Saucer-shaped intrusions of tens of meters to tens of kilometres across have been observed both from surface geological mapping and geophysical observations. However, there is only one location where they have been reported to extend c. 100 km laterally, and emplaced both in a sedimentary basin and the crystalline basement down to 12 km depth. The legacy BABEL offshore seismic data, acquired over the central Fennoscandian Shield in 1989, have been recovered and reprocessed with the main goal of focusing on this series of globally unique crustal-scale saucer-shaped intrusions present onshore and offshore below the Bothnian Sea. The intrusions (c. 1.25 Ga), emplaced in an extensional setting, are observed within both sedimentary rocks (<1.5 Ga) and in the crystalline basement (>1.5 Ga). They have oval shapes with diameters ranging 30–100 km. The reprocessed seismic data provide evidence of up-doming of the lower crust (representing the melt reservoir) below the intrusions that, in turn, are observed at different depths in addition to a steep seismically transparent zone interpreted to be a discordant feeder dyke system. Relative age constraints and correlation with onshore saucer-shaped intrusions of different size suggest that they are internally connected and fed by each other from deeper to shallower levels. We argue for a nested emplacement mechanism and against a controlling role by the overlying sedimentary basin as the saucer-shaped intrusions are emplaced in both the sedimentary rocks as well as in the underlying crystalline basement. The interplay between magma pressure and overburden pressure, as well as the, at the time, ambient stress regime, are responsible for their extensive extent and rather constant thicknesses (c. 100–300 m). Saucer-shaped intrusions may therefore be present elsewhere in the crystalline basement to the same extent as observed in this study some of which are a significant source of raw materials. A major focus was therefore on imaging the Moho and its geometry and crustal thicknesses across various tectonic domains to gain information that could indicate collision or accretion of different crustal units or micro-continents during the Paleoproterozoic-Archean eras. Consequently, the original processing was geared towards imaging the lower and middle crust, leaving a major portion of the top 10 km less well imaged. This also hindered connecting the upper crust with deeper structures and past geological processes that may have affected the whole crust. The BABEL project was a significant collaborative geoscientific project in Europe seeking evidence that indicated that plate tectonic processes were active already in the Paleoproterozoic2. Additionally, intriguing features such as 100-km scale dolerite intrusions in the upper crust3 were observed and tentatively interpreted. The BABEL data have been revisited earlier by others6, however mainly for reinterpretations using the original processed data (stacked or migrated sections) when new geological and/or geophysical data became available; for example, gravitational7 or magnetic data8. The BABEL data are an extraordinary resource and have tremendous potential for improvements and for revising our understanding of past tectonic processes.Almost three decades ago in 1988–1989), a research team from Denmark, Finland, Germany, Sweden and the United Kingdom, through the BABEL  project, acquired 2,268 km of marine reflection seismic data and wide-angle refraction data in the Baltic and Bothnian Seas  acquired in the Bothnian Sea penetrate Paleoproterozoic rocks, two major faults and subsidence-controlled depressions filled with Mesoproterozoic and younger sedimentary rocks. By convention the Mesoproterozoic sedimentary rocks in the Fennoscandian Shield are referred to as Jotnian sandstones Fig. . Preserv15 and the Mesoproterozoic sediments were deposited unconformably on this peneplain in alluvial, fluvial or aeolian environments. In places, these units are overlain by Neoproterozoic, possibly Ediacaran, to Lower Palaeozoic sedimentary rocks  occur in five separate clusters with slightly different ages, ranging between 1.27–1.25 Ga, and represent the largest manifestation of non-orogenic mafic magmatism in Europe21. They intruded both the Paleoproterozoic crust, as well as the Mesoproterozoic sedimentary rocks, providing an upper age limit for deposition of the latter. Individual intrusions range in thickness from less than ten metres to about one kilometre and in the three central clusters they are mainly present as horizontal to gently dipping saucer-shaped or oval-shaped intrusions, whereas dykes dominate the clusters to the north and south19. The CSDG is considered to the product of the most significant Mesoproterozoic extensional event in the Fennoscandian Shield. Isotope signatures (Hf and Nd) indicate an asthenosphere source for the magma that interacted with the subcontinental lithospheric mantle upon ascent. It has been inferred that the long-lived magmatism is related to either a mantle plume (hot spot) or discrete extensional events behind an active continental margin21.Until the onset of the rapakivi magmatism, the Paleoproterozoic crust was uplifted and eroded close to the present-day levelscks Fig. . These