the dissociation constant and the Hill coefficient, respectively.where Competitive binding curves were measured as described above, but the dilution series contained unlabeled EGF mixed with a constant concentration of labeled EGF. The binding curves were fitted to the equation below:whereKd and Ki are the dissociation constants of TAMRA-EGF and unlabeled EGF, respectively, and cT is the concentration of TAMRA-EGF.ErbB1 homoassociation and ErbB2 homoassociation were measured with Ab11 antibody and trastuzumab, respectively. ErbB1-ErbB2 heteroassociation was measured with AlexaFluor546-Ab11 (against ErbB1) and AlexaFluor647-trastuzumab (against ErbB2). The measurements were carried out with a FACS Aria III flow cytometer  according to principles described in et al.An Olympus FV1000 confocal microscope running in pseudo photon-counting mode was used to carry out N&B analysis according to Digman How to cite this article: Kovács, T. et al. The Dipole Potential Modifies the Clustering and Ligand Binding Affinity of ErbB Proteins and Their Signaling Efficiency. Sci. Rep.6, 35850; doi: 10.1038/srep35850 (2016).


The El Niño-Southern Oscillation is the dominant mode of interannual climate variability across the Pacific Ocean basin, with influence on the global climate. The two end members of the cycle, El Niño and La Niña, force anomalous oceanographic conditions and coastal response along the Pacific margin, exposing many heavily populated regions to increased coastal flooding and erosion hazards. However, a quantitative record of coastal impacts is spatially limited and temporally restricted to only the most recent events. Here we report on the oceanographic forcing and coastal response of the 2015–2016 El Niño, one of the strongest of the last 145 years. We show that winter wave energy equalled or exceeded measured historical maxima across the US West Coast, corresponding to anomalously large beach erosion across the region. Shorelines in many areas retreated beyond previously measured landward extremes, particularly along the sediment-starved California coast. ENSO end members El Niño and La Niña are linked to elevated coastal hazards across the Pacific region. Here, the authors show that the wave conditions and coastal response for the 2015–16 El Niño indicate that it was one of the most significant events of the last 145 years. The El Niño-Southern Oscillation (ENSO) explains much of the interannual variability in sea surface temperature, sea-level pressure and atmospheric forcing across the equatorial Pacific, affecting global climate patterns4104512131415By various metrics, the 2015–2016 El Niño winter was one of the three strongest events in the historical record22Here we provide a detailed assessment of wave conditions, water levels and coastal response during one of the most significant El Niño events of the Industrial Age: the 2015–2016 El Niño. The study analyzes two decades of winter oceanographic forcing across the US West Coast, focusing on the response of 29 beaches along the California, Oregon and Washington coasts, fronting a population of ∼25 million. The region experienced substantial increases in coastal hazards during previous El Niño winters, and has been shown to broadly represent conditions across the Central and Eastern North Pacific5The wave climate in the Eastern North Pacific varies seasonally, with larger waves in the fall and winter months driven by the development and passage of extra-tropical cyclones across the mid-latitudes, as well as episodic Eastern Pacific tropical storms in the summer and fall. High pressure dominates in the spring and summer months, with prevailing northwesterly winds and southern hemisphere storms typically resulting in lower wave energy conditions424As a key driver of coastal change, mean and elevated  wave energy flux , were ∼50% above normal averaged across all regions during the 2015–2016 El Niño winter. During the 19 years of analysis, mean wave energy flux was only exceeded by the 1997–1998 El Niño , but elevated wave energy flux in 2015–2016 was the highest on record  measured on the Oregon coast . The anoBeach morphology responds to the seasonal modulation in forcing across the Eastern North Pacific, with beaches tending to build seaward (prograde) during the low wave energy summer months and retreat landward (erode) in the stormier winter months2128Seasonal beach behaviour was assessed for 29 beaches along a ∼2,000 km span of the US West Coast that have been surveyed using aerial Light Detection and Ranging (Lidar), global positioning system-based (GPS) topographic beach surveys with All-Terrain Vehicles and backpacks, and/or discrete measurements of sand levels . TemporaAveraged across the six regions of the US West Coast, the winter shoreline retreat of 2015–2016 was the highest on record, with erosion 76% above the normal winter shoreline retreat, 27% higher than any other winter and easily eclipsing the El Niños of 2009–2010 (+12%) and 1997–1998 (−9%) . At the During the winter of 2015–2016, highly elevated winter wave energy flux , coupled with seasonally elevated water levels (+11 cm), drove unprecedented levels of winter shoreline retreat , including the most landward shoreline positions measured for the majority of beaches in California since topographic data collection began 20 years ago. The historical significance of this El Niño can be determined by analysing the relatively consistent record of wave energy, water levels and beach behaviour across the study area (available since 1997), and buoy records that date back to the mid-1970s31The primary difference in wave energy flux between the most powerful El Niño events of the past two decades  appears related to a latitudinal shift in the primary storm tracks and resulting wave generation location. Elevated wave energy flux during the winter of 2015–2016 exceeded the 1997–1998 event by 29% in Northern California and the Pacific Northwest, including a 44% increase off the coast of Washington. Conversely, higher mean +37%) and elevated wave energy (+27%) was measured during 1997–1998 for Central and Southern California compared to the 2015–2016 winter. The distinct northerly wave direction anomaly and the smaller elevated wave energy flux anomaly in Southern California during 2015–2016 relative to 1997–1998 are likely related to storm tracks taking a more southerly route during the 1997–1998 El Niño134% and ele3840While projections of El Niño frequency and magnitude for the 21st century are variable42Although erosive conditions were clearly amplified in 2015–2016, large landward shifts in shoreline positions did not translate to pervasively severe erosion of the dunes and bluffs that back the beaches in the Pacific Northwest. This is likely due to the fact that these beaches have generally been accreting since the previous 2009–2010 El Niño, and were in a significantly prograded state in summer 2015 due to the previous two mild winters  and 4a. The potential for even more extreme coastal erosion during the 2015–2016 El Niño was also moderated by the earlier onset of peak annual high tides and the seasonal water-level anomaly associated with El Niño. The fall 2015 peaks in California, for example, were significantly earlier than the winter peaks that occurred during the El Niños of 1982–1983 and 1997–1998, thereby reducing the probability for the coincident arrival of the largest waves and water levels during