Microstructures from deep ice cores reflect the dynamic conditions of the drill location as well as the thermodynamic history of the drill site and catchment area in great detail. Ice core parameters , grain size, grain shape), mesostructures  as well as borehole deformation were measured in a deep ice core drilled at Kohnen Station, Dronning Maud Land (DML), Antarctica. These observations are used to characterize the local dynamic setting and its rheological as well as microstructural effects at the EDML ice core drilling site (European Project for Ice Coring in Antarctica in DML). The results suggest a division of the core into five distinct sections, interpreted as the effects of changing deformation boundary conditions from triaxial deformation with horizontal extension to bedrock-parallel shear. Region 1 (uppermost approx. 450 m depth) with still small macroscopic strain is dominated by compression of bubbles and strong strain and recrystallization localization. Region 2 (approx. 450–1700 m depth) shows a girdle-type LPO with the girdle plane being perpendicular to grain elongations, which indicates triaxial deformation with dominating horizontal extension. In this region (approx. 1000 m depth), the first subtle traces of shear deformation are observed in the shape-preferred orientation (SPO) by inclination of the grain elongation. Region 3 (approx. 1700–2030 m depth) represents a transitional regime between triaxial deformation and dominance of shear, which becomes apparent in the progression of the girdle to a single maximum LPO and increasing obliqueness of grain elongations. The fully developed single maximum LPO in region 4 (approx. 2030–2385 m depth) is an indicator of shear dominance. Region 5 (below approx. 2385 m depth) is marked by signs of strong shear, such as strong SPO values of grain elongation and strong kink folding of visual layers. The details of structural observations are compared with results from a numerical ice sheet model  for comparison of strain rate trends predicted from the large-scale geometry of the ice sheet and borehole logging data. This comparison confirms the segmentation into these depth regions and in turn provides a wider view of the ice sheet.This article is part of the themed issue ‘Microdynamics of ice’. Ice sheets possess the highest potential to cause drastic changes within the global water cycle owing to their large water reservoir. This horizontal movement of ice is expressed in the surface velocities measured locally with ground-based GPS surveys  or by rec-axis distributions (LPO) with depth is an indicator of the changing dominance of the deformation modes )figure 3d) and shape parameters (SPOs). The magnitudes of grain elongations given as aspect ratios (e) and grain elongation directions (f) are calculated from the principal axes of an ellipse fitted to each segmented grain. The distributions of the directions are shown as circular histograms (‘rose diagrams’) in figure 5b . The raw data of inclination measurements from the elongation directions from vertical sections reflect a jump between 1655 and 1758 m . Altted from . In the 58 m cf.  owing to58 m cf.  and sign(c)Visual stratigraphic layering was recorded continuously along the core with a line scanner (LS) developed at the Alfred Wegener Institute in Bremerhaven, Germany, and the Niels Bohr Institute at the University of Copenhagen, Denmark ,49.a).Typical LS samples are 1 m ice slabs that have been cut lengthwise from the EDML core, with their upper and lower surfaces polished with a hand-held microtome blade. Oblique dark-field, indirect illumination of each slab is scanned along the core axis by a digital line-scan camera. The line-scan camera can capture only the light that has been scattered by inclusions in the ice matrix, such as microscopic particles or air bubbles, because ice itself is transparent. Thus, the degree of brightness recorded by the camera is proportional to the concentration of inclusions in the sample, in such a manner that a clear piece of ice, free of inclusions, appears dark a.Images recorded with the LS system reveal the visual stratigraphy of an ice core in great detail (1 pix = 0.1 mm) because of the high resolution and sensitivity of the digital line-scan camera. Strata as thin as 1 mm can be easily detected with this method. Any small change in the optical properties of the ice matrix, usually caused by a change in the mean size or concentration of inclusions, gives rise to a new horizon.(d)a) owing to the local ice deformation between two measurements (January 2004 and November 2005) to estimate the strain regime at the EDML site. Changes in diameter of the borehole, which do occur, are not evaluated in this study.During the EDML drilling campaign, the borehole was logged at several stages of drilling progress. The logging system continuously recorded the tilt of the borehole with respect to the vertical (inclination) as well as the heading of the borehole with respect to magnetic north (azimuth) by means of a compass . Additiofigure 4(e)To derive a velocity–depth profile at the EDML site, we used the parallel ice sheet model (PISM v 0.6.1) –53.et al. [The deformation of polycrystalline ice is modelled, using the Nye generalization  of the Get al. , to whicAt each time step of a PISM simulation, the geometry, temperature, water content and basal strength of the ice sheet are included into momentum balance equations to determine the velocity of the flowing ice.Instead of solving the full set of Stokes equations for the momentum balance, PISM solves, in parallel, two different shallow approximations: (i) the non-sliding shallow ice approximation (SIA ), which et al. [et al. [−2 according to the SeaRISE-Antarctica recommendations [The present-day state of the Antarctic ice sheet was computed based on the present-day geometry Bedmap2 , and varet al. . The ori [et al.  has beenndations . Using andations  data forndations  heat flundations ), where Each simulation was conducted in a series of subsequent grid refinements  using 40, 20 and 10 km horizontal resolution and 41, 81 and 101 vertical layers, respectively. We used the flux correction method provided by PISM in addition to a prescribed (present-day) calving front position to result in an ice sheet that is close to the observed present-day geometry. After initialization (1 a), a short relaxation period (100 a) and a purely thermal spin-up with the geometry held fixed (200 k years) on the 40 km grid using only the non-sliding SIA, the model ran for 100 k years, 20 k years and 4 k years on the 40, 20 and 10 km grid, respectively, in the hybrid (SIA + SSA) mode. The individual boundary conditions that have been chosen for one ensemble member were held constant over time, thus forming a present-day climate equilibrium model realization.De (c) and the three principal strain rates, D1, D2 and D3 , have been derived from the simulated velocity field diagnostically on the PISM grid.All components of the strain rate tensor as well as the effective strain rate De c and the3.