Here, we re-investigate the role and nature of cattle husbandry at Neolithic Schipluiden using mortality profiles and stable isotope analysis  of animal bone collagen and tooth enamel. The age-at-death analysis suggests that cattle were managed for both meat and milk production. The δ18O and δ13C analysis of tooth enamel provide evidence that calving spread over five-and-a-half-months, which would have led to a longer availability of milk throughout the year. Cattle were grazing in open, marshy environments near the site and winter foddering was practiced occasionally. The faunal isotopic data also reveal that the high 15N in human bone collagen is more likely to signal the consumption of products from cattle that grazed on 15N-enriched salt marsh plants around the site, rather than a marine-oriented diet. This undermines the previous interpretation of the dietary practices at Schipluiden by showing that human diet in mid-4th millennium BC Rhine-Meuse area was fully “Neolithic”, based primarily on products from domesticates, especially cattle, with some input from wild terrestrial and aquatic resources available in their surroundings, contrary to what has been proposed before. Collating these results demonstrates a high level of investment in cattle husbandry, highlighting the social and economic importance of cattle at the lower Rhine-Meuse Delta during the 4th millennium BC.Schipluiden , the earliest known year-round settlement in the Rhine-Meuse Delta in the Netherlands, is a key site for addressing the nature of Neolithic subsistence in the wetlands of northwestern Europe. A preliminary zooarchaeological study suggested that cattle husbandry was a major activity at Schipluiden. In contrast, stable carbon and nitrogen isotope analyses of human remains from the site indicated a marine-oriented diet, implying that the Mesolithic-Neolithic dietary transition continued well into the mid-4 Bos taurus) and its subsequent spread into Europe caused unprecedented economic, biological, and social transformations in both animal and human history [The domestication of cattle  and carbon (δ13C) isotope values in cattle tooth enamel to investigate cattle diet and calving season at the site. To infer cattle pasture usage and foddering practices, we use stable carbon (δ13C) and nitrogen (δ15N) isotope analysis of bone collagen. Furthermore, we explore the role of terrestrial protein in the human diet by integrating our isotopic dataset from the Schipluiden animal bone collagen with previously published isotopic data from human remains recovered from Schipluiden and the Late Mesolithic sites of Hardinxveld-Giessendam-Polderweg (hereafter Polderweg) and Hardinxveld-Giessendam-De Bruin (hereafter De Bruin), which provides us with a glimpse into the Mesolithic dietary patterns in the region [Here, our objective is to redress this deficiency by investigating cattle husbandry at Schipluiden in detail, through zooarchaeological and stable isotope analyses. Using long bone fusion and tooth wear stages, we analyse cattle mortality profiles to identify slaughter management practices. We use the sequential analysis of stable oxygen  and naked barley (Hordeum vulgare var. nudum) [Sus cf. scrofa) and red deer (Cervus elaphus) were the most commonly hunted species. Fur-bearing animals, namely beaver (Castor fiber), otter (Lutra lutra), and wildcat (Felis silvestris) were identified in low numbers. Sea mammals, such as common seal (Phoca vitulina) and bottlenose dolphin (Tursiops truncatus), made a very small contribution to the mammal assemblage. [Anguilla anguilla), flounder (Pleuronectes flesus), and Atlantic sturgeon (Acipenser sturio)–comprising 97% of the identified fish remains. Freshwater and marine fish were rare (c.3%) [Canis familiaris) skull [The human subsistence strategies have been based on the exploitation of a wide range of natural resources, supplemented by crop cultivation and animal husbandry, which Louwe Kooijmans and Jongste viewed as an “extended broad-spectrum economy” . Archaeo. nudum) . Among a. nudum) . A broad. nudum) ,41. Sheeemblage. . Waterfoemblage. . The iche (c.3%) . A notews) skull . The deps) skull .15N and low δ13C values in eight samples of human bone collagen and the associated zooarchaeological evidence for fishing and fowling [A palaeodietary study on the human remains from Schipluiden suggested that the exploitation of marine resources was substantial, based on elevated δ fowling ,25. Thes fowling .Reconstructing the age-at-death and the sex ratio of livestock in the zooarchaeological record is mostly reliable through standardized observations on tooth eruption and wear