the 2015–2016 eventWhile natural or artificial increases in beach volumes may reduce erosion-related hazard risk during extreme El Niños at some beaches, hazard risk on many US West Coast beaches may be worsened by historical and possible future reductions in watershed sediment supply to beaches. Even with major reductions in the coastal sediment supply due to dam construction, which has reduced pre-historical riverine sediment supply by 50% in Southern California5052In California, the 2015–2016 El Niño serves as proxy for this potential trend: a multi-year drought34Water levels anomalies of 7–17 cm above normal were measured across the US West Coast during the El Niño winter of 2015–2016, similar to anticipated global mean sea-level increases expected within the next few decadesE) was calculated as the difference between the summer/fall (August–November) maximum and subsequent winter/spring (January–April) minimum to coincide with oceanographic forcing fluctuations. The annual shoreline erosion anomaly for each region was calculated as:Coastal change data sets collected between 1993 and 2016 were compiled from 29 beaches, representing the six regions of Southern California, Central California, North-central California, Northern California, Oregon and Washington USA; . Represey is the erosion anomaly (%), Ey is the erosion in year y and avg(Ey) is the mean of this quantity over the entire record. Hence, positive values of the anomaly correspond to erosion larger than the mean.where EAMean monthly sand height values from Isla Vista beach, within the Central California region, were calculated from near-daily observations taken at the vertical face of a concrete staircase in the intertidal zone (∼MSL) from 1993 to 2016 ref. . IndividF, was calculated using:For each of the six study regions, wave  and water-level data  were used to assess interannual variability in wave forcing and water-level anomalies to characterize conditions across the US West Coast. Wave energy flux, Hs is the significant wave height and T is the wave period. Wave directional anomaly was calculated as the number of degrees clockwise (+) or counterclockwise (−) of the peak direction from the average peak direction. Water-level data were gathered from nearby tide stations, which are usually located in semi-enclosed harbours and sheltered from waves. All data were binned into boreal winter (December 1–February 28) and fall/winter (October 1–March 31) averages. A summary of the oceanographic forcing for each region is presented in where The codes used to generate the results for this project are available upon request from the corresponding author.All relevant data used in the production of this manuscript are available upon request from the corresponding author.How to cite this article: Barnard, P. L. et al. Extreme oceanographic forcing and coastal response due to the 2015–2016 El Niño. Nat. Commun.8, 14365 doi: 10.1038/ncomms14365 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary FigureData inventory for study site information.Summary of oceanographic forcing  and coastal response . Numbers in bold and highlighted in yellow are the maximum values of the time-series for each region. For wave direction the most negative (southerly shifts) are bolded/highlighted.


Moraine-dammed lakes at debris-covered glaciers are becoming increasingly common and pose significant outburst flood hazards if the dam is breached. While moraine subsurface structure and internal processes are likely to influence dam stability, only few sites have so far been investigated. We conducted electrical resistivity tomography (ERT) surveys at two sites on the terminal moraine complex of the Ngozumpa Glacier, Nepal, to aid assessment of future terminus stability. The resistivity signature of glacier ice at the site (100–15 kΩ m) is more consistent with values measured from cold glacier ice and while this may be feasible, uncertainties in the data inversion introduce ambiguity to this thermal interpretation. However, the ERT data does provide a significant improvement to our knowledge of the subsurface characteristics at these sites, clearly showing the presence (or absence) of glacier ice. Our interpretation is that of a highly complex latero-terminal moraine, resulting from interaction between previous glacier advance, recession and outburst flooding. If the base-level Spillway Lake continues to expand to a fully formed moraine-dammed glacial lake, the degradation of the ice core could have implications for glacial lake outburst risk. Ngozumpa Glacier is located in the Eastern Himalaya , an area9Surface ERT experiments were acquired at (A) a partially healed breach through the western lateral moraine, (B) an area of moraine ridges and hummocky topography on the terminal moraine and, (C) the debris covered glacier surface ~1 km from the terminus . Site A At all three sites on the Ngozumpa terminus , bulk reThe lower resistivity values (~1–10 kΩ m), occurring in the surface layer of all profiles  and 3 ar3336In all profiles the transitions between areas of different resistances are smooth  and 4a, When liquid water in the ground freezes, the mechanism of electrical conduction remains dependant principally on the porosity and saturation and electrical conductivity of the pore waters until the size of pore space decreases to less than a few micrometres3649In situ measurement of bulk resistivity of ice typically varies over several orders of magnitude, dependant on the temperature and character of the ice. Bulk resistivities for perennially frozen ground and Alpine rock glaciers have been reported in the range of 10–100 kΩ m525355564852535557The internal character and structure of Alpine-style rock glaciers has been characterised at numerous sites, the majority of which report combinations of glacier ice, ice lenses and sediment and debris cemented by interstitial ice52536091562Despite significant uncertainty in the actual electrical conduction mechanism, the resistivity signature of glacier ice at Ngozumpa Glacier is a magnitude lower than values typically recorded for temperate glacial ice314864Evidence from the European Alps suggests that very few mountain glaciers are temperature thoughout679112, the continental global average is ~65 mW / m22122) as indicated by a lack of hot springs, a feature synonymous with higher geothermal heat-flux elsewhere in the HGB11 and 2.1 W/m/KGeothermal heat-flux is poorly known in the Khumbu Himal, but better constrained in the Himalayan Geothermal Belt (HGB) where heat fluxes range between 60–90 mW / mTwo main effects introduce significant uncertainty into the inversion outcomes and thus, the interpretation of the resistivity signatures at Ngozumpa Glacier. Firstly, all ERT data are collected along 2-D profiles and then inverted assuming that the medium is effectively invariant in the third direction. As a consequence, the 2-D representation of an actual 3-D distribution of electrical subsurface properties leads to an uncertainty in the inverted image which is not generated by data error or noiseAs little is known about the subsurface characteristics at Ngozumpa, negating the use of a priori information, we develop a synthetic resistivity data model (See Methods). The model, based on the inversion results and ground truthing from profile 13 location , allows The inverted synthetic resistivity model, like profile 13, shows evidence of resistivity signature