(a)c-axis distributions show a typical evolution with depth for a drill site located on an ice divide: a broad distribution represented by all three eigenvalues around one-third (b) appears uniform in the pole figure display  down to 1650 m depth. In a transitional region (approx. 1700–2030 m), the LPO evolves towards an elongated single maximum well defined by the inflection point of the e2-trend (b): above approximately 1650 m e2 grows; below 1650 m e2 decreases. Although increasing strictly monotonically with depth in the upper two-thirds of the core, also the e1-trend shows a change in slope at approximately 1650 m depth. The gradual evolution of the single maximum is generated by a gradual concentration of c-axes within the girdle, observable in panel 5 of figure 4b), which rises at this depth level towards unimodal distributions. This is followed by a collapse of e3 and e2 into a single maximum at approximately 2030 m depth (figures 4c). In a narrow layer (from 2345 to 2395 m depth), the LPOs become highly diversified with tendencies of single maximum to girdle and back to single maximum distributions represented by a wide range of values in e3 and e2 (0–0.5) and in e1 (0.5–0.9) (figure 4b). This is the lower half of the Eemian (marine isotope stage 5.5 (MIS5.5)) layer, characterized by lower δ18O values (figure 4d), which indicates higher precipitation temperatures than derived from the overlying ice of the last glacial period. Statistical evaluation is difficult, as the Eemian ice exhibits the largest grain sizes observed in the EDML core, except for the basal layer (figure 4d).The ne-third b appears display  and is ae2-trend b: above 2 and e1 between vertical and horizontal sections  are due to the ambiguity of measurement of c-axes lying close to the observation plane. Data at the periphery of a pole figure (projection onto a thin section plane) are of low quality and thus partly excluded from the population in display and LPO data processing, leading to the described bias. The automatic fabric analysers used here calculate the extinction angle, where no light gets through the system, by fitting the light amplitude values for each step of the polarizers to a sinusoid curve [c-axes in a plane normal to the observation axes is caused by the G20 instrument often being unable to resolve the quadrant pair of the azimuth of these orientations. The G20 used a rotating prism to realize three viewing axes and images are taken from three directions. This includes a fourfold symmetry of the extinction angles for the viewing directions. The automatic fabric analyser software philosophy excludes any possibly false information, thus any ambiguous data values are not included. The G50 instrument uses a quarter-wave (λ/4) plate to determine the azimuth, so that fast/slow directions by adding or subtracting λ/4 resolve the azimuth symmetry of the quadrants . Compar(b)2), but is strongly affected by the impurity content of ice from different climatic stages (d). Over the first 700 m, the grain size increases and decreases again with depth until reaching the last glacial maximum (LGM) ice , which is identified by the most depleted stable water isotope values . Below this, grains increase again down to approximately 2300 m , but show a higher variability within samples than in all stages above. A sharp decrease down to the smallest measured grain size values is observed just below the MIS5.5 ice (approx. 2400 m), followed by a sudden increase (approx. 25 times) to grains of similar or larger size than the section size in the deepest basal ice layers. Further doubling to tripling of grain size occurs in this basal layer estimated from the deepest three samples measured (‘square’ symbol and separate bottom axis in figure 4d).The grain size generally increases with depth . Thus, the fully automatic segmentation may not capture all (slightly weaker) grain boundaries. These weaker boundaries, in principle, are an indication for newly formed grain boundaries developing from subgrain boundaries during dynamic recrystallization . Both methods give diverging results at various levels with the manually corrected segmentation giving approximately 10% smaller values in most depth levels d. Thus, ization b,c as circular histograms , which show the magnitude and direction of elongation. Statistical evaluation  is not possible for horizontal sections owing to few samples. In general, only moderate mean aspect ratios up to 1.8 are observed (e). Large standard deviations in all samples indicate that only a few grains are actually elongated, whereas many exhibit an equi-dimensional habitus. However, for vertical sections, we observe clear trends in the mean values: first, increasing elongations from 1 to 1.5 down to 1100 m depth, followed by a decrease down to approximately 1700 m depth. Between 1700 and 2200 m, aspect ratios remain stable at small average elongations of approximately 1.2, with an increased variability observed among samples below approximately 2050 m depth. Low values approach approximately 1 and indicate equi-dimensional grain shapes on average. Most intensely elongated grains in the EDML ice core are observed at a sudden transition of aspect ratio from 1.1 to 1.7, located between 2356 and 2393 m depth. Below approximately 2575 m depth range statistical analysis is not reliable owing to the very large grain size.A visualization of SPO given as grain elongations in the vertical and horizontal sections is shown in figure 5aluation e,f is noobserved e. Large f). Statistical analysis, however, reveals elongation of grains in the horizontal direction (0°) down to approximately 1000 m depth. This is remarkable as between 500 and 1200 m the relative azimuthal core orientation was lost repeatedly in the brittle zone. The geographical azimuthal  orientation of the drill core controls the actual observed angle of inclined features. Three-dimensional-sectioning effects of orientations of lines or planes, e.g. during sample preparation as in this study or in geological outcrops, are well known in structural geology [Grain elongation directions, defined as angles of the long axis of a fitted ellipse with the horizontal, show large variations within individual sections f. Statis geology . These sc) with grain elongations in horizontal sections  