stages, as well as biometric data. These methods enable estimating the relative abundance of smaller females and larger males in the adult population and the modalities of slaughtering management which are associated with animal exploitation strategies ,43. Care18O values provides additional insights into the length of calving season, which determines the duration of milk availability for human exploitation [Investigating cattle birth seasonality through modeling of δoitation . Cattle oitation . In tempoitation ,48. Physoitation ,49. Yearoitation .18O) of sequentially sampled tooth enamel bioapatite [18O of meteoric water reaches its highest value in summertime and decreases in the winter [18O values are measured (X0) relative to the tooth enamel–root junction (erj), the inter-individual variability of the birth season within the sampled population can be measured [18O values  and modeled based on the equation proposed in [r) to measure the proximity between the calculated (modeled) and measured δ18O sequence.The birth season of prehistoric animals has been widely investigated through looking at intra-tooth variation in oxygen isotope ratios  isotope values measured in tooth enamel bioapatite are a proxy for the animal’s total diet [18O values provide the seasonal framework to interpret the results. Sequential sampling of high crown herbivore molars can be used to investigate diet during tooth development at an individual scale as well as the seasonal variation of animal diet, grazing environment, and foddering practices [3 plants, the dominant plant type in Europe, have δ13C values between –27.5‰ to –23.5‰ (−25‰ on average) when growing in open environments [13C between tooth enamel bioapatite of consumers and their diet [13C values of about –11‰ in bioapatite. Plants growing in closed forests can yield δ13C values as low as −31‰ due to recycling of 13C-depleted CO2 and reduced photosynthesis due to low light levels, known as the canopy effect [13C values, depending on the degree of canopy density [Stable carbon  value reflects the average δ13C values of the dietary protein during the last years of an individual's life, whereas the stable nitrogen (δ15N) values indicate the protein intake level of an individual in a food web [13C and δ15N values and herbivores bone collagen δ13C and δ15N are about 5.1‰ and 3.5‰ respectively [13C values is about 1‰ [3 plants in open environments would lead to δ13C values of –22.5‰ to –18.5‰ (–20‰ on average) in herbivores’ bone collagen, considering a 5.1‰ spacing between diet and collagen δ13C [13C and δ15N values in bone collagen, whereas freshwater/riverine components will be reflected in low δ13C values compared to the terrestrial ecosystems [15N values significantly (approximately 3‰) compared to other terrestrial resources as a result of salinity and sea-spray effect [Stable carbon and nitrogen isotope compositions of bone collagen are widely used to reconstruct past human and animal dietary patterns e.g. ,8,57,63),57,638,5food web . The enrectively . In carnabout 1‰ . In animabout 1‰  and the gen δ13C . Contribosystems ,70. Salty effect . Therefoy effect ,25.13C values of ca. –12 ± 1‰ are representative of a diet with 100% marine protein component, whereas δ13C values of ca. –20 ± 1‰ mirrors a 100% terrestrial-based diet [In human palaeodietary studies, faunal isotopic reference baselines are used to distinguish between terrestrial versus marine protein consumption. Overall, in human bone collagen δsed diet . The valStudy and sampling of the faunal remains were conducted under the permit no. 2019–10, which complied with all relevant regulations. This permit is granted by the Zuid-Holland Provincial Archaeological Depot in Alphen aan den Rijn, Netherlands, where the assemblage is stored.To reconstruct the cattle mortality profile, we used mandibles with teeth embedded and loose lower teeth (NISP = 138) recovered through hand collecting and wet-sieving (4mm). We distinguished loose first (M1) and second (M2) mandibular molars based on the cervical length (CervL) relative to the anterior width (WA) . We usedGiven that the fusion of long bones occurs within a certain age range , we usedFor the sequential sampling of tooth enamel, we selected only the lower third molars to make the data comparable with previous studies on cattle birth seasonality ,68. We a–root junction. A total of 151 samples were obtained from the eight cattle molars. The distance of each sample from the tooth enamel–root junction was recorded. Sampling was undertaken at the Centre for Isotope Research (CIO), University of Groningen, following the protocol proposed by Balasse [After