p22. Until this study, the processes producing the emplacement of these intrusions, linking shallow and deeper structures observed in the seismic data, have been left unexplained. The improved seismic reprocessing results presented here are key in helping elucidate the intrusion process.Along BABEL line 1, the CSDG intrusions generate strong, concave reflections and their presence below the Bothnian Sea is further indicated by the correlation with their occurrences at the Swedish and Finnish coasts in addition to direct observations on the sea floorAll the reprocessed seismic lines show improved reflectivity from near surface down to the Moho, as well as in sub-Moho reflections, Figs –S4 due t22 and exposed on the sea floor. A footprint of the fault (dyke filled) is also visible in the bathymetry data as well as shipborne magnetic data22 acquired during the same time as the seismic data were acquired. The sea floor suddenly drops by more than 50 m from south to north due to this faulting. Smaller faults can also be observed in this region based on discontinuities in the sea-floor reflection. The three sets of reflections (S1–S3) project to, or near, the surface at the margins of the Meso- to Neoproterozoic sedimentary basin and form a concentric feature (hence referred to as saucer-shaped), clearly observed in the sea-floor morphology  are observed from CDP 8400 to 12700 in the 1.5 km to 12 km depth range. These features are saucer-shaped (at least in the 2D seismic sections) with a sharp and steep seismically transparent zone intersecting them around CDP 9300 Figs  and 3a. ogy Fig. . We argu22 and Axberg16 on shallow marine seismic data. At depth, a highly reflective lower crustal up-doming is well imaged (L1) under which a clear sub-Moho reflection (SM1) dipping to the south is observed. Similarly, a sub-Moho reflection is also observed in the parallel line 6 (SM2), although deeper (c. 80 km) in the upper mantle. The lower crust is also reflective along line 6 and up-domed on its northernmost part (L2), as observed along line 1. Interestingly, along both lines 1 and 6, nearly seismically transparent areas between 5 to 12 km depths (T1 and T2) are present, with reflectivity increasing substantially below these zones.On the southern half of line 1, between CDP 3600 and 5700, a basin  is observed (B1) that was previously masked in the original processing. The existence of the basin is known from the work of Winterhalter24. The eastern part of line 7 shows a higher crustal reflectivity (even distinct ones such as R2) than the western and central parts of the line.Along line 7, two mainly opposite dipping reflectivity patterns are observed. On the westernmost part of the line, the dipping reflectivity  extends to near the surface and appears to be flattening out in the lower crust at around 35–40 km depth. Where lines 1 and 6 intersect line 7 Fig. , a good The most striking feature along line C is an increased band of southeast-dipping reflectivity and a strong southeast-dipping sub-Moho reflection (SM3) extending down to about 80 km depth Fig. . A set o3 and have been interpreted as intrusions in other publications too7. The high reflectivity of the anomalies leads us to this interpretation as well, since we have a high-impedance contrasts between the intrusion and the surrounding rocks because of their density and velocity difference. Their thickness (50–200 m) is also a further support for their strong amplitude. However, the process resulting in their emplacement both in the sedimentary rocks and in the crystalline basement over a lateral extension of 100 kilometres has been a matter of debate. A general model of emplacement was proposed by Korja et al.7. Several intrusions crosscut the present margin of the sedimentary rocks (<1.5 Ga) as evidenced by the multiple (at least four), inward dipping, oval intrusions on land that are mainly found in the crystalline basement , which in the end led to several connected intrusions28. Emplacement of these magmas in the upper crust was associated with caldron subsidence that subsequently and successively was filled with Mesoproterozoic and younger sediments. The up-doming at the Moho  in the offset interval of 140–220 km. This effect has also been observed in other regions in the world31, and interpreted as a layered intrusion in the lower crust in the form of individual sills that may have formed magma chambers for the volcanic activity at the surface32.The saucer-shaped intrusions are interpreted to result from the same process, i.e. asthenosphere upwelling, as for the rapakivi magmatism although separated by nearly 300 Myoho Fig.  with a q36. Presently, the largest areas covered by these sedimentary rocks are in the Baltic and Bothnian Seas and Gulf of Bothnia where they are underlain to a large extent by rapakivi intrusions37. At least in the Baltic and Bothnian Seas these intrusions also coincide with the 1.27–1.25 Ga CSDG19, both emplaced in extensional settings.While the Meso- to Neoproterozoic sedimentary rocks are only preserved locally, mainly in fault-controlled grabens, they probably covered large parts of the Fennoscandian Shield at the time of depositionet al.7, we suggest the following scenario for the formation of the dyke