shows that the mean orientation of the girdle plane, that is, the average plane containing all c-axes, and the orientation of the grain elongations in the horizontal are perpendicular to each other.Comparison of the vertical girdle LPO presented in the standard glaciological stereographic projections into the horizontal c with gr(d)a). The last feature results from the fact that air bubbles in this zone are by far the largest and most efficient light scatterers, so that the visual stratigraphy is dominated by depth variations in the number and size of air bubbles. The ice core in this zone appears so bright under the LS that the recorded images seem partially washed out, with a faint stratigraphy. Within the bubble–hydrate transition zone , strati0–1200 m ,86. Owina). Just a few cloudy bands occasionally show minimal undulations with amplitudes not larger than a couple of millimetres. Below 1700 m depth, these undulations gradually increase in intensity and frequency, to such an extent that below 1700 m depth microscale folds develop and some flimsy cloudy bands become slightly inclined or disrupted (a). However, it is only beneath 2050 m depth that well-defined mesoscale folds (with amplitudes up to a few centimetres) and sloped strata  become dominant (a). Further down, the intensity of such disturbances increases notably: cloudy bands appear ragged and fuzzy at diverse inclinations, sometimes up to 30° with respect to the horizontal.From the lower part of the BHT zone at approximately 950 m depth down to around 1600 m depth, the EDML stratigraphy remains undisturbed and horizontal a. Just aisrupted a. Howevedominant a. Furtheet al. [d). It coincides with conspicuous changes in ice microstructure, e.g. an abrupt reduction in grain size (5 mm to less than 1 mm) and an increase in grain elongation (aspect ratios 1.2–1.7), within a depth interval of less than 10 m .Below 2200 m depth, the strong layer mixing and low concentration of micro-inclusions make cloudy bands too faint and disrupted to be identified. Notwithstanding, at 2386 m, cloudy bands suddenly reappear neatly parallel, horizontal and well defined again. Faria et al. ,78,86 hahan 10 m d,e.z-fold and other serious stratigraphy disturbances become frequent. As a general trend, the intensity of the cloudy bands gradually reduces with depth until they completely disappear below 2600 m, where the temperature rises above −7°C and the average grain size increases dramatically (d).Drastic variations in intensity, inclination and folding of cloudy bands are recognized below 2400 m depth. Intense cloudy bands inclined up to 45°, kink folding, multiple atically d.(e)x, y, z of the borehole track by integrating the data along the path. Owing to the integration, the possible error in the borehole track increases with depth. The data presented in this study were recorded in January 2004 and November 2005. Both logs reached a depth of approximately 2550 m, which is still about 200 m above the bedrock. Later, re-logging data could not be included here owing to a failure of the azimuthal measurement, preventing the calculation of the shape of the borehole. However, these latter data would not have included the lower approximately 200 m, because the intrusion of subglacial water made the lowermost part of the borehole inaccessible. As the water rose faster than it could be removed, this also caused the end of the drilling [The measured borehole inclination and azimuth were smoothed, using a Butterworth low-pass filter to eliminate the influence of movements of the cable and erratic movements of the logger owing to unevenness of the borehole walls. The logger depth is taken as the paid out cable length. This set of three coordinates  was then transformed into three-dimensional Cartesian coordinates drilling ,25.a. The gradient of the borehole displacement is largest below approximately 2050 m depth (figure 4a), indicating the depth where shear becomes dominant. Above, the displacement is fluctuating around approximately 0.5 m, which is partly owing to the rather high noise of these measurements compared with the small displacement signal owing to the slow deformation. The absolute total displacement is in accordance with the measured surface velocity a. Comparison with the measured borehole temperature (same figure 4a) reveals a distinct difference, as modelled temperatures are always higher  than the observations. In the lowest part of the borehole, temperature observations are missing, but observed water at the ice–bed interface indicates temperate ice conditions. Thus, the simulated basal temperature of approximately −1.4°C (pressure corrected) underestimates the temperature at the base. The overall curvature of the simulated temperature–depth profile is smaller (more linear) than the observation, most likely indicating that the model underestimates the downward advection of cold ice from the surface. Assuming that the zeroth-ordered stress terms from the momentum balance equations are still the largest contributions to the effective stress at the EDML site, we expect the model to overestimate the deformation of ice, mostly in the ice column except for the basal zone.The simulated temperature–depth profile extracted from the PISM grid at the grid location next to the EDML site (approx. 1.5 km away) is shown in figure 4−1, and is thus twice the observed value  is always positive (tensile) along the depth profile and increases towards the base up to approximately 420 × 10−5 a−1  along the depth profile with the minimum of approximately −412 × 10−5 a−1 at the base. This component mostly compensates D1 . The second principal strain rate D2 is one magnitude smaller than D1 or D3 and reaches the maximum (14 × 10−5 a−1) at approximately 2400 m depth. Although relatively small, the differences between D1 and D3 towards the bed result in the slightly positive (tensile) component D2. D2 increases at approximately 1700 m depth evolving from compressional towards tensile and crosses the compressional/tensile border at approximately 2020 m depth, further intensifying its tensile nature. With depth, D2, decreases towards zero again. Additionally, the vertical gradients of the strongest principal strain rates D1 and D3 are shown in figure 4c to highlight the onset of the evolution of changing deformation at approximately 1700 m depth.Figure 410−5 a−1 . The thi3  is vertical, D1  is orientated perpendicular to the ridge, and D2 lies parallel to the ridge (a). Down to approximately 1000 m depth, the overall deformation can thus be described as a classical extensional regime as expected on ice divides: extension normal to the ridge, almost no deformation, that