cleaning the surface of the tooth, sequential sampling of the enamel powder was conducted on the anterior lobe by drilling with a diamond bur, perpendicular to the tooth growth axis. Approximately, 7–10 mg of enamel powder was taken for each sample. Depending on the length of the tooth, 16–22 samples were taken from each tooth, starting from the crown and continuing to the enamel Balasse . Stable Esox lucius, n = 1), and thinlip mullet . All individuals are adults based on their morphology and/or skeletal fusion. Among the cattle remains, we only selected the elements which are smaller than the wild female aurochs from Denmark to ensure that only domesticated animals were analyzed , red deer (n = 6), dog (n = 3), suids (n = 6), sturgeon (n = 4), flounders , eel (n = 1), pike  of 16 cattle metacarpals are between 40 and 71mm, showing a bimodal distribution. The peak of smaller metacarpals (12 out of 16) reflects the higher number of mature females slaughtered .13C and δ18O measurements in enamel bioapatite are plotted in 13C values vary between −13.8‰ and −10.3‰. Using a +14.1‰ 13C-enrichment factor between diet and enamel bioapatite [13C values of –27.9‰ to –24.4‰. The degree of intra-tooth carbon isotopic variation is less than 1‰ in most cases which reflects seasonal changes in the isotopic composition of the animal's diet throughout tooth formation. BOS94 has the highest amplitude (1.4%) and consumed plants with δ13C values between −27.9‰ and −25‰ with the lowest value coinciding with a low δ18O value (−6.6‰).Results from δoapatite , these i18O values vary between −6.7‰ and −1.9‰. The amplitude of intra-tooth oxygen isotopic variation is between 0.7‰  and 1.8‰ (Bos209). All the sampled individuals yielded a sinusoidal δ18O sequence representing at least one complete seasonal cycle, except for Bos170. The periodic cycle measured for this specimen was too short to be modeled; hence we did not use this individual to infer the birth season.The δ18O sequences are shown in X) varies from 34.5 to 54.4 mm, reflecting inter-individual differences in annual tooth growth rate. To remove the influence of this variability, we normalised the locations where the highest δ18O value is measured (x0) to the period , to express the duration of the annual cycle over which all individuals were born (x0/X). These values are distributed between 0.13 and 0.58 (range of 0.45) in five specimens, meaning that the analysed cattle were born within 45% of a yearly cycle (12 months), corresponding to a birthing season of approximately five and a half months at Schipluiden . Red deer returned δ13C values between −21.8‰ and −20.4‰ (–21‰ on average) and suids between −22‰ and −20.8‰ (–21.4‰ on average). In comparison, dogs exhibit average δ13C values higher than cattle by 3.6‰, with values ranging between –18.9‰ and –16.5‰. Atlantic sturgeons display a wide range in δ13C values for anadromous (migratory) fish (−20.1‰ to −14.2‰).Cattle bone collagen show δd −20.2‰ . Bos1738collagen , Schiplu15N values measured in cattle bone collagen are exceptionally high, ranging from 7.5‰ to 11.6‰, with the majority clustering between 8.5‰ and 9.2‰. BOS17407 stands out with the lowest δ15N value of 4.9‰. Red deer and suids display average δ15N values slightly lower than that of cattle (9‰) by ~1‰, representing a clear overlap in the range of measured δ15N values for herbivores and omnivores. Red deer δ15N values range between 5.5‰ and 11.2‰ (8‰ on average). Suids have a range of δ15N values between 6.5‰ and 9.5‰ (8.1‰ on average). δ15N values as high as 12.3‰ to 14.9‰ were measured in dog bone collagen. Sturgeon display the highest δ15N values within the dataset, ranging between 11.7‰ and 14.8‰ (14.1‰ on average). The δ15N values measured in the faunal remains align with expected trophic levels, with fish occupying the highest trophic level, followed by dogs, as carnivores. Omnivores and herbivores show relatively lower δ15N values, however, their elevated δ15N values to what would be expected is notable.The δth millennium BC.The established role of cattle husbandry in Schipluiden, as the earliest known Neolithic settlement in the Lower Rhine-Meuse area, is demonstrated by the dominance of morphologically domestic cattle in the archaeological assemblage . The difAge-at-death analysis suggests sophisticated cattle husbandry in Schipluiden in which animals were managed for meat and milk production. Meat exploitation appears to be one of the central foci of Schipluiden cattle husbandry, through the slaughtering of a large proportion of animals at their peak weight. The limited number of morphologically wild cattle bones may represent aurochs