suppression, likely a result of the equivalence problem. The subsurface glacial ice layer (150 kΩ m) extends along the full length of the synthetic model profile at depth , under tThe profiles acquired at site A, near the latero-terminal moraine   suggestiThe presence of glacier ice within the breach moraine complex in the western lateral moraine (Site A) may have consequences for the stability of a future moraine dam. Despite ambiguity in the thermal interpretation of the high resistivity values (>100 kΩ m), the identification of the debris-ice interface in profile 13 confirms that this signature is indicative of glacier ice at this site. If the glacier ice identified in the latero-terminal moraine ridge is indeed cold (>30 m), it suggests the ice core is currently well insulated. Seepage and piping are phenomena known to undermine the longer-term stability of moraine damsERT surveys at Ngozumpa Glacier reveal a highly complex latero-terminal moraine, resulting from interaction between previous glacier advance, recession and outburst flooding. The work highlights the compound nature of debris-covered glacier moraines and the complex range of processes that can occur within them. The presence of ice within the re-advance moraine could pose an increased risk of outburst flood if the base-level Spillway Lake continues to expand to a fully formed moraine-dammed glacial lake. Better prediction of longer-term moraine-dam stability necessitates investigation of the entire moraine structure. At this stage, focusing efforts in one area alone cannot give a reliable prediction of subsurface characteristics in another, even where surface morphology is similar. Further, it is not currently possible to determine the longer-term stability of a moraine dam from remote sensing or by analogy to other moraine dam sites.The ERT technique clearly identified glacier ice in the re-advance moraine at site A, based on the characterisation of the ice signature at site C. However, the limitation of the 2-D survey and inversion, combined with the surface topography and high resistivity contrasts causing equivalence, posed significant uncertainty in interpretation. Such uncertainty in interpretation is clearly illustrated by the result of the inversion of the synthetic data set , based oFurther investigation is urgently required to better constrain the results and interpretation of the ERT surveys in this environment and, to determine the spatial and temporal extent of their applicability. The highly variable surface topography and significant resistivity contrasts, render a 2-D representation of the 3-D distribution of electrical subsurface properties subject to uncertainty, thus future surveys should be collected and inverted in 3-D. In addition, the effect of a thick, conductive debris layer must be quantified to allow further use of the technique for moraine dam applications. Laboratory experiments investigating the effect of a debris layer of differing characteristics on the resistivity signature from underlying glacier could reduce the uncertainty in the interpretation of the presence or absence of ice at different sites. If a more robust characterisation of the resistivity signatures of debris-covered ice could be developed, the large difference in the resistivity signature between cold and temperate ice could be used to aid investigation the thermal regime of debris-covered glacial ice.−1 and 0.5–2.6 km s−1 respectively)83Geophysical surveys are typically constrained by some prior knowledge of the subsurface; debris thickness and morphology data, as well as ice temperature measurements are key to further investigation. However, the high altitude, isolated locations of glaciers such as the Ngozumpa, provide significant challenges to such an approach. A common approach in Alpine glacier and permafrost applications has been to employ two or more geophysical techniques at a single survey location32787980322741In November 2010, 2-D ERT surveys were carried out at three locations on the Ngozumpa Glacier with the aim of identifying the ice margin at two key locations . All ERT33To minimize uncertainty caused by ground heterogeneity and significant local topography, rigorous error analysis was adopted from our previous workAll resistivity data were inverted using the finite-difference code DCIP2D870 are the current and reference models respectively. The constant αs (damping factor) controls the closeness between the current model and the reference model, while the constants αx and αz control the model roughness in the horizontal and vertical directions respectively . Minimization is achieved iteratively by updating m with respect to m0 based on the misfit functional, ϕdwhere m and mpred are the measured and computed data respectively, and εi are the measurement errors. In practice this is not straight forward, the inverse problem is non-unique; an infinite number of models will fit the data to a required level of uncertainty89919394969587where s is the location of the current source. The potential ϕσ, is the potential due to a single current.where σ is the electrical conductivity (S/m), ∇ is the gradient operator, I is the strength of the input current (A), and rThe synthetic data set was based on the same Wenner Alpha array configuration with 24 electrodes at 3 m spacing as the real Profile 13 location  data cols = 0.05 is used to ensure closeness between the reference and iteratively updated current models. The DOI index is then calculated fromConsidering the high resistivity contrast and the significant topography in the survey areas, a further two complementary reliability checks were carried out on the inverted real data. The aim of which was to eliminate any remaining inversion artefacts and assess the data resolution at depth. First, the depth of investigation (DOI) index was adopted48951 and m2 are the resistivity values in the two final models, and m01 and m02 are the values of the corresponding reference models. Rb are the resistivity values at the base of the two final models, expected to be equal to, or at least very close to, the corresponding value of the reference model. In areas where the final models are well constrained by the data, and the inversion process considered reliable, the DOI index is close to zero. Any cells with a DOI index greater than 0.24895where m2 = 0.90–0.98). Where the intersection occurs close to the end of one or both profiles, values are poorly correlated (R2 = 0.52–0.63). This is likely to be a result of the lack of sensitivity towards each end of the region beneath the electrodes in the Wenner array configuration. The application of the DOI index does not remove the regions as there is sufficient data to alter the resistivity value from the reference model. The magnitude of the values in these end regions will be subject to greater uncertainty as they are based on fewer measurement points. This effect is likely to be exacerbated at sites, such as Ngozumpa Glacier, where the subsurface can be very heterogeneous. While confidence can be held in the magnitude and broad spatial pattern of resistivity values due to rigorous error quantification, greater uncertainty is present towards the ends of the profiles and in the exact location and nature of resistivity boundaries.Secondly, the inverted final resistivity values along the one-dimensional intersection between any two ERT profiles were plotted against each other where possible . Sci. Rep.7, 46766; doi: 10.1038/srep46766 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Picea abies, despite a frozen shoot, stay ice free down to −50 °C by a mechanism termed supercooling whose biophysical and biochemical requirements are poorly understood.Bud primordia of Bud architecture was assessed by 3D—reconstruction, supercooling and freezing patterns by infrared video thermography, freeze dehydration and extraorgan freezing by water potential measurements, and cell‐specific chemical patterns by Raman microscopy and mass spectrometry imaging.