is, very small D2  and D2 begin to tilt by rotation around the D1 direction perpendicular to the ridge (b). At approximately 1700 m depth, D2 starts to increase  regime towards the basal regime (simple shear). This is also visible in the increasing obliqueness of D3 away from the vertical (figure 7b versus c) and a further decreasing value of D2  is now approximately 45° inclined (figure 7c). At approximately 2360 m depth, D1 (tensile) finally rotates away from the direction perpendicular to the ridge. D1 (tensile) rotates quickly between approximately 2370 and 2380 m depth (figure 7d) to a NW–SE orientation. Below this D1 (tensile) and D3 (compressive) the axes are both approximately 45° with the basal bedrock plane (figure 7e) and D2 is now tensile, though still very small. This describes the situation as expected for bed parallel shear.The three-dimensional orientations of strain rate eigenvectors are presented in he ridge a. Down tsmall D2  along thhe ridge b. At appue of D2 . At appr, values . D3  from the surrounding macroscopic geometries and balances. The combined evaluation of these data shows that the structural observations can be interpreted as the effects of the transition from vertical compression with transverse extension to horizontal shear. The depth horizons are indicated as grey regions in By the chosen model spin-up, we ensure that the ice sheet geometry corresponds to the current state. This is essential for realistic strain rates at the borehole location, as the main drivers for ice flow are local ice thickness and surface slope. However, our model set-up results in a temperature versus depth profile that deviates from the observation. Nevertheless, to the best of our knowledge, no Antarctic-wide modelling study and free evolving geometry with palaeoclimate forcing has shown to result in realistic present-day geometry as well as realistic temperature–depth profiles as observed at ice core locations. This is the subject of ongoing research (e.g. ice sheet model intercomparison project (ISMIP6) ).a) shows the temperature derived from the modelling results in comparison with the temperatures measured in the borehole. The reasons for the difference between modelled and measured temperatures are threefold. First, the present-day climate forcing does not include variations in accumulation rates or surface temperatures over time. Second, the relatively smooth bedrock topography on the 10 km model grid may hinder the model to generate realistic horizontal flux divergence that influences the amount of downward advection at the EDML location. Last, without the flux correction method applied to the surface mass balance the resulting ice thickness tends to be too large at the EDML site c-axes, which are observed to have an almost random distribution, and to produce SPO of grains. This missing SPO with the long axis of grain elongation directions pointing to various directions in shallow depths has been similarly reported for the Dome Fuji and Dome C ice cores [b,d,e,f), but higher-resolution analyses reveal that highly localized recovery and recrystallization occur already in these shallow depths [c), which is in accordance with the average small change with depth of borehole displacement down to 2000 m depth. The rather large wiggling of the borehole data in this upper depth range could in principle be attributed to changing rheology or temperature; however, the borehole displacement data are rather noisy as expected owing to the small surface velocity. With repeated borehole logging after 10 years, we expect more robust data with a better noise–signal relation.In the upper part of the ice core, the still small strain rates do not suffice to align the ce cores ,46,88. Dce cores . The comce cores . At firsw depths ,81,91. Tw depths ), as prew depths . Flow moUnder these conditions, grain boundary migration dominates the microstructure evolution and masks the deformation habitus of grains ,19. Thisa).In this depth range, the overall deformation yields a classical extension regime as expected on ice divides: extension normal to the ridge, almost no deformation along the ridge and vertical compression b) and simultaneous strengthening of grain elongation perpendicular to the LPO girdle  and along the horizontal plane suggest the dominance of a horizontal extension as described before by Lipenkov et al. [The progressive evolution of a vertical girdle LPO b and simO girdle b,c and av et al. . Grain e3) starts to incline away from the vertical (b), which is indeed reflected in the SPO as the dip of grain elongation becomes inclined from the horizontal to several degrees (f), whereas the borehole was inclined by only approximately 1.5° throughout this region and below down to approximately 1700 m depth (see borehole inclination in red in figure 4a). The microstructure develops aspects of ‘oblique foliation’, typical for shear zones in quartzite rocks [a).Flow model calculations predict that at approximately 1000 m depth the compressive direction , which has also been described for most other deep ice cores ,95,96.(c)c) indicates a transition from dominating triaxial deformation towards horizontal shear. Flow model calculations show that D2 starts to increase  describe the change from the extensional regime typical at ice divides towards an increasing shear contribution. The transition from dominating triaxial deformation towards horizontal shear causes an increase in the dipping angle of grain elongations in vertical sections up to 10°, forming a microstructure close to the ‘oblique foliation’ observed in 3) at approximately 1700 m depth , whereas the tensile component D1 remains approximately constant. This deviating evolution of D1 and D3 is clearly represented by the increasing D2, which develops from slightly compressional towards extensional behaviour in this depth region b left) show a broad, but distinct distribution (cone angle of 45°) with a slight tendency towards double/multiple maxima. This can be interpreted as grains being partly elongated perpendicular to the main compression direction in shear, thus lining up in the instantaneous stretching direction [2 (3) is now approximately 45° inclined. The increasing component of bed-parallel shear readily causes millimetre-scale z-folding by amplifying small undulations, which leads to local tilting of stratigraphic layers (10–15°). RES reflectors fade out in this depth (‘echo-free zone’) owing to the loss of layer coherency [The single maximum LPO along the vertical core axis is fully developed after a sudden final collapse of the girdle between 2035 and 2045 m. This sudden change in the LPO was also detected as a clear reflector