and sill system in the crust. The CSDG magma, with an asthenosphere source21, intruded through existing zones of weakness in the lithosphere, such as faults and shear zones that developed or were reactivated during extension related to the rapakivi magmatism. Through these zones enormous amount of melt migrated as dykes, and later as sills, at different levels in the upper crust. Thus, a network of several dyke zones originating from different parts of the underlying thermal anomaly is expected. The seismically transparent zone crosscutting reflections S1–S3 in the seismic section represents eventually such a network of dykes. When the pressure of the overburden and magma was nearly equal, the feeder dykes  with their peripheral areas connected to the saucer-shaped intrusions exposed on land , explaining why the saucer-shaped intrusions on land are mainly encountered in the crystalline basement. The fact that the sill reflectors display a general gentle dip towards the zone of dykes and faults (crosscutting transparent zone) may be a further indication that the fault zone and those near the shoreline must have been active during and even after the emplacement of the sills.A major fault, located offshore close to the Swedish coast, was initiated and/or reactivated during emplacement of the saucer-shaped intrusions, displacing the dykes and sills and their Mesoproterozoic sedimentary host-rocks for over 2 kmThe saucer-shaped appearance of the intrusions is less likely to be due to post-emplacement tectonics, i.e. deflation of the underlying and feeding magma chamber, because (1) it would require deflation of many individual magma chambers at different levels, or (2) deflation of a hot-spot-scale to form such an extensive  saucer-shaped intrusions, which would have resulted in a massive LIP-scale melt; an interpretation, which is difficult to argue for with the current evidences available on surface geology, and (3) deflation would not suit presence of overlapping and cross-cutting sills, which have different center points and curvature.40, several reasons led us to reject this scenario. First, the intrusions are not only confined to the sedimentary basins, on the contrary they are mainly emplaced in the crystalline basement. These inward dipping intrusions show undulated contacts as opposed to those found in the sedimentary rocks. Second, there are smaller saucer-shaped intrusions onshore that do not follow the outline of the subsided basin along seismic line 1. Third, the 1.27–1.25 Ga CSDG is also emplaced as steeply dipping dykes, both in the sedimentary rocks and in the crystalline basement. Sedimentary rocks may have covered the latter at the time of the CSDG emplacement. These points suggest a multiple batch, nested, emplacement mechanism unrelated to basin formation with the intrusions connected to one another as a multi-set of 20–100-km wide saucer-shaped bodies at different levels in the upper crust. Thus, the observed saucer-shaped intrusion along BABEL line 1 is part of a nested intrusion system, which was fed by a magma chamber in the lower crust/upper mantle as indicated by the L1/SM1 features  might support a generic relationship between the two, as has been suggested elsewhereet al.28 suggested a dynamic emplacement process characterized by inflation and deflation cycles. Whereas, Galland et al.41 used modelling results to conclude that layering controls the formation of these types of intrusions and that the ones emplaced at deeper levels have larger diameters. This model is consistent with the data presented in this study, where the deeper intrusions have far larger lateral extent than the ones at shallower levels.Different mechanisms have been proposed for emplacement of saucer-shaped intrusions. For the Golden Valley sill in South Africa, Polteau 42. Magmatic layering in the up-to 350 m thick intrusions implies that the bodies cooled slowly owing to heat generated by repeated and stacked intrusions, allowing time for development of the layered structures. It has been suggested that stacked saucer-shaped intrusions may form by magma that moves from deeper intrusions that become steeper as they propagate and act as feeder dykes for the shallower bodies43. This scenario is often depicted in a 2-dimensional schematic illustration  saucer-shaped intrusions with the same lateral and depth extent (>100 km) as observed in this study can be found in the crystalline basement elsewhere. Some of these intrusions like those in South Africa are significant resources of critical raw materials such as Fe, Co, Cr and Ni. For example, the saucer-shaped intrusions of this study onshore were partly mined for their iron content in the past. (2) Legacy crustal-scale seismic data are valuable and should be kept safely with adequate documentations for reprocessing as they may provide unprecedented images for new knowledge and geological understanding that may be impossible or require high cost for obtaining through new acquisition work.23. Table 23, excellent quality seismic data were acquired with minimum water-wave noise and noise from other ships passing by during the survey. Much of the reported issues were associated with the used equipment. For example, defective depth controller, power