−1) caused accumulation of extraorgan ice masses that by rupture of the shoot, pith tissue are accommodated in large voids. The barrier tissue has exceptionally pectin‐rich cell walls and intercellular spaces, and the cell lumina were lined or filled with proteins, especially near the primordium. Primordial cells close to the barrier accumulate di, tri and tetrasaccharides.A bowl‐like ice barrier tissue insulates primordia from entrance by intrinsic ice. Water repellent and densely packed bud scales prevent extrinsic ice penetration. At −18 °C, break‐down of supercooling was triggered by intrinsic ice nucleators whereas the ice barrier remained active. Temperature‐dependent freeze dehydration (−0.1 MPa KBud architecture efficiently prevents ice penetration, but ice nucleators become active inside the primordium below a temperature threshold. Biochemical patterns indicate a complex cellular interplay enabling supercooling and the necessity for cell‐specific biochemical analysis. Picea abies are able to supercool down to −50 °C. This is achieved by a sophisticated bud architecture, which insulates the bud primordium from external and internal ice penetration. The ice barrier tissue and adjacent cell layers show a highly differentiated chemical composition facilitating supercooling of Picea abies bud primodia.Mechanisms of freeze avoidance and supercooling are poorly understood, although they are widely present frost survival mechanisms in tissues of woody plants. Despite a frozen shoot, vegetative bud primordia of  Karst. were collected in the Botanical Garden of the University of Innsbruck  from five randomly chosen individuals out of 60 potted trees cultivated outdoors under natural field conditions. Investigations were conducted on terminal and lateral vegetative buds of P. abies when the overall bud had at least 2 mm in size.Twigs with buds of 2.2After sampling, differential thermal analysis (DTA) measurements were conducted according to Burke, Gusta, Quamme, Weiser, and Li . On 2–3 −1 to below the frost killing temperature of the buds.Dead reference samples that had been dried at +80 °C for 72 h, were frozen in the same way as living bud samples. The aluminium solids with samples were put into the cooling compartment of a temperature‐controlled commercial freezer . Controlled cooling (software developed by Othmar Buchner in LabView 2012) started at +5°C, and then the freezer temperature was lowered at 3 K.hThe temperature was monitored using thermocouples connected to a multiplexer  and recorded with a data logger (CR10 Campbell Scientific) at an interval of 10 s. DTA values were obtained by subtracting the temperature of the reference twig from the temperature of the sample twig. The high temperature exotherm (HTE) and the low temperature exotherm (LTE) were determined graphically from the DTA plot as the starting temperature of the respective freezing exotherm.2.3P. abies was determined by exposing shoots bearing buds to a set of different freezing temperatures. At least 10 buds randomly chosen from twigs were taken at 2‐week intervals from five individual potted trees. These twigs were placed on wet paper towels inside sealed plastic bags. A set of twigs was kept in a cooling chamber at +4 °C to +6 °C as an untreated control. Bud samples that were meant to be frozen were put inside sealable plastic bags into eight freezers  and exposed to different target temperatures  between −5 °C and −50 °C. From +2 °C cooling proceeded at a rate of 3 K h−1, down to target temperatures, which were held for 4 h. Thawing took place at a rate of 3 K.h−1.Frost resistance of vegetative buds of Viability of bud primordia was once assessed by triphenyltetrazolium chloride (TTC) staining and in parallel by measuring chlorophyll fluorescence of bud primordia, using a MINI PAM . Until now, chlorophyll fluorescence has been used as a viability assay on leaves  was fitted to the data using OriginPro 7G . From the parameters of the logistic function, the LT2.4P. abies were monitored with a digital infrared camera . This infrared camera has a thermal resolution of 0.2 mK. We used a close‐up lens  to obtain a spatial resolution of 25 μm. Infrared images were recorded at an interval of 10 frames per second. Subsequent processing of the images by infrared differential thermal analysis , the three buds were arranged in a focal plane. Additionally, six thermocouples were mounted close to the buds. Thermocouple output was recorded every 10 s by a data logger .A longitudinal section of the terminal part of a twig of 2.5P. abies, twigs were detached from 10 randomly selected trees in December 2014 and January 2016. From these twigs, about 20 buds and subtending shoots were selected and fixed in methanol‐formaldehyde‐acetic acid‐alcohol  at 20 μm and triple stained with safranin, fast green, and orange G . Images were aligned manually and background color was removed using color‐clearing subroutines within After Effects as described . The chambers were connected to a PSYPRO water potential system (Wescor Inc.). The PSYPRO system measures water potentials on the basis of psychrometric recordings. The C‐52 chambers were used with sample holders with a diameter of 7 mm and a depth of 2.5 mm.Total water potential of the bud primordia of detached vegetative buds of −1. These standards were applied to 6 mm diameter filter paper discs .The C‐52 chambers were calibrated with NaCl solutions  with concentrations of 100, 290, and 1,000 mmol kgWater potential was measured on isolated bud primordia, which were dissected from the shoot tissues with a razor blade. To ensure that only primordial cells were measured, all other tissues below the primordium were removed. This was done by cutting off layer after layer from the direction of the subtending shoot to the primordium, until the nearly transparent cells of the crown tissue got visible. Bud scales were removed by a gentle twisting and squeezing motion.2.8−1, UHTS spectrometer, WITec Germany) and a deep depletion charge‐coupled device camera  to record the Stokes scattering. Cosmic ray removal and the average spectra, as well as the Raman images, were calculated using the WITec Project software. Different samples, positions, and scan sizes were measured, and the biggest and longest scan, representing the most comprehensive one, is shown in this paper. Within the selected area , 78,125 spectra (125 × 625 pixel) were acquired with an integration time of 1.2 s and a laser power of 100 mW. Although many bands are overlapping, integration over some characteristic bands allowed us to follow overall chemical composition as well as differences within the investigated region.Raman imaging reveals the chemical composition of plant cell walls in context with the microstructures with a spatial resolution smaller than 0.5 μm in a nondestructive manner . MALDI‐time‐of‐flight (TOF) MSI was performed using a Bruker Ultraflex MALDI TOF mass spectrometer equipped with a smartbeam Nd:YAG‐laser with an effective spot size of 35 μm; the laser power was adjusted to 80% (manufacturer's arbitrary units). In positive ion reflectron mode, the sample was rastered at a lateral resolution of 100 × 100 μm and 100 MS spectra in the mass range of m/z 100 to m/z 2,000 were averaged at every single spot. Data were analyzed using FlexImaging 2.1 software . The mass filter ranges for the generation of ion images were set to ± 0.025% of the monoisotopic molecular ions' m/z value.From 10 collected twigs, single buds were randomly chosen and longitudinally cut (about 20‐μm thick) by the use of a microtome  and frost resistance of the primordia for each season  were calculated . The supercooling ability of bud primordia depending on different treatments was tested on 44 controls, 24 damaged (“cut”), and 24 infiltrated buds . Differences between mean values with homoscedasticity were tested with a one‐way analysis of variance (anova) and a subsequent Duncan post hoc‐test. In case of a negative Levene‐test, the Mann–Whitney U‐test was used as a nonparametric post hoc‐test . All analyses were conducted at a significance level of p ≤ .05.Mean values of ice nucleation temperatures . Under the experimental conditions, all three bud primordia supercooled and remained ice‐free down to at least −18.2 °C. After separate initial ice nucleation events  and the frost resistance of bud primordia of r Figure . Althoug3.3Mechanical damage or substitution of air cushions between bud scales and the primordium surface by vacuum infiltration with water, caused an immediate loss of supercooling ability Figure . This de3.4−1), total water potential values indicated successive moderate freeze dehydration  and dehydration . Raman images elucidate the cell wall as well as deposits within the cell lumina, with a diffraction limited spatial resolution  and to less extent cellulose signals (band at 1090 cm−1 and 375 cm−1)  as well as strong bands at 859, 939, 1,089, 1,122, 1,337, and 1,455 cm−1 . Proteins, as visualized by integrating the Amide I region from 1,523 to 1,700 cm−1  of a disaccharide , a trisaccharide , and a tetrasaccharide .Moreover, 2D chemical mapping of longitudinal slices of m Figure . Accordi44.1P. abies is facilitated by extrinsic and intrinsic ice barriers and the absence of active ice nucleators. After mechanical destruction of the bud scales or vacuum infiltration with water, supercooling ability was completely lost. Isolated primordia of larch (Larix kaempferi) were also unable to supercool  and did not enter via the crown tissue. Evidently below a certain temperature threshold, ice nucleators inside the primordium become active and trigger intracellular ice nucleation. Similar observations were reported for supercooling reproductive organs of other woody plants  by 19.3 K , then the bud scales and the bark tissue become transparent (starting from second 16 on). This reveales the leave primordia, which surround the shoot primordium. The resin ducts are colored yellow (second 20 to 35), while the outer vascular tissues and bud scales are transparent. After fading of the resin ducts, the crown tissue is highlighted in red color (second 36 to 41), followed by the ice voids, which are colored blue (second 42 to 68).Video S1. 3D reconstruction of a bud of Click here for additional data file.Video S2. Sequence of the 341 individual images of cross sections of a whole bud of P. abies starting from the tip of the bud scales (section no. 1) to the apex of the primordium (section no. 38) and the beginning of the shoot primordium (section no. 52). The whole primordium extends from section no. 38 to no. 166. The bowl‐like ice barrier starts to get visible at section no. 155, from section no. 167 to no. 181 the crown tissue (red) can clearly be seen. The ice voids (blue) and the resin ducts (yellow) were found in all sections starting from no. 175 and no. 153 onwards, respectively.Click here for additional data file.


Ice-albedo feedback due to the albedo contrast between water and ice is a major factor in seasonal sea ice retreat, and has received increasing attention with the Arctic Ocean shifting to a seasonal ice cover. However, quantitative evaluation of such feedbacks is still insufficient. Here we provide quantitative evidence that heat input through the open water fraction is the primary driver of seasonal and interannual variations in Arctic sea ice retreat. Analyses of satellite data (1979–2014) and a simplified ice-upper ocean coupled model reveal that divergent ice motion in the early melt season triggers large-scale feedback which subsequently amplifies summer sea ice anomalies. The magnitude of divergence controlling the feedback has doubled since 2000 due to a more mobile ice cover, which can partly explain the recent drastic ice reduction in the Arctic Ocean. In the Arctic Ocean, recent observations have revealed major reductions in summer ice extent3, thinning of sea ice5, and a shift from perennial to seasonal sea ice8, particularly after the 2000s. It is well established that climate change signals are amplified in the Arctic11 and that such “polar amplification” is associated with ice albedo feedbacks14.Ice-albedo feedback is a key aspect of global climate change. In the polar region, a decrease of snow and ice area results in a decrease of surface albedo, and the intensified solar heating further decreases the snow and ice area15. In the seasonal ice zone, presence of an open water fraction with a much lower albedo results in high solar radiation absorption by the upper ocean17, which in turn serves as the dominant heat source for sea ice lateral and bottom melt19. Since the seasonal ice zone is dominated by thin and undeformed first-year ice, the melting of sea ice immediately increases the fraction of open water in the ice-covered area and thus drives up absorption of solar energy in the upper ocean. Hence, in regions dominated by seasonal ice such as the Southern Ocean and the Sea of Okhotsk, ice-albedo feedback due to the albedo contrast between water and ice surfaces, termed ice-ocean albedo feedback, enhances summer sea ice retreat20 and partly controls interannual variability of the ice cover22.Until recently, the Arctic Ocean has been characterized by a thick multiyear ice cover that persisted throughout the summer, with melt confined to its upper surface26 and the shift from perennial to seasonal sea ice. Satellite observations indicate a significant positive trend in solar heating of the upper ocean associated with recent changes in sea ice concentration and/or increase in ice-free area19. However, key questions, such as how much of the variation in sea ice retreat and the recent sea ice reduction are explained by heat input through the open water fraction, or the specific physical processes at work in triggering and translating the feedback, remain unanswered.Recently, such feedback effects have also received attention in the context of drastic reductions in summer Arctic sea ice extentn = 36, p < 0.001; Supplementary Fig. Here we show the dominance of heat input through the open water fraction on sea ice loss and its variation, which is a necessary condition for ice-ocean albedo feedback, based on the relationship between sea ice retreat and heat budget over the ice-covered area. Then we explore the