occasionally hunted as a supplemental meat to domestic cattle. Evidence for a high number of adult females in the assemblage based on biometric analysis of metacarpals, together with slaughtering animals in the age class of 6‒8 years indicates milk production. This inference is supported by the abundance of slaughtering “post-lactation” calves between 6–15 months, implicating that a high number of calves were kept alive to ensure milk let-down. Based on the fusion data, among 321 cattle long bones studied previously , 124 areth to 4th millennium BC in central and northern Europe [The high frequency of infants in age class 0–1 month at Schipluiden may be due to natural mortality, which mainly occurs within the first month of life during winter calving, primarily due to exposure . The lacn Europe ,81,82. Pn Europe ,82 stronth millennium BC, Bohemia, Czech Republic), calving season was short (2–3 months) [th Millennium BC, Paris Basin, France) calving season is reported to be about six months [r measurements, however, suggest the high accuracy of the modeled data based on the measured δ18O sequence.The length of cattle birth season at Schipluiden, based on stable oxygen isotopes was around five and a half months, corroborating the inference that milk production was an important purpose for cattle farming at Schipluiden. This result makes the calving season in Schipluiden much longer than that of modern primitive cattle breeds, for example in Scotland, spanning over 2–3 months . Cattle  months) , whereasx months . Variatix months . Environx months . To whatx months . The lacx months . The extx months ,85 durinth millennium BC. The recent discovery of milk protein in human dental calculus in Neolithic Britain has also provided direct evidence for the consumption of dairy products [Milk processing can also be evidenced by the organic residue analysis of ceramics ,86. So fproducts . While w3 plants were abundant. Four cattle individuals , had δ13C values as low as –27.9‰ for their diet. This is lower than the δ13C value of –27.5‰ as the endpoint for grazing on pre-industrial C3 plants in open areas [13C values (–27.2‰ to –26.4‰) have been recorded from the Late Mesolithic aurochs in Balkweg (5680–5520 BC) and Jardinga (5570–5370 BC) in the northern Netherlands [Alnus sp.), willow , apple (Pomoideae), sloe (Prunus spinose), juniper (Juniperus communis), and ash (Fraxinus sp.) have been recovered at the site in the form of branches and twigs [Pinus), oak (Quercus), lime (Tilia), and hazel (Corylus) albeit in smaller proportions [13C values which coincide with low δ18O values, suggesting leaf foddering occurred during the cold season. The absence of an observed canopy effect in the δ13C values of red deer and suids makes foddering some cattle with leafy hay a more plausible scenario than animals grazing in waterlogged areas.Schipluiden cattle appear to have pastured in an isotopically similar environment to red deer and suids, possibly at the beach plain where Cen areas . Similarherlands . Tree spnd twigs . Cattle portions . This po13C values suggest that cattle herding predominantly took place in open areas and /or open forests, with occasional use of dense forest resources. Low δ13C values, which reflect the use of forest resources, have been previously observed elsewhere in temperate Europe including the LBK sites of Bischoffsheim , and Vaihingen , ,97, 96,9 Germany ,98). At  Germany ,100. Col Germany .15N values measured in cattle and red deer bone collagen in our dataset are exceptionally high in comparison with contemporary populations of cattle [15N enrichment include suckling [15N values of infants by one trophic level (2‰–3‰) in comparison to their mothers [15N value (11.6‰) was measured in a cattle femur with fused distal epiphysis . Another cause of elevated stable nitrogen isotopic values may be stress or starvation [The δf cattle . Factorssuckling ,103, strsuckling , soil masuckling ,105,106,suckling ,107. Thr mothers . In our arvation , which marvation , which i15N value of plants by 4‰ or higher, depending on the intensity of manuring. This process in return elevates the δ15N values in humans and animals who consume the plants growing in this environment [15N values of cattle grazing in this environment. The positive correlation between soil salinity and the 15N ratio of coastal and salt marsh plants is well documented [Long-term incorporation of manure into croplands to increase the fertility of the soil can increase the ironment ,105,109.ironment . We cannironment ,111, is cumented ,112. Ove13C values between –24.0‰ and –20.0‰ and δ15N values between 9.9‰ and 16.7‰  and one