failures or autopop of the airgun46.For the seismic data acquisition year 1988–1989), the S.V. Mintrop vessel from Prakla-Seismos was used. The ship towed an array of 42 airguns with a total volume of 120.6 litters at a depth of 7.5 m. A 3-km-long hydrophone streamer with a receiver group interval of 50 m was towed behind and used for seismic data recording. The streamer consisted of 60 groups of 64 hydrophones placed at a depth of 15 m in the water. To improve spatial resolution, different shot spacings were used for different lines. Shots along lines 1, 7 and B had 75 m spacing and a recording length of 25 s while along lines 6 and C, a shot spacing of 62.5 m and a recording length of 23 s or 22 s, respectively was used. Nonetheless the sampling rate was 4 ms for all the BABEL lines988–1989,TM. First, poor quality shots and receivers (misfires or gun drop-outs) were found and removed from the processing flow.Data were recovered in standard SEGY (Society of Exploration Geophysicists seismic data processing format) thanks to a number of individuals who transferred them from tapes to digital disks and to standard seismic processing format. However, not much information was available in the headers in terms of geometry and acquisition set-ups. A significant amount of time was spent to gather this information in order to use them for reprocessing of the data. For example, some of the files we had in the beginning were corrupted. The first few hundred shots seemed to be fine, but at some point, the traces had no amplitude and the header information was missing. After we figured out that we were not able to recover this lost information, we could luckily find the complete files, redundant files saved, in between all the 87 BABEL files without clear structuring. In order to find the right files, we had to compare the original field record number in the header with the ones in the original observer logs. Furthermore, some of the lines were split up into several files and had to be merged together into one file before we could start with the processing work. Seismic Unix was used for this purpose. Fortunately, we also had the shot coordinates given in an ASCII file in the UKOOA P1 1984 format. Given these locations of the shots and fixed hydrophone arrays and distance from the ship marine-type geometry was set up and used with a CDP (common depth point) spacing of 25 m. A conventional poststack migration was used to carefully process the data and make sure no unwanted noise can be introduced. The reprocessing focused mainly on attenuating water-surface multiples and enhancing the Moho boundary through a careful velocity analysis and noise filtering approach. Most of the processing was done using the reflection processing software GLOBE Claritas4 also showed that FK-filtering on these types of data is in most cases sufficient to improve the shallow features. A carefully conducted velocity analysis linked to NMO  correction helped to image near-surface features. Bandpass filter, coherency filter (FX-deconvolution: frequency space) and FK-filter were applied post-stack to suppress artefacts and noise coming from the pre-stack processing and stacking.During the preparation of the data, we realized an approximately 41 ms delay on the recorded data (estimated using the nearest receiver and a water velocity of 1500 m/s). This delay was compensated using a static shift before the actual processing began. Trace balancing, compensation of spherical divergence, band-pass filtering as well as a predictive Wiener deconvolution and AGC (automatic gain control) (window 3000 ms) were used to reduce the noise and enhance the reflections. Additionally, a set of FK-filtering (frequency-wavenumber) was applied to reduce the multiples. Earlier reprocessing work47 filter preserving 15% of the highest curvelet coefficients at each scale was applied to remove any remaining noise from the data and enhance the coherency of large-scale reflections. This processing step was performed with a MATLAB toolbox developed by Górszczyk et al.48 for the purpose of legacy seismic data processing and employing Discrete Curvelet Transform implementation of Candes et al.49 Finally, a finite-difference migration algorithm, which is based on an implicit 45° migration in the time domain developed by Claerbout50, was used for post-stack time migration using GLOBE ClaritasTM. It provided better results compared with other tested methods such as Kirchhoff or Phase-shift migrations. For the time-to-depth conversion, published wide-angle data were utilized ranging from 5500 m/s near the surface to 7500 m/s at the Moho level around 14 s.Before the migration, a curvelet denoisingAdditionally, different velocities were tested in the upper crust to check the effect on the apparent up-doming of the lower crust along line 1. Figure 23, the new processing scheme differs in a few stages. Different frequency windows were used for the filtering, and no down sampling was performed during the reprocessing thanks to the improved computing power, which was not available when the original processing work was conducted (Fig. In comparison to the former processing workflowSupplementary information