specific trigger of the feedback effect, and examine whether ice melt is in fact amplified significantly by this feedback, and whether the drastic reduction in summer ice extent can be explained by this feedback, based on the combined analysis of satellite observations and a simplified ice-ocean coupled model. We selected the Pacific Arctic Sector  is much larger than that at the ice surface (Fi), because shortwave radiation is the dominant component of heat budget in the analysis area , and compare this heat with the volume of sea ice melt (Qm) which was calculated from the observed decrease of ice area multiplied by mean ice thickness, accounting also for the decrease by ice advection (Adv)  is comparable to Qu and thus contributes significantly to ice melt. In this paper, we assume that heat input at the sea ice surface resulting in surface melt is exclusively used to reduce ice thickness. We calculated the seasonal evolution of mean ice thickness which decreases from the initial thickness of 1.4 m, as observed by ICESat . While this simplification is a zeroth-order approximation, it is valid at least for examining interannual variability, considering that the relative standard deviation of yearly Qi (6%) is much smaller than that of yearly Qu (32%).For the ice-covered area defined by ice concentrations >15%, we have analyzed the daily heat budget separately for the water and ice surfaces from 1979 to 2014 (see Methods). During the summer season, net heat flux at the water surface  as 0.77, 0.85, 0.92, and 0.91 for the monthly mean from May to August, respectively . The error in Qu caused by melt ponds is shown by red shading in Fig. Qm mostly due to the uncertainty of mean ice thickness is also indicated by gray shadings. It is noted that ice export from the fan-shaped area and its interannual variation  using the ice drift velocity derived from satellite observations (see Methods). Comparisons between Div and ice retreat conditions  has the highest correlation with the sea ice retreat lagged by 1–2 months, with high correlation persisting through the end of August. Thus, this early Div is also well correlated with the yearly value of Qm . These suggest that the divergent ice motion in the early melt season can be a trigger of ice melt acceleration through ice-ocean albedo feedback. After the 2000s, such relationship has likely become stronger, suggested by a much higher regression coefficient than that prior to 2000 , respectively (C = CFY + CMY). Here we assume that the melt of first-year ice and that of multiyear ice are proportional to their respective areal fraction. However, only first-year ice can be reduced in area through melt, not multiyear ice. The local heat balance of the upper ocean is given bycw (=3990 J kg−1 °C−1) is the specific heat capacity of seawater, ρw (=1026 kg m−3) and ρi (=920 kg m−3) are the density of seawater and sea ice, respectively, Lf (=0.276 MJ kg−1) is the latent heat of fusion for sea ice, and hi is the mean ice thickness. The melt rate of first-year ice is parameterized asKb (=1.2 × 10−4 m s−1) is the bulk heat transfer coefficient between ice and ocean27, and Tf (=−1.86 °C) is the freezing temperature. Equations  eventually melts completely and hence bottom melt contributes indirectly to decreases in sea ice area in addition to lateral melt, the simplified model considers these processes in a bulk fashion. In all the model runs, the initial ice concentration is set to 99%, the initial distribution of multiyear ice  is the influence of heat stored in the upper ocean. We obtain an e-folding time for this solution of Lfρihi/Fw, which serves as a measure of the inherent time scale of this coupled system. The estimated time scale of ~1.4 months during the early melt season  explains the suggested lag time of enhanced melt relative to the trigger of ice divergence . This increased heat uptake can explain about 70% of the observed 2.1-fold increase in total sea ice melt (from 121 MJ m−2 to 257 MJ m−2). This contrast in the increase in annual ice melt compared to heat input is also evident in Fig. 5.Findings from this study show that the feedback effect triggered by early-season divergent ice motion plays a key role in the seasonal evolution and interannual variation of sea ice retreat in the Pacific Arctic, particularly since the early 2000s. Below, we consider the contribution of such feedback to recent reductions in ice extent and volume, based on a comparison between mean states before and after 2000 Fig. . In the 38, influence of cloud cover40, longwave radiative forcing due to anthropogenic CO2 emission41, melt pond distribution in the early summer season42, release of the solar heat stored in a near-surface layer of the ocean43, and increases in the heat inflow through Bering Strait44 may also contribute to drastic ice reductions. However, we note that these factors are intrinsically linked to divergence in the ice pack, because increased heat input from any source may enhance sea ice mobility. Thus, this study provides a new perspective on the observed drastic ice reduction in demonstrating, through modeling and analysis of remote sensing data, that ice divergence in the early melt season is a key trigger for amplification of ice retreat through ice-ocean albedo feedback. This finding also suggests that early-season ice divergence is associated with a substantial skill for seasonal ice prediction. A detailed analysis for the impact of individual processes on sea ice retreat at the local scale is beyond the scope of this study. Future work will require comprehensive analysis of output from fully coupled climate models along with time series of relevant sea ice quantities.Other factors such as changes in atmospheric circulation patterns46 are provided by National Snow and Ice Data Center (NSIDC) and sea ice concentration is derived using the Bootstrap Algorithm47. For sea ice drift velocity, we use the dataset48 derived from a wide variety of sensors such as AMSR-E, SSM/I, AVHRR, IABP Buoys, and etc., provided by NSIDC. Spatial distribution of multiyear ice is estimated using the ice age data which is based on the particle tracking method31. Air temperatures and dew point temperatures at 2 m, wind speed at 10 m, and total cloud cover are obtained from the ECMWF Interim Re-analysis (ERA-Interim49), and then interpolated onto the 25 km polar stereographic grid with a Gaussian weighting function. For significance test of the statistics in this paper, we have performed the two-tailed t-test throughout the paper. Here n and p indicate sample number and significance probability, respectively.In this study, daily sea ice data are derived from the Nimbus 7 Scanning Multichannel Microwave Radiometer (SMMR) and the Defense Meteorological Satellite Program (DMSP) Special Sensor Microwave Imager (SSM/I and SSMIS) from 1979 to 2014. The vertical and horizontal brightness temperatures50, over the ice-covered area of the Pacific Arctic  is calculated fromFw is the net heat budget at the water surface, C is the ice concentration, Sg is a unit grid cell area (≈25 km × 25 km), with subscript k denoting all sea ice pixels with Fw > 0 W m−2 in the analysis area, and Se (=ΣkSgk) as the extent of sea ice cover. Similarly, heat input at the ice surface and melt ponds are calculated as,Fi and Fp are the net heat fluxes at the ice and melt pond surfaces, respectively, and fp is the areal fraction of melt ponds on