cormorant  exhibit high δ13C (–14.1‰ and –13.7‰ respectively) and δ15N (15.2‰ and 18.7‰ respectively) which supports our interpretation that they did not play an important role in the Mesolithic diet. Evidence for this interpretation derives from the location of the sites along a river and far away from the contemporaneous seashore and the abundant zooarchaeological evidence for freshwater species [13C depletion in these individuals [With this study, we have extended the faunal stable isotope baseline for Schipluiden, which enables us to revisit the previous inferences on human palaeodiet at the site. Interesting patterns arise when we compare our faunal dataset with previously reported human and faunal isotopic data from the Mesolithic sites of De Bruin and Polderweg and those from Schipluiden ,25. We mnd 16.7‰ . These d species ,34. The ividuals .13C values between –21.6‰ and –18.7‰ (–19.9‰ on average), scattering between the endpoints for pure terrestrial and marine diet [15N values are as high as 10.1‰ to 16.0‰ (14.2‰ on average). Excluding the data from a 2-year-old child that exhibits the highest δ15N (16.4‰) in the dataset, possibly as a result of suckling [15N values of the human bones from Schipluiden are unusually higher than other Neolithic populations documented in temperate Europe [15N values, compelling a reconsideration of the original interpretation. Schipluiden human remains exhibit on average δ15N values that are 5.2‰ higher than those from cattle remains. In part, we can suggest that the elevated δ15N values measured in human remains can be explained by the consumption of terrestrial mammals, mainly cattle with elevated δ15N values. Human bone collagen from Neolithic Orkney (4th millennium BC) exhibit similar δ13C values which have been interpreted as a result of consumption of products from animals grazing on marine seaweed which in turn would increase their δ13C values substantially [13C values of Schipluiden cattle bone collagen and tooth bioapatite are in the expected range of terrestrial C3 plants and does not suggest any seaweed consumption. Overall, in contrast to the previous study, we suggest that Schipluiden had a mixed terrestrial and marine protein diet but that the marine protein input was relatively small in comparison to terrestrial resources. The high δ15N values observed at Schipluiden resulting not only from consuming marine resources but also from substantial consumption of terrestrial plants and animals, such as cattle, from a 15N-enriched environment.In comparison, seven human adults from Schipluiden exhibit δine diet . The δ15suckling , the δ15e Europe –116. Thee Europe ,25. In tantially . The δ13th millennium BC.This interpretation of how human palaeodiet at Schipluiden was constructed is consistent with previous and current zooarchaeological results from Schipluiden, which both indicate a substantial consumption of cattle protein since the onset of farming at the site . This pi3 plants were abundant with leaf-fodder providing a limited contribution to the diet of some cattle. The major investment in maintaining this animal would have also strengthened its social importance for the community, as is evidenced by the special attention paid to cattle body disposition.Schipluiden, the earliest known year-round settlement at the Rhine-Meuse delta, is a key site for investigating the Neolithic way of life in Northwest Europe. Using zooarchaeological remains we show that cattle husbandry was an integral component of Schipluiden subsistence. Cattle meat and milk production were achieved by culling male cattle near or at their optimum weight while probably maintaining female cows until older ages. Post-lactation slaughtering of calves guaranteed lactating cow’s milk let-down. Farmers could control the birthing season of cattle through fodder provision, extending the availability of milk for human consumption during food scarcity or crop failure. Cattle were mainly kept in the vicinity of the site where C15N in human bone collagen is more likely to signal the consumption of products deriving from cattle which grazed on 15N-enriched salt marsh plants around the site than a marine-based diet. This contradicts the previous interpretation of the subsistence at Schipluiden being a continuation of marine food consumption long after the introduction of farming. Results presented in this study suggest that human diet in the mid-4th millennium BC Rhine-Meuse area was primarily based on products from domesticates, especially cattle, with some input from the available wild terrestrial and aquatic resources. This changes the current narratives of Neolithic lifeways in the Rhine-Meuse Delta and urges further interdisciplinary research for understanding the nature and role of early animal husbandry in this region.Using the faunal isotopic data, we show that the high S1 File(DOCX)Click here for additional data file.