Few land plants can synthesize and accumulate the osmoprotectant glycine betaine (GB) even though this metabolic trait has major adaptive importance given the prevalence of drought, hypersaline soils or cold. GB is synthesized from choline in two reactions catalyzed by choline monooxygenases (CMOs) and enzymes of the family 10 of aldehyde dehydrogenases (ALDH10s) that gained betaine aldehyde dehydrogenase activity (BADH). Homolog genes encoding CMO and ALDH10 enzymes are present in all known land plant genomes, but since GB-non-accumulators plants lack the BADH-type ALDH10 isozyme, they would be expected to also lack the CMO activity to avoid accumulation of the toxic betaine aldehyde. To explore CMOs substrate specificity, we performed amino acid sequence alignments, phylogenetic analysis, homology modeling and docking simulations. We found that plant CMOs form a monophyletic subfamily within the Rieske/mononuclear non-heme oxygenases family with two clades: CMO1 and CMO2, the latter diverging from CMO1 after gene duplication. CMO1 enzymes are present in all plants; CMO2s only in the Amaranthaceae high-GB-accumulators plants. CMO2s, and particularly their mononuclear non-heme iron domain where the active site is located, evolved at a faster rate than CMO1s, which suggests positive selection. The homology model and docking simulations of the spinach CMO2 enzyme showed at the active site three aromatic residues forming a box with which the trimethylammonium group of choline could interact through cation-π interactions, and a glutamate, which also may interact with the trimethylammonium group through a charge-charge interaction. The aromatic box and the carboxylate have been shown to be critical for choline binding in other proteins. Interestingly, these residues are conserved in CMO2 proteins but not in CMO1 proteins, where two of these aromatic residues are leucine and the glutamate is asparagine. These findings reinforce our proposal that the CMO1s physiological substrate is not choline but a still unknown metabolite. Land plants are sessile organisms that have evolved a great variety of strategies to escape from, or contend with, the many kinds of abiotic and biotic stresses to which they may be exposed during their lives. Osmotic stress—caused by drought, saline soils or low temperatures—constitutes the major limitation of agricultural production worldwide . To copered) to form betaine aldehyde hydrate, which in aqueous solution is in equilibrium with betaine aldehyde—the BADH substrate  Rieske and mononuclear non-heme iron centers were built using QM calculations  , ACPYPE e center . The vale center  was usedmodelled . The Rosmodelled .SoCMO2 model with AutoDock Vina  Rieske and mononuclear non-heme iron centers, which correctly predicted the geometries found in X-ray structures of oxygenase enzymes with the same kinds of iron centers.Given that to date no three-dimensional structure of any CMO protein has been reported, to analyze structural features that could provide insight into the possible differences in substrate specificity between CMO1 and CMO2 proteins, we constructed and analyzed homology models of the SoCMO2 model because this is the best characterized CMO to date. This model was made without the putative chloroplast peptide signal, which was proposed to consist of 60 residues [SoCMO2 model was the X-ray structure of a bacterial Rieske-mononuclear non-heme dioxygenase from Rugeria sp. (PDB: 3N0Q), which has only 24% identity with SoCMO2; other Rieske/mononuclear non-heme oxygenases have even lower identity with SoCMO2. This low degree of identity between the protein model and the protein being modeled made very difficult the construction of a good quality model. Nevertheless, after following the steps described in the Methods section, we obtained a SoCMO2 homology model that complied with the expected functional requirements for a Rieske/mononuclear non-heme oxygenase enzyme. As the first SoCMO2 model obtained with MODELLER was not satisfactory, we improved its quality by repeated MD simulations. A comparison of the final SoCMO2 model with the initial one is shown in We started building the residues , and witresidues . TherefoSoCMO1 sequence was not yet available, we then followed the same procedure to obtain the Arabidopsis thaliana (AtCMO1) homology model using as template the, SoCMO2 model. The AtCMO1 model was constructed from residue 58 to residue 422, eliminating the N-terminal region that contains a putative signal peptide (47 residues) and an additional 11 residues that were predicted to be disordered. When we had a reasonable good AtCMO1 model, we became aware that the SoCMO1 sequence had been reported and so we decided to use this AtCMO1 model as template to make the SoCMO1 3D-structure prediction. For the same reasons as before, only residues 103 to 426 of the complete SoCMO1 sequence (426 residues) were included for the model. Considering this region, AtCMO1 and SoCMO1 share a 63% of sequence identity.As an example of CMO1 proteins, and because at the time that we were doing these experiments the SoCMO2 amino acid sequence (XP_021849509), the AtCMO1 amino acid sequence (NP_194718) and the SoCMO1 amino acid sequence (XP_021866412) were the first retrieved when using the SoCMO2, AtCMO1 and SoCMO1 homology models, respectively, indicating