in radio-echo sounding (RES) data, which is caused by the dependence of the electromagnetic wave velocity from the crystallographic orientation –100. Grafigure 5irection , and grairection ,19. Thesction [2 . The prioherency  caused boherency .d). However, being approximately 400 m above bedrock, this layer is nearly at the same altitude as the bedrock heights just downstream of EDML, as depicted in figure 4d) at this transition is classically explained by impurity influence on grain size [δ18O taken as the ‘proxy’ for ice types) in some layers seem to allow grain size to increase locally. The difference in these ice types is characterized by varying impurity concentrations. We use a ‘proxy’ for the ice types however, because picking one impurity is not meaningful with our current knowledge. This may be due to the combined effects of several impurities, as suggested by Fitzpatrick et al. [δ18O). Direct microstructural evidence on the microstructural scale, such as impurity accumulation along grain boundaries, is difficult to prove [figure 4e) suggests an increased influence of deformation on grain topology moving the microstructure from a recrystallization dominated one towards a deformation microstructure (see a comparison of end members in figure 4c) as well as a qualitative observation of a closing borehole [The bottom layer of region 4 (approx. 2365–2385 m depth) is characterized by the lower border of the Eemian ice (MIS5.5) layer at approximately 2360–2390 m depth d. Howeveain size –106, whiain size . Just abk et al. . Impuritk et al. ,106. Thito prove ,110. Thito prove ,79. The borehole . To asse1) has left the direction perpendicular to the ridge and rotates very quickly to an along-ridge orientation (d).The boundary zone between regions 4 and 5 at approximately 2380 m depth is to some extent a remarkable layer with respect to the overall deformation predicted by the model: the principal tensile direction (Dentation d.(e)z-fold that may suggest that folding has occurred in the past or on other scales which are difficult to evaluate from ice core information. Possible changes of the predominant deformation mechanism showing microshear deformation have been interpreted from striking microstructure observations  around −10°C, the grain size in the depth range 2385–2450 m does not increase, possibly owing to the high impurity content and shear rate, but increases to very large sizes below 2500 m depth.The top of region 5 (approx. 2385–2405 m depth) is characterized by its stratigraphy, which is well defined, ordered and seemingly straight. This is slightly surprising in contrast to the significantly disturbed layers just above. However, we also observe small but sudden changes in the layer inclination and flattened  pattern ,111). Thperature a around e) are also indicative for high shear rates. Shear shows stronger effects on microstructure as under higher strain rates the balance between deformation-induced and recrystallization-induced effects is changing significantly in favour of deformation (figure 4c).Only below approximately 2405 m depth, we observe strong kink folding of the stratigraphic layers, which is a clear indication for high shear rates in recent times. The single maximum LPO slightly inclined from the vertical  observedfigure 4The bottom of region 4 as well as the top of region 5 represent a very interesting, but complex, boundary zone. We want to emphasize the ambiguous depth of this boundary, in the sense that different parameters differ strongly in the zone between region 4 and region 5: (i) the LPO changes and shows anomalous behaviour between 2365 and 2380 m depth, (ii) grain shapes elongate suddenly between 2385 and 2395 m depth, (iii) grain sizes change at the lower Eemian layer border (2356–2386 m depth) and show (iv) grain growth only from 2520 m depth again, and stratigraphy shows anomalous straight banding between 2385 and 2405 m depth. The processes and predominating deformation regimes in this region are not yet understood as they are strongly punctuated and probably catalysed by strong changes in material properties owing to different impurity contents in glacial–interglacial–glacial ice layering. Further investigations on impurity effects on rheology and microstructure are needed.2  along the ice column. We use an isotropic model to confirm this interpretation by considering the overall three-dimensional flow field driven by ice sheet geometry. By combining several parameters from SPO and LPO, we find indications for the deformation and recrystallization processes being active at the EDML site. We show that it is the balance of both which determines microstructure and possibly flow behaviour.Recent microstructural modelling studies ,19,112 cImpurities can have an impact on the balance between deformation- and recrystallization-induced changes in grain topology. Impurities are postulated to affect recrystallization, by slowing down grain boundary migration through pinning or dragging, and deformation itself by providing dislocation sources. Polar ice is always at a high homologous temperature (Evaluation of the LPO, grain elongation distributions and visual stratigraphy leads to a division into five distinct regions along the core. Here the results are interpreted as the effects of triaxial deformation with horizontal extension changing towards bedrock-parallel shear. This is in good agreement with modelled strain rate trends as well as borehole deformation observations.Down to approximately 1000 m depth, triaxial deformation with vertical compression and horizontal extension clearly dominates. The influence of shear on the grain structure and fabric starts at approximately 1000 m depth and becomes more prominent between 1700 and 2030 m depth, intriguingly observable in the smooth transition between girdle and single maximum LPO and in the borehole geometry. A final collapse of the eigenvalues in a narrow zone between approximately 2030 and 2050 m depth marks the transition to bed-parallel shear. Shear is the dominating deformation down to the base, but is interrupted by a narrow layer with changing conditions, with abnormal LPO leading into a region of high shear activity, with extreme values in grain shapes.Owing to relatively small strain rates at the drilling location on the ice divide, only subtle changes in SPO can be observed at EDML, but we suggest including these analyses in future ice core studies especially in mechanically more active regions such as the forthcoming East Greenland ice core project. These analyses can help to assess the share of ice deformation and basal sliding with respect to transport of ice towards the ocean.