ETRR), obtained from chlorophyll fluorescence parameters by short illumination periods (10–30 s) at each light level, can provide a rapid and easy measurement of photosynthetic light response in plants. However, the relationship between ETRR and the steady-state light response of CO2 exchange rate (AS) of terrestrial plants has not been studied in detail. In this study, we compared the ETRR and AS for five woody and four fern species with different light and/or water adaptations. Under well-watered conditions, a constant temperature (25 °C) and with stomatal conductance (gs) not being a main limiting factor for photosynthesis, ETRR and AS were closely related, even when merging data for regression analysis for a species grown under different light conditions and measured under different light intensity and air humidity. However, when Alnus formosana was treated with low soil water and air humidity, because of the decrease in AS mainly due to stomatal closure, the ETRR–AS relation was not so close. In addition, at both 100 and 2000 μmol m−2 s−1 photosynthetic photon flux density (PPFD), ETRR and AS were significantly correlated within a plant group  regardless of the broad difference in SA due to different species or environmental factors. The results indicate that the relationship between the ETRR and AS is varied by species. We concluded that 1) ETRR could reflect the variation in AS at each irradiance level within a species under well-watered conditions and 2) ETRR at 100 μmol m−2 s−1 PPFD (as the efficiency of light capture) or 2000 μmol m−2 s−1 PPFD (as a maximum photosynthetic parameter) could be used to compare the photosynthetic capacity within a plant group, such as woody plants and ferns.The rapid light response of electron transport rate ( LC are widely used to describe the physiological plasticity of plants. Thus, the LC of photosynthesis is fundamental for plant ecophysiological research [Photosynthesis is a major determinant of biomass production and terrestrial carbon budgets . Sunlighresearch ,2,3,4.Traditionally, the LC of photosynthesis has been measured by the rate of steady-state photosynthesis under a range of relevant light intensity. Thus, the measurement is limited by the long measurement time and cumbersome leaf gas exchange techniques, especially in the field . RecentlETRS) is under steady-state conditions at a given strength of illumination. Because CO2 fixation (As) is a major sink for electrons from PSII, when A is inhibited by environmental and/or physiological factors, leaves may downregulate their PSII efficiency, mainly by xanthophyll-dependent non-photochemical quenching to avoid damage caused by excessively absorbed energy [A and other alternative sinks remains unchanged under many conditions. Examples are C4 plants (with photorespiration mostly restricted) and C3 plants under conditions with approximate temperature as well as CO2 and O2 concentrations but varied light intensity [2 fixation and photorespiration are major sinks for electrons from PSII in C3 plants, the ratio of ETR to As (or PSII efficiency/photosynthetic rate per absorbed quantum) greatly increases with decreasing CO2 partial pressure [2 partial pressure [ETR obtained by SLC methods  involves short illumination periods (10–30 s) at each light level, so the RLC can be measured within 1.5–2 min, but leaves do not achieve steady-state conditions during each light step [ETRR can provide reliable information about cardinal points of photosynthesis [ETRR and steady-state photosynthetic rate (AS); thus, ETRR is widely used to assess the photosynthetic activity and biomass productivity [In contrast to ght step ,24. Neveynthesis . It can uctivity ,27,28,29riods 10– s at eacuctivity ,32,33.ETRR is used to study environmental acclimation [gs) is another important limiting factor in the photosynthesis of terrestrial plants. To prevent water loss and facilitate CO2 diffusion to mesophyll cells, guard cells may monitor the plant water status and the CO2 demand from the mesophyll [A and gs may decrease with decreasing light intensity [2 concentration and decreases to water, for lower As/gs ratio, than angiosperms [AS and ETRR may decrease together [As and gs requires several minutes , and, andETRRs  for three tree species measured at 80% and 40% relative humidity (RH). AS and gs for four ferns and two understory shrubs, measured under well-watered conditions