that the models indeed can accommodate the respective sequences better than the sequences of any other protein in the NCBI RefSeq database. The results of the validation of the homology models shown in Fig 6D) show the absence of abnormally strained, high-energy residues, and that the overall energy of the models was negative.Analyzing the three homology models with the Rd.HMM protocol  we foundSoCMO2 model. Also, the carboxyl group involved in the transfer of electrons between the iron centers in the Rieske/mononuclear non-heme oxygenases [SoCMO2 and SoCMO1 models is shown in In the three homology models, the residues involved in iron coordination in the Rieske and mononuclear non-heme centers are properly positioned and the distance between them was similar to the one found in the known crystal structures and the appropriate one for the transfer of electrons from the Rieske to the mononuclear non-heme iron center, as exemplified in ygenases , which bSoCMO2 model (SoCMO1—as well as in that of the AtCMO1 model (not shown)—there are no negatively charged or aromatic residues. In SoCMO1, Glu346 is Asn327, Tyr295 is Leu275, and Phe301 is Leu281 . We selected the best pose —are conserved in every known CMO2 sequence, while the residues at equivalent positions in SoCMO1—Leu275, Leu291 and Asn327 (SoCMO1 numbering)—are also highly conserved in all known CMO1 sequences  or tyrosine (in S. penelli and S. lycopersicum). Also, only in these three enzymes Asn327 has been changed for a serine residue. The conservation of such different active site residues within the two kinds of CMO enzymes strongly suggests differences between them in substrate specificity. In addition to these active-site residues and to those involved in coordinating the mononuclear non-heme iron, there are several other residues totally conserved among the CMO enzymes in both the Rieske and mononuclear non-heme iron domains , which suggests that, although it is not needed for the oxygenase activity, it may be important for substrate specificity, not only of the CMO2 but also of the CMO1 enzymes.Interestingly, three of the residues suggested by the equences . Out of  domains . Their lsine qua non condition for the plant to become a GB-accumulator. But the current experimental evidence obtained with wild-type and transgenic plants suggests that the acquisition of the ability to synthesize GB by evolving a BADH activity should have been accompanied by other adaptations, such as the gain of a significant CMO activity. In the present work we provided phylogenetic and structural evidence of how the CMO activity might have evolved in some of the CMO proteins.During evolution of land plants some of them gained the ability to synthesize the osmoprotectant GB, which allowed their growing in very adverse environments where saline soils are prevalent, like in coastal highly saline lands, or in dry or cold climates. This metabolic trait confers a great adaptive advantage not only to halophytes plants, but also to mesophytes, which may experience sporadic episodes of water deficit or freezing temperature. As have been previously shown , the chaCMO1 gene duplication event that seems to have taken place early in the evolution of the Amaranthaceae family. Although we found other episodes of CMO gene duplication in other plants, these events seem to be more recent, as suggested by the few residues that have been changed in the resultant proteins, and probably they did not give rise to new functions. CMO2 proteins clearly evolved at a much higher rate than CMO1 proteins, which led to the formation of a new clade within the CMO subfamily. Of the two CMO protein domains, the mononuclear non-heme domain, where the active site is located, is the one that diverged faster. Moreover, the active site residues that probably are important for determining substrate specificity—as indicated by the homology models and docking simulations—are different in CMO1 and CMO2 enzymes, which strongly suggests functional differences between them. Based on the kind of these active site residues—aromatic and glutamate in CMO2 enzymes versus glutamine and non-polar, non-aromatic in CMO1 enzymes—, we think that only CMO2 are bona fide choline monooxygenases, i.e. their physiological activity is choline monooxygenase, while most of the CMO1s, including those of the Amaranthaceae species, are not. Indeed, our results hint at the possibility of CMO1s acting on aromatic substrates. It is probable that CMO1s retained the original and so far unknown physiological activity of the ancestor, an activity that seems important for the plant given the conservation of the CMO gene in every terrestrial plant of known genome. Interestingly, in the fungal and bacterial CMO-like enzymes the active site residues at the equivalent position of the ones that we propose in this work to be characteristic and possibly determinants of the substrate specificity of the CMO2 enzymes—Tyr295, Phe301 and Glu346 (SoCMO2 numbering)—are changed to non-aromatic, non-charged residues identical or similar to the ones in CMO1 proteins, suggesting either that choline is not the substrate of none of these enzymes or that the reaction that they catalyze is different than the one catalyzed by CMO2 