This review deals with phytocystatins, focussing on their potential role as defence proteins against phytophagous arthropods. Information about the evolutionary, molecular and biochemical features and inhibitory properties of phytocystatins are presented. Cystatin ability to inhibit heterologous cysteine protease activities is commented on as well as some approaches of tailoring cystatin specificity to enhance their defence function towards pests. A general landscape on the digestive proteases of phytophagous insects and acari and the remarkable plasticity of their digestive physiology after feeding on cystatins are highlighted. Biotechnological approaches to produce recombinant cystatins to be added to artificial diets or to be sprayed as insecticide–acaricide compounds and the of use cystatins as transgenes are discussed. Multiple examples and applications are included to end with some conclusions and future perspectives. In angiosperms, a broad range of members is found, with extensive species/clade specific duplications leading to 26 members in the grass species Brachypodium distachion [Peptidase inhibitory proteins are a complex group of molecules involved in the regulation of the protein degradation caused by peptidases. The MEROPS database  .Tetranychus urticae detected four different cathepsin-L like enzymes, indicating that they represent the most abundant proteases in the gut lumen that are ultimately incorporated into the mite faecal pellets [C13 CysProt genes (legumain-like) was also identified in the T. urticae genome [CysProt that catalyse the hydrolysis of dietary proteins have been characterized in the digestive tract of several groups of phytophagous arthropods, including coleopterans, hemipterans, homopterans and mites B. The ma pellets . A unique genome  proteolye genome . Their fLeptinotarsa decemlineata [T. urticae mites reared on maize showed significantly higher cathepsin B-like activity than when reared on beans [Expression levels and activity profiles of CysProt in phytophagous arthropods vary through development and depending on the type of plant consumed. Quantitative changes in activity were reported when comparing larvae and adults of the beetle mlineata  and whenmlineata . Midgut on beans . Transcron beans  and thaton beans . The repon beans . The proon beans . Secretion beans . In conton beans .Cystatins, like other proteins involved in plant-pest interactions, include conserved amino acids affecting their biological activity. These residues have been subjected to a positive selection during the course of their evolution to generate cystatin variants with improved inhibitory potency and specificity towards herbivorous ,31. A coC. maculatus gut proteases and substantially delayed its growth and development in feeding-diet bioassays, soyacystatin L was essentially inactive as an insecticide. Soyacystatin N variants containing mutations in essential motifs were constructed. Kinetic inhibitory analysis of mutated variants allowed the identification of a novel soyacystatin N isoform with higher affinity to C1A proteases than the wild type [In 1998, Koiwa et al.  describeild type . These oild type ,56. Likeild type .Kiggundu et al.  identifiL. decemlineata CysProt was assessed by biotinylated forms of SlCYS8 and SlCYS8 variants [L. decemlineata larvae, which showed a differential readjustment of functional protease families after feeding single variants of tomato multicystatin SlCYS8 mutated at positively selected amino acid sites [The binding affinity method used to select PhyCys variants with greater insecticidal activity was already described by Koiwa et al. in 1998 . For thevariants . These tvariants . Howeverid sites .The potato multicystatin PMC is another cystatin composed of eight repeating units, each one capable of inhibiting CysProt . Biochem68→G, mutation adjacent to the conserved QxVxG motif, was better inhibitor for papain and cathepsin B-like activities than the wild type. The HvCPI-1 and its C68→G variant were tested in vitro against the midgut protease activities from L. decemlineata. In consequence, the mutant was selected for potato transformation [68→G cystatin, supporting the improved properties of C68→G substitution against the CysProt of this pest.In barley, several punctual mutants of the HvCPI-1 cystatin were generated and their inhibitory properties against C1A proteases investigated . The varormation . A decreAll these studies underline the complexity of protease/inhibitor interactions in plant-arthropod systems and point out the potential of positively selected amino acids as target sites to tailoring the inhibitory specificity of the cystatins towards CysProt of agronomic significance in crop protection.Plant-herbivore coevolution has demonstrated that both partners have developed sophisticated mechanisms to overcome defences elaborated by each other.Ceutorhynchus assimilis to oilseed rape plants overexpressing OC-I cystatin. Zhu-Salzman et al. [Callosobruchus maculatus due to soya cystatin was only observed in its earlier stages of the development.The impact of dietary PIs in the arthropod depends on their inhibitor properties but also on the arthropod species, on its developmental stage and even it varies within different strains from the same species. Girard et al.  describen et al.  demonstrPhytophagous pests rapidly adjust their battery of digestive enzymes in response to the accumulation of defence compounds, mainly to PIs. Comparisons of gut proteolytic activities in the presence or absence of specific PIs have revealed multiple adaptation strategies to circumvent anti-nutritional effects of diet ,62. AmonL. decemlineata larvae on OC-I-transgenic potato leaves resulted in larger foliage consuming due to the production of OC-I-insensitive proteases as a compensatory response to the nutritional stress [L. decemlineata larvae fed on potato plants expressing the HvCPI-1 C68→G cystatin from barley was probably the result of the metabolic cost associated with the hyperproduction of digestive cathepsin B-like proteases [C. maculatus overcame the growth retardation in its earlier developmental stages caused by dietary soya cystatin by increasing the total proteolytic activity and an enzymatic profile shift toward cystatin-insensitive proteases. Consequently, larvae recovered their feeding habits and growth, even in the presence of the cystatin in the diet. Similarly, the spider mite T. urticae responded to the ingestion of the barley HvCPI-6 cystatin by increasing the expression of both inhibitor-target and non-target proteases [L. decemlianeata larvae in response to feeding on single mutated cystatin variants. Curiously, predatory insects can also