and 75% RH, were described previously [ETRR and Ci/Ca for Pyrrosialingus, Aspleniumantiquum, and Diplaziumdonianum, measured at 80% and 40% RH, were selected, as shown in AS and gs, these two variables for three ferns measured at 2000 μmol m−2 s−1 photosynthetic photon flux density (PPFD) are also shown in AS and ETRR for all tested species under different PPFD, RH, and soil water conditions is shown in AS, gs, and ETRR for all tested species showed a hyperbolic increase with increasing PPFD. However, these LCs varied by species and environmental conditions during cultivation and measurement. Under well-watered conditions, a pioneer tree, Alnus formosana, had the highest light saturation point and maximal value of photosynthesis, followed by a hemiepiphytic tree, Ficus microcarpa, and a hygrophytic tree, Salix warburgii, then by two understory shrubs, Ardisia crenata and Ardisia cornudentata  and P. lingus, a slight-shade-adapted fern, showed a hyperbolic relation with ETRR: the AS–ETRR relation could be best fitted by the equation Y = aX/(b + X) . The AS of A. formosana was inhibited largely by low soil water content and low RH, but its ETRR was not as inhibited as AS; thus, the AS–ETRR relation was not as close as for the other tested species. At both 100 and 2000 μmol m−2 s−1 PPFD, the leaves with high AS always had high RETR, regardless of species or environmental factors. However, the slope of the RAS–ETR regression line was higher for woody plants than ferns  four ferns, (2) two understory shrubs, (3) Ficus microcarpa and Salix Warburgii, and (4) Alnus formosana  and found a similar association of AS and ETR regardless of the use of α = 0.84 or 0.80–0.89  to calculate ETR . In addition, our plants featured no specific anatomical structures. So we chose the empirical mean α of 0.84 [ETR is calculated as the product of PSII efficiency and absorbed light. Many studies used the empirical mean of α (0.84) to calculate ETR and compare differences in ETR among species  and unde of 0.84 .AS between high- and low-light-grown materials can be defined by ETRR [ETRR also depends on the short-term (min) light history of photosynthetic organisms immediately before measurement as well as illumination time for each light level during measurement. Maximum ETRR value is very low after long-term dark adaptation, but when organisms are exposed to light immediately before RLC measurement, maximum ETRR increases with increasing illumination time and to a stable level within 8 to 15 min of illumination [ETRR increases with increasing light intensity immediately before measurement [−2 s−1 PPFD) pre-irradiance [ETRR increases with increasing illumination time at each light level [Measurement of SLC requires light steps long enough to allow for stabilization of the photosynthetic processes under each irradiance level. RLC only requires 10 to 30 s at each light level; nevertheless, the difference in  by ETRR ,31,35. Hmination . In addiradiance . During ht level ,53.ETRR and AS, we used overnight dark-adapted leaves for measurement of ETRR and then measured leaves were kept in the dark until measurement of AS. Even for leaves not exposed to light until the measurement and with short (10-s) steps of increasing irradiance, ETRR was still high  and closely related to AS , whe, wheETRRnditions .ETRR–AS relationship varies by species. The increase in LC in the light-limiting region, as well as light-saturation and maximum photosynthetic variables, have been used for research into plant ecophysiology [ETRR for some species did not reach saturation until 2000 μmol m−2 s−1 PPFD, we could not compare the light-saturation variable among species. Here, we used only data obtained at 100 μmol m−2 s−1 PPFD for the efficiency of light capture and data obtained at 2000 μmol m−2 s−1 PPFD as a maximum photosynthetic variable to elucidate the interspecific relationship between ETRR and AS. A significant ETRR–AS relation could be found within a plant group   used to calculate ETR for all tested materials. Moreover, slopes were higher for woody plants than ferns for both AS–RETR , there was only a 1.1-fold difference between ETR with α = 0.81 and 0.89 used for calculation. However, the difference in slopes for the AS–RETR regression between woody plants and fern was much higher than 1.1-fold  . Therefo AS–ETRR  and AS–Epathways ,22, suchpathways , water–wpathways , and cycpathways  as well pathways  and sulfpathways  assimilaETRR could reflect the variation in AS at each irradiance level within a species under well-watered conditions and 2) ETRR obtained at 100 μmol m−2 s−1 PPFD (as the efficiency of light capture) or 2000 μmol