enzymes and requires a different mode of binding of choline at the active site. Unfortunately, none of the plant CMO1 or of the bacterial or fungal CMO-like proteins have been biochemically characterized as yet, so their substrate specificity and the reaction that they catalyze are unknown. The aromatic nature of the tyrosine residue at position 281 (SoCMO2 numbering) is also conserved in the CMO-like proteins, which suggest that this aromatic residue is characteristic of the cd08883 domain and may be important for their general function. Regardless of their specificity, these findings suggest that photosynthetic eukaryotic CMO1 proteins and fungal CMO-like proteins evolved in parallel from an ancestral bacterial CMO-like protein, forming independent protein subfamilies with similar architecture and putative catalytic properties, which will be analyzed in a forthcoming work. On the other hand, it is also possible that CMO1 proteins do have a low CMO activity. Even more, this activity might have been present in the ancestral CMO1 protein. To date, most of the experimentally characterized enzymes exhibiting CMO activity belong to the CMO2 clade: spinach [A. caudatus [The results of our phylogenetic analysis show that plant CMO proteins are monophyletic and clearly form two clades indicating the existence of two kinds of CMO proteins: CMO1 and CMO2. Limited phylogenetic studies have been previously reported ,19,57 an spinach ,14,58, s spinach , and A. caudatus . Only a CMO gene copies was free to undertake multiple mutations, which, under the strong pressure of the osmotically stressed environment, eventually lead to the acquisition and fixation of the CMO activity in these proteins. The low number of known CMO1 sequences from the Amaranthaceae family (just three sequences) contrasts with the higher number of known CMO2 sequences from the same family (17 sequences). This numerical discrepancy can be explained by the few known genomes of Amaranthaceae species to date (only those of the same three species) and by the fact that the CMO2 gene sequences were obtained from cDNA when the plants were subjected to osmotic stress, indicating that in this family the CMO2 gene, and not the CMO1 gene, is the one up-regulated by these adverse environmental conditions. In these studies, the increase in the levels of the CMO2 gene transcripts and/or CMO2 protein correlated with significant increases in the levels of GB, indicating that this is the protein that exhibits CMO activity and participates in the synthesis of this osmoprotector, at least in the high GB-accumulator species of this plant family. Indeed, osmotic stress response elements for salt, dehydration and cold were found in the CMO2 gene promoter of the Amaranthaceae halophyte, GB-accumulator plant Suaeda liaotungensis [Many plants of the Amaranthaceae family are halophytes that tolerate salty and dry soils, and probably the ancestor that gave origin to this plant family also colonized osmotically stressed habitats. The acquisition of a functional CMO together with the acquisition of a functional BADH, which also occurs in the Amaranthaceae family , grantedungensis .in vivo and that the CMO1 proteins do not. But then the question is, which enzyme produces betaine aldehyde from choline in those GB-accumulators plants that do not have the CMO2 protein and only have CMO1? Antibodies raised against CMO from spinach did not detect any protein in mangrove leaves (Avicennia marina) [Arabidopsis (dicot GB-non-accumulator plants) [SoCMO2; therefore, their results may be explained by the fact that all the other CMO proteins tested against this antibody were CMO1 proteins, which, as shown here, have low sequence identity with the CMO2 proteins. It seems therefore that the likely explanation of these results is the lack of cross-reactivity of the polyclonal anti-CMO2 antibodies against CMO1 proteins, due to the important differences between the two kinds of CMO proteins. Regarding the proposal of the possible existence of homologs of CDH or choline oxidase in land plants, we did not find any in pBLAST searches. It may be possible to hypothesize that another, non-yet described enzyme able to convert choline to betaine aldehyde was recruited for GB synthesis. In this respect, it results interesting that a recent report described a plant oxidase named GB1 that greatly increased the levels of GB when overexpressed in maize and soybean plants [Together, our results prompted us to propose as a hypothesis that only CMO2 proteins have CMO activity  marina) , barley  marina) , sunflow plants) , a findi plants) . Howevern plants . We founn plants , since tS1 Fig(PDF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Cyclic alternating pattern (CAP) is a neurophysiological pattern that can be visually scored by international criteria. The aim of this study was to verify the feasibility of visual CAP scoring using only one channel of sleep electroencephalogram (EEG) to evaluate the inter-scorer agreement in a variety of recordings, and to compare agreement between visual scoring and automatic scoring systems. Sixteen hours of single-channel European data format recordings from four different sleep laboratories with either C4-A1 or C3-A2 channels and with different