adapt their digestive metabolism to the presence of PhyCys ingested by their herbivorous preys. Cystatins from rice and barley independently expressed in transgenic potato plants induced digestive compensation in the natural predators Perillus bioculatus and Podisus maculiventris, respectively, via their herbivorous prey feeding on the plant [Regarding PhyCys, there are some examples describing different physiological mechanisms adopted by insects and acari to avoid the deleterious effects provoked by these PIs. Feeding bioassays conducted with l stress ,70. In croteases . Zhu-Salroteases  demonstrroteases . By usinroteases  demonstrhe plant ,72,73.C. maculatus larvae reared with a diet enriched in a cystatin revealed a substantial rearrangement in its proteome [The ingestion of a PI not only induces changes on the pest digestive proteases but also on the global gut content proteome. Proteomic data of the intestinal tract of the coleopteran proteome . These fSome biotechnological strategies have been developed to analyse the effect of a known molecule with insecticide/acaricide properties on phytophagous arthropods. One of them is the production and purification of the molecule of interest as a recombinant product in heterologous systems. The alternative approach is the plant transgenesis through the integration of a desirable gene in the genome of a crop species to confer resistance to a specific pest.Nicotiana benthamiana, allows the isolation of the recombinant protein for further studies or applications [Recombinant cystatin production using biological systems such as bacteria, yeast or plant cultures, either to perform in vitro inhibitory assays, to be added into artificial diets or even to be sprayed as defence compounds, constitutes an important biotechnological approach. The heterologous expression of PhyCys fused to specific tags to be easily purified from microbial and yeast cultures, or their transient expression in ications ,75. Mostications ,38,76,77Another application of recombinant cystatins is based on the fact that some insects, at least certain laboratory strains, may feed on artificial diets. Thus, feeding bioassays can be performed by adding recombinant cystatins to diets to evaluate insect susceptibility. Many reports have tested differences in insect growth, development and survival as well as alterations in digestive physiology in response to a cystatin-artificial diet under confined conditions .M. persicae and Acyrthosiphon pisum, and two mites, T. urticae and Brevipalpus chilensis, which rely on CysProt for digestion [L. decemlineata and Diabrotica virgifera, whose major digestive proteases also belong to the C1A peptidase family [D. virgifera resulted more susceptible to be inhibited by barley cystatins than the enzymes from Colorado potato beetle. The inhibitory profile indicated that cathepsin L-like activity of L. decemlineata was high inhibited by barley cystatins than the B-like. Both activities were similarly reduced in the case of the western corn rootworm. Recombinant HvCPI-6 was the strongest inhibitor and reduced about 70% and 50% cathepsin L- and B-like activities in L. decemlineata, respectively, and almost produced a complete inhibition of both activities in D. virgifera extracts. The cystatins HvCPI-1, -2, -3 and -5 also inhibited both cathepsin activities in extracts from both coleopterans whereas HvCPI-8 only reduced the cathepsin L-like activity of Colorado potato beetle. No significant inhibition was detected when HvCPI-4, -7, -10, -12 and -13 proteins were used  after purifying the 13 recombinant cystatins (HvCPI-1 to -13). First, in vitro inhibitory experiments were performed against protein extracts from two aphids, igestion ,38. Now,e family . Proteinere used .The whole set of inhibitory results obtained with the recombinant barley cystatins and the protein extracts from pest species demonstrated an inhibitory specificity of the different PhyCys against C1A proteases from different arthropod origin. This specificity is essential to select the best inhibitor for controlling each arthropod species, or even a particular strain, in an integrated pest management program. Our findings also indicate that the HvCPI-6 cystatin resulted especially efficient, which has been confirmed using artificial diets  and afteBacillus thuringiensis toxins to control insect pests and reduce insecticide spray practises supports the usefulness of crop biotechnology [T. urticae fed on Bt-plants is able to accumulate higher levels of Cry toxins than that expressed by transgenic plants without suffering any alteration in their behaviour [M. persicae that fed on Bt or non-Bt plants, although Bt toxin residues have not been found in their guts [The experience of more than twenty years of commercialization of Bt-crops expressing chnology . Neverthchnology ,81. For ehaviour . Similareir guts . In thisArabidopsis, potato and taro have been generated with the aim of controlling insects and acari whose digestive physiology is mainly based on CysProt. As is shown in 68→G have been transgenically expressed in Arabidopsis, potato and maize to determine how they affect insects and mites. In addition, Arabidopsis and taro cystatins and potato multicystatin have been ectopically expressed in poplar and solanaceous species to analyse their inhibitory properties against insects.There are many examples of transgenic plants expressing cystatin encoding genes as well as engineered cystatin genes with improved potency or specificity to control pests . NumerouT. decemlineata after feeding on potato plants independently expressing OC-I, OC-II and HvCPI-1 cystatin [M. persicae has been also used to perform bioassays with potato, oilseed rape and eggplant expressing the OC-I or its mutated form OC-IΔ86 [M. persicae after feeding Arabidopsis expressing HvCPI-6 [From the insect side, coleopteran species are probably the most frequently employed pests to perform bioassays with cystatin-transgenic plants. Among them, many studies highlighted changes in the digestive physiology, performance and adaptive effects of the Colorado potato beetle cystatin ,73,84,85cystatin ,86,87. T OC-IΔ86 ,90,91,92 HvCPI-6 . In addiFrankiniella occidentalis.To avoid pest adaptation to PI-plants, the gene pyramiding approach by stacking various defence genes in a transgenic plant has been developed as an excellent biotechnological method. Besides, the creation of hybrid PIs by combining PI genes or derived partial sequences may enhance their activity or increase the potential number of interactions in the target pest . PlastidT. urticae, an important polyphagous pest that feeds on more than 150 crop species and presents