sea ice. Detailed procedures for estimations of ice albedo and fp are described in the next section.Using sea ice observation data and meteorological reanalysis data, we have calculated the net heat budget at the water and ice surfaces which is expressed as the sum of shortwave and longwave radiation, sensible and latent heat fluxesαi) which varies in response to sea-ice surface conditions. Based on data for ice age31, we classified sea ice as first- and multiyear ice and then parameterized their albedos separately52. Surface albedos for each ice type are determined from the number of days elapsed since onset of melt as derived from satellite microwave radiometer data53. Supplementary Fig. α = αiC + αw (1 − C)) with that derived directly from AVHRR satellite observations54. These two independently derived albedos show similar seasonal evolutions, validating this approach. Then, we estimated the fraction of melt ponds (fp) as a function of αi, based on in situ observations55 as follows:FY and MY denote the first- and multiyear ice, respectively.Since the distribution of melt ponds, a key factor in the heat budget of ice-covered area, is difficult to derive from the satellite observation directly, we have estimated the time evolution of melt pond fraction from a combination of several empirical approaches. First we estimated the ice albedo (Vi) and ice export from the boundary of analysis area B (ΔVo) during the time step Δt as follows,hi is the mean ice thickness in the analysis area, and t. To reduce the influence of errors in sea ice concentration data, we set Δt as 10 days. Among the sea ice properties, ice thickness is by far the least observed. Here we calculate the reduction of ice thickness due to surface melt. According to the comparison of ice mass balance measurements with heat budget analysis56, ~40% of the heat absorbed into the ice surface contributes to the reduction of ice thickness. Based on this work, we can obtain a time-dependent ice thickness as,h0 is the initial ice thickness, Lf (=0.276 MJ kg−1) is the latent heat of fusion for sea ice with salinity of 6 psu57, and ρi (=920 kg m−3) is the density of sea ice. We use an initial ice thickness of 1.4 m, corresponding to the mean ice thickness in the analysis area in the spring season (February through May), obtained from ICESat observations58 from 2003 through 2008  during Δt is obtained fromThe volume of ice melt and ice divergence are estimated from ice concentration and ice drift data as schematically shown in Supplementary Fig. Then the volume of sea ice melt per day is converted to the corresponding heat according toDiv) during Δt is estimated as the ice area export from the boundary of analysis area B and ice edge E (defined as the 15% ice concentration contour), standardized by the sea ice extent Se;The ice divergence (Qu, Qm, and Div, derived from satellite observations.Sea ice quantities obtained from satellite observations are subject to varying levels of error. Here, we assess the uncertainty of results by considering the error for key variables, Qu is obtained as the product of open water fraction and net heat flux at the water surface. Since the shortwave radiation, which does not vary as much from year to year, is dominant in the net heat flux during the summer season, the error in Qu is mostly due to the uncertainty in the sea ice concentration dataset. Previous studies have reported that derived ice concentrations are less reliable over melting and ponded sea ice28. Also, melt ponds act as a conduit for heat input into the upper ocean. Hence, calculations of Qu and its error need to consider the pond coverage of sea ice. Regarding the treatment of melt ponds in the bootstrap algorithm, we assume two extreme, bounding cases. Namely, all melt ponds are classified as either part of the sea ice or the open water fraction (Supplementary Fig. Qu as the sum of heat absorbed through the open water fraction and transmitted from melt ponds. Note that the transmittance of melt ponds τ strongly depends on the ice thickness. Following previous studies which estimate the typical τ based on in situ observations, we use the value of 0.55 for the first-year ice59 and 0.17 for the multiyear ice60. The obtained Qu of the lower and upper bounds are shown by red shadings in Fig. The volume of ice melt is calculated as the product of ice area loss and mean ice thickness. The uncertainty of change in ice area obtained from the ice concentration is much smaller than that of ice thickness, for which much fewer data are available. In this paper, we assume that the uncertainty of ice melt is controlled by the uncertainty in mean ice thickness which is evaluated from the assumed initial ice thickness. From the 6-year observations by ICESat (Supplementary Fig. Div is due to errors in ice drift velocity and ice concentration. Based on error propagation, an uncertainty of Div can be represented asδDiv, δu, and δC are errors in Div, u, and C, respectively. Here we assume δu ≈ 0.02 m s−1, based on documentation provided by the dataset originators48. In the early melt season, the analysis area is mostly covered by sea ice with δC of only a few percent; with a typical value of −1, the relative error Div mainly originates from that of ice drift velocity and equation (Div, The uncertainty in estimating Supplementary Figures


Ralstonia solanacearum, several of these effectors contain domains with predicted enzymatic activities, including acetyltransferases, phosphatases, and proteases, among others. How these enzymatic activities get activated inside plant cells, but not in the bacterial cell, remains unknown in most cases. In this work, we found that the R. solanacearum effector RipAY is phosphorylated in plant cells. One phosphorylated serine residue, S131, is required for the reported gamma-glutamyl cyclotransferase activity of RipAY, responsible for the degradation of gamma-glutamyl compounds (such as glutathione) inside host cells. Accordingly, non-phosphorylable mutants in S131 abolish RipAY-mediated degradation of glutathione in plant cells and the subsequent suppression of plant immune responses. In this article, we examine our results in relation to the recent reports on the biochemical activities of RipAY, and discuss the potential implications of phosphorylation in plant cells as a mechanism to modulate the enzymatic activity of RipAY.Most bacterial pathogens subvert plant cellular functions using effector proteins delivered inside plant cells. In the plant pathogen  Ralstonia solanacearum causes the devastating bacterial wilt disease in numerous hosts, constituting an important threat to agriculture . These proteins  are key determinants of the interaction between pathogen and host, contributing to the alteration of plant cellular processes to promote infection . T3E acticulture , but is iculture . Strainsffectors , and theffectors . Remarkaffectors . After dffectors . This raffectors .R. solanacearum T3E RipAY is present in all the R. solanacearum strains sequenced to date  was purchased from Abclonal, United States. Sequencing-grade modified trypsin was purchased from Promega . The Nanosep centrifugal filter units with Omega membrane (MWCO 10 kDa) were bought from Pall Corporation . All other chemicals were purchased from Sigma-Aldrich  unless otherwise stated.The flg22 peptide . After washing, IPed proteins were stripped from the beads and separated on precast SDS-PAGE