sampling frequencies were used in this study. Seven independent scorers applied visual scoring according to international criteria. Two automatic blind scorings were also evaluated. Event-based inter-scorer agreement analysis was performed. The pairwise inter-scorer agreement (PWISA) was between 55.5 and 84.3%. The average PWISA was above 60% for all scorers and the global average was 69.9%. Automatic scoring systems showed similar results to those of visual scoring. The study showed that CAP could be scored using only one EEG channel. Therefore, CAP scoring might also be integrated in sleep scoring features and automatic scoring systems having similar performances to visual sleep scoring systems. Cyclic alternating pattern (CAP) in non-rapid eye movement (NREM) sleep is a neurophysiological pattern that can be visually scored by international criteria, and has been described in several conditions over a period of 20 years . Visual CAP analysis has been performed by detection of electrocortical events with regular intervals in a range of seconds during NREM sleep . These eOne limitation for the application of CAP analysis in sleep medicine has been the extra time needed to apply such a scoring system in clinical practice. Namely, the classification of short duration events in sleep EEG like the A phases of CAP is a laborious and error-prone task. Nonetheless, the clinical meaning has been linked to the detection of sleep instability in mild sleep disorders . The effAnother limitation is the concern about the feasibility, accuracy, and reliability of CAP classification independent of sleep stages using a single central EEG lead (C4-A1 or C3-A2). According to Rosa et al. , CAP scoThe main objective of this work was to assess the viability and performance of visual and automatic scoring on a single EEG channel, as suggested by the published CAP scoring rules in Terzano et al. , using rSixteen hours of single-channel European data format (EDF) in eight polysomnographic recordings from four different sleep laboratories with either C4-A1 or C3-A2 channel and with different sampling frequencies  were used. In order to speed up the visual classification process, 2 h of NREM sleep were extracted from each recording, 1 h epochs of NREM from the first half and the other 1 h epochs from the second half of the night, totaling 16 files.Seven scorers  performed visual scoring according to CAP scoring criteria described in Terzano et al. , and twoTwo types of analysis were performed: the first was the time-based agreement of the scorings, where the scorings were compared for every sampling point and the second was event-based, where an agreement was scored whenever 2 events have an intersection longer than 0.5 s. In this report, only the event-based analysis is presented.Following the above example, a specific agreement analysis can be proposed as shown in The total number of events scored by the 2 automatic scoring systems and the 7 visual scorers are presented in We calculated the MA between laboratories . The senAll file averages are reported in This is the first study to show that CAP can be scored using only a single EEG channel. Although the CAP scoring experience of different scorers varied considerably (from newcomer to very experienced), the results were in ranges seen for inter-scorer agreement in similar studies . An inteOur results demonstrated that the automatic analysis of CAP has a clinical application. Because the number of events to be visually scored was usually large , it was necessary to mark the events boundary accurately, otherwise the classification of the events into different A subtypes and the precision of the phase durations, cycles, and alternating conditions could be compromised. There is already some published data on visual CAP scoring agreement with automatic scoring systems ,38 for eMoreover, ambulatory monitoring is increasingly performed in clinical practice and often one EEG lead is the only available EEG information. Our results showed that just one EEG lead was well correlated between human and automatic scoring. Development of automatic scoring will allow greater usage of CAP scoring in clinical practice, particularly when using ambulatory monitoring with few recording leads. Considering the potential large clinical usage of such automatic scoring systems and our encouraging results, there is no doubt that CAP scoring could be easily integrated into conventional sleep scoring. Our findings further suggest that usage of a single EEG channel for automatic scoring could be more refined by using visual analysis of several EEG leads.Some limitations of the CAP analyses, however, have to be addressed, particularly the clinical meaning of the CAP rate. For now, it has only been proposed as a NREM sleep tool to evaluate sleep instability. Probably due to the amplitude criteria, it is not applied to REM sleep. However, the automatic scoring of CAP following the default decisions made by specialists in CAP analyses may assist the users in improving their recognition of NREM sleep. In conclusion, once several NREM sleep disturbances can be assessed by CAP analyses, the automatic scoring system or CAP might be applied for research purposes to better understand sleep phenomena.