a great record of pesticide [Arabidopsis resistant plants expressing HvCPI-6 cystatin showed a significant reduction of the mite cathepsin L- and B-like activities. In parallel, the development and reproductiveperformance of mites was negatively affected [T. urticae infestation by co-expressing two barley proteases inhibitors (the HvCPI-6 cystatin and the trypsin inhibitor CMe) in Arabidopsis plants corroborated the impact of the inhibitors on the endogenous mite peptidase activities and on the mite survival. Interestingly, double transgenic plants showed significantly less damaged leaf area than plants from single transformation events and much less than non-transformed controls [The importance of PhyCys to combat phytophagous tetranychid mites is remarkable. Few approaches have been useful to control the two-spotted spider mite esticide . Howeveraffected ,71. The The widespread use of transgenic plants with cystatin alone, stacked with other genes, or with engineered cystatin specifically targeting pest digestive proteases has demonstrated its potential for pest control.Many advances have been achieved on the characterization and function of PhyCys in different physiological processes of the plant involving the regulation of endogenous or heterologous proteases. Their defensive function has pushed researchers to consider PhyCys as proteins of particular value with big potential to be integrated as a new tool in Pest Control Management. Most of the strategies developed up to now have been based on their transgenic expression in plants. Nevertheless, some biochemical characteristics of cystatins, such are their small size or their stability, have redirected the interest for their production as recombinant molecules. The large-scale production of PhyCys has already demonstrated their utility as nutraceuticals, food additives or stabilizers of other recombinant proteins . Probabl


Bacillus subtilis and Staphylococcus aureus are known to be inhibited by elevated iron concentrations. The work herein reports a Km (coproporphyrin III) for S. aureus ferrochelatase of 1.5 µM and it is shown that elevating the iron concentration increases the Km for coproporphyrin III, providing a potential explanation for the observed iron-mediated substrate inhibition. Together, structural modelling, site-directed mutagenesis, and kinetic analyses confirm residue Glu271 as being essential for the binding of iron to the inhibitory regulatory site on S. aureus ferrochelatase, providing a molecular explanation for the observed substrate inhibition patterns. This work therefore has implications for how haem biosynthesis in S. aureus is regulated by iron availability.The majority of characterised ferrochelatase enzymes catalyse the final step of classical haem synthesis, inserting ferrous iron into protoporphyrin IX. However, for the recently discovered coproporphyrin-dependent pathway, ferrochelatase catalyses the penultimate reaction where ferrous iron is inserted into coproporphyrin III. Ferrochelatase enzymes from the bacterial phyla Firmicutes and Actinobacteria have previously been shown to insert iron into coproporphyrin, and those from  However, bacterial species in the Firmicutes and Actinobacteria Phyla  have been shown to utilise a ‘coproporphyrin-dependent’ pathway  iron–sulphur cluster in this region, which is not present in the S. cerevisiae enzyme. This cluster is ligated by one residue in the core of the protein (Cys 196) and three in the extended C-terminal region , and at the time was thought to be responsible for anchoring the extended C-terminal to the rest of the monomer, and was also suggested to be indirectly involved in stabilising the dimer form of the protein ) were fitted to the Michaelis–Menten equation using non-linear regression. Ki,app values were calculated by fitting v vs. [inhibitor] data to a single-binding site model described by eqn (1)  is the inhibitor concentration, Ki,app is the apparent inhibition constant defining the inhibitor concentration that elicits 50% of the total inhibition, and n is the Hill coefficient.Purified CpfC was assayed using coproporphyrin III (Frontier Scientific) as the porphyrin substrate and ferrous iron as the inserted metal ion. Coproprophyrin III powder was solubilised using a few drops of ammonium hydroxide and was then diluted in assay buffer . Coproporphyrin III stock solutions were quantified in 0.1 M HCl spectrophotometrically using an extinction coefficient of M−1 cm−1 . FerrousM−1 cm−1 . Iron chM−1 cm−1 . Activit eqn (1) , where nS. aureus CpfC was generated using the RaptorX server  was varied  values for wild-type and E271S CpfC as 66 µM and 11 mM, respectively, highlighting a dramatic loss of sensitivity to Mg2+ for the mutant enzyme. Together, these data strongly indicate that in wild-type S. aureus CpfC, the binding of Mg2+ or Fe2+ at this metal site results in enzyme inhibition. In addition, the data in S. aureus CpfC including iron levels well above the inhibitory threshold for the wild-type enzyme  were of a similar magnitude to those presented herein. We report the first measurement of Kapp (coproporphyrin III) for S. aureus CpfC as 1.5 ± 0.2 µM, which is slightly lower than previous measurements of 10.5 and 7.8 µM for CpfC enzymes from M. tuberculosis and B. subtilis, respectively [Kapp (Fe2+) is comparable with a previous estimate of 0.6 ± 0.1 µM for this enzyme [Preliminary kinetic analysis of ectively . While aKm for coproporphyrin III  at the inhibitory site. Substitution of this glutamate residue with serine resulted in two dramatic outcomes: substrate inhibition by Fe2+ was lost and Mg2+ was no longer a potent inhibitor of metal chelation    that is rin III)  and E271rin III) , which sS. aureus to tightly regulate how this nutrient is used within the cell. The current work is consistent with a model whereby at very low Fe2+ concentrations ferrochelatase is able to insert iron into coproporphyrin III, whereas at higher iron concentrations flux through this step of the pathway is down-regulated. There may be many reasons for this substrate inhibition, including avoiding accumulation of Fe-coproporphyrin III, or perhaps once the haem requirements of the cell are satisfied Fe2+ is made available for other processes (e.g. Fe–S cluster assembly). Future studies on bacteria that perform coproporphyrin-dependent haem biosynthesis are required to reveal the hierarchical channelling of iron for different uses in the cell, which will be important during infection  and growth in microbial communities (B. subtilis).It is well-known that there is fierce competition for iron both during infection resulting from nutritional immunity , and dur