gels . Western blot was performed using the anti-FLAG (Sigma) and anti-GFP  primary antibodies, respectively. Blots were stained with Coomassie Brilliant Blue to verify equal loading. Protein digestion, liquid-chromatography coupled to tandem mass-spectrometric analysis and peptide identification were performed as described  equipped with LEICA LAS X image acquisition software. The green fluorescent protein (GFP) was excited by a 488 nm argon laser at a strength of 20% and detected at 501–528 nm (Em).Leaves from 4- to 5-week-old Agrobacterium-mediated transient expression of RipAY  fused to a green-fluorescent protein tag (RipAY-GFP) in Nicotiana benthamiana cells . Among these residues, S131 and S251 are located within the ChaC domain , and are conserved in RipAY sequences from several R. solanacearum strains representative from different phylotypes . An additional phosphorylated residue, S32, was only conserved in strains from the phylotype I, although RipAY sequences from other strains present serine residues in the position 30 or 31 .In order to identify RipAY residues post-translationally modified in plant cells, we previously performed na cells . GFP immN. benthamiana is able to suppress the rapid generation of reactive oxygen species (ROS) after treatment of plant tissues with the immune elicitor flg22 . Strikingly, although the single S32A mutation did not affect RipAY suppression of flg22-triggered ROS, expression of the double S32A/S131A mutant consistently increased this response in comparison with GFP-expressing control tissues .RipAY-GFP transiently expressed in or flg22 . In ordeN. benthamiana tissues expressing RipAY showed a dramatic reduction of glutathione content (10–15%) compared with GFP-expressing control tissues . Mutants containing the S131A mutation were partially impaired in glutathione degradation in comparison with wild-type RipAY . Besides S131A, additional mutations in other phosphorylated residues did not significantly affect RipAY ability to degrade plant glutathione .It has been previously shown that the ability of RipAY to suppress immune responses is associated to the degradation of plant glutathione through its GGCT activity . To testSupplementary Figure S3).We next tested whether S131 phosphorylation could contribute to the activation of RipAY function. Mutation of serine to aspartic acid (D) sometimes resembles the addition of a phosphate group on a serine residue, mimicking a phosphorylation event. However, a S131D mutation abolished RipAY ability to suppress flg22-triggered ROS burst . As we reported before, we consistently detected a double band corresponding to RipAY in Western blot assays , which may be caused by PTMs of the protein. This double band became more obvious when using acrylamide gels containing Phos-Tag, which binds phosphorylated residues, slowing down the migration of phosphorylated proteins , suggesting that this band-shift may be caused by phosphorylation. Interestingly, the S32A mutation altered the ratio between these bands, increasing the concentration of the fast-migrating band , suggesting that phosphorylation in S32 accounts for most RipAY phosphorylation, or that phosphorylation of this site is required for the subsequent phosphorylation of others.The mutagenesis of the phosphorylated residues did not affect RipAY accumulation  in planta . Similarly, none of these mutations altered the reported RipAY-GFP association with NbTRX09 and NbTRX15 in Co-IP assays .Protein phosphorylation can affect protein-protein interactions and the subcellular localization of phosphorylated proteins. We previously reported that RipAY-GFP localizes at the cytoplasm and nucleus of n planta . Associain vitro . Since tN. benthamiana cells, we were able to detect phosphorylation in several RipAY peptides . Mutagenesis of the phosphorylated residues to alanine indicated that S32A alters the putative phosphorylation-associated bandshift of RipAY, and mutations in S131 abolish its function. Interestingly, although S32 seems to be the major site responsible for the RipAY bandshift , mutations in that residue did not affect the RipAY-mediated degradation of glutathione or suppression of immune-related ROS burst . Therefore, it remains unclear whether phosphorylation in S32  is not relevant for RipAY activity or required for other activities not measured in our assays. Moreover, although the S131A mutation completely abolished the ability of RipAY to suppress the immune-related ROS burst, it only partially compromised its ability to degrade glutathione , indicating that S131 has a quantitative contribution, but is not fully required, for the GGCT activity of RipAY.Upon transient expression in in planta by substituting S131 to an aspartic acid residue. It is still possible that phosphorylation of S131 is important for activity, but S131D cannot mimic phosphorylation. Of note, phosphorylation is a dynamic process, and the addition/removal of a phosphate group may need to be regulated in spatiotemporal manner. In such situations, the substitutions to negatively charged amino acids might not mimic phosphorylation, but rather result in loss of function, as it has been observed for phospho-mimic mutants before , it would be difficult for the pathogen to alter their sequence in order to avoid inactivation in resistant hosts.Importantly, S131 has been reported to be present within the putative substrate-binding site of the RipAY ChaC domain . A S131At growth . It is p AvrPtoB . In that AvrPtoB . In the in vitro, although the underlying mechanism is still unknown , suggesting that phosphorylation of these sites is not required for TRX binding. Interestingly, although TRXs catalyze the reduction of disulphide bonds of target proteins, a mutation in the sole cysteine (C) residue of RipAY (C333S) did not abolish TRX binding or any of RipAY activities in vivo and in vitro . These findings suggest that phosphorylation may be an extended mechanism for the modulation of the catalytic activity of R. solanacearum T3Es in plant cells, although the validation of this hypothesis will require the specific biochemical characterization of each T3E independently. Furthermore, the development of more sensitive proteomic approaches will help determining whether these phosphorylation events take place in natural conditions during the infection and whether other T3Es injected simultaneously inside plant cells may play a role in these modifications.Phosphorylation has been found in an increasing number of T3Es from different plant pathogens , such asactivity . R. solant cells . Additio1 predicted that S32 and S251 are phosphorylated by kinases from the proline-dependent serine/threonine kinase group, such as cyclin-dependent kinases. However, we were not able to detect such kinases among RipAY interactors in our IP+LC-MS/MS approach in planta (R. solanacearum T3Es will be essential to fully understand their biochemical activation, and will be an important step toward the biotechnological application of this fundamental knowledge to the development of disease-resistant plants. Given the transient nature of kinase-substrate interactions, this constitutes a challenge for future studies.Using the RipAY amino acid sequence, a search in the Scansite3 servern planta . To daten planta . The ideYW, YS, and AM designed experiments, performed experiments, and analyzed data. AM wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.