m−2 s−1 PPFD (as a maximum photosynthetic parameter) could be used to compare the photosynthetic capacity within a plant group, such as woody plants and ferns. Because ETRR can be measured within 1.5–2 min, it might be a useful tool for ecophysiological research. However, we investigated only five woody plants and four fern species. The number of species may not be enough to argue the taxonomic distinctions, and more comparisons might be needed. In addition, photorespiration is another major sink for electrons from PSII in C3 plants. The AS/ETRR ratio may vary on changing the CO2 and O2 concentration as well as temperature because the allocation of electrons between CO2 fixation and photorespiration may vary.In the present study, we found that 1) Pyrrosia lingus (Thunb.) Farw., Asplenium antiquum Makino, Diplazium donianum (Mett.) Tard. -Blot., and Archangiopteris somai Hayata), 3 broad-leaved tree species with different water adaptation (Alnus formosana (Burkill) Makino, a pioneer tree; Salix warburgii O. Seem., a hygrophyte, and Ficus microcarpa L., a hemiepiphyte) and 2 broad-leaved understory shrubs (Ardisia crenata Sims. and Ardisia cornudentata Mez.) in this study [A. formosana and S. warburgii are usually distributed near the rivers or in gullies; P. lingus and F. microcarpa can survive in the dry environment; whereas D. donianum, Arc. Somai, and S. warburgii are sensitive to drought [A. formosana  were the same as we used previously, and collected from central Taiwan [As. antiquum, and 1 rhizome with 3–4 leaves per pot for the other 3 ferns) filled with organic soil and maintained outdoors in the nursery of the Endemic Species Research Institute, Chichi Township, Nantou County, Taiwan . Materials were regularly watered and fertilized  and received up to 3 levels of light intensity ), according to the light condition of their habitat, i.e., 3 trees received 100% sunlight; 2 slight- to medium-shade ferns, P. lingus and As. antiquum, received 100%, 50%, and 10% sunlight; 1 medium-to-heavy shade fern, D. donianum, and 2 understory shrubs received 10% and 50% sunlight; and 1 heavy-shade fern, Arc. somai received 10% sunlight. The average elevation and temperature were about 250 m and 20 °C. The average annual rainfall and air humidity were about 2200 mm and 80%. During the growth period of the materials (March–November), the average hourly values of daily maximum photosynthetic photon flux density (PPFD) ranged from 1296−1456 μmol m−2 s−1 (Mar.−Aug.) and 1150−770 μmol m−2 s−1 (Sept.−Nov.) (data from the Endemic Species Research Institute). Only A. formosana was treated with mild and severe drought immediately before photosynthetic measurement by withholding water, until AS values were reduced to about 70% and 30%, respectively, of the maximum (AS under well-watered conditions: 100%) [We examined 4 fern species with different light adaptation  .ETRR was at dawn at room temperature and involved the software of the PAM-2000 fluorometer . Nine steps of active light from about 60–2300 μmol m−2 s−1 PPFD were applied at each irradiation step for 10 s [F) and maximal (Fm’) levels of fluorescence were measured at the end of each irradiance level. The F was determined under each PPFD level, and the Fm’ was determined by applying a 0.8-s pulse of saturating flashes of approximately 6000 μmol quanta m−2 s−1. Actual PSII efficiency (ΦPSII) was calculated as (Fm’−F)/Fm’, and ETRR was calculated as ΦPSII × PPFDD × 0.5 × α [ETRR at 200, 400, 800, 1200, and 2000 μmol m−2 s−1 PPFD was calculated from linear interpolation between the 2 nearest values. After the measurement of ETRR, the measured leaves were kept in the dark until the measurement of the steady-state light response of CO2 exchange. From 09:30 h to 15:00 h, photosynthesis and stomatal conductance were measured by use of a portable, open-flow gas exchange system , and an integrated fluorescence LI-6400-40 chamber head stepwise from low to high levels of PPFD . The values of AS (net photosynthetic rate), gs, and intercellular CO2 concentration/ambient CO2 concentration (Ci/Ca) were recorded when the gas exchange was stable (about 4 min in the dark and 10–20 min under each level of illumination). Throughout the measurements, leaf temperature and CO2 concentration were kept at 25 °C and 350–400 μmol mol−1 (no control), respectively, for all materials. Relative humidity (RH) in the chamber was taken at 75% and 50% (air entering chamber controlled by passing temperature-controlled water) for ferns and understory shrubs, and 80% and 40% for trees.Measurements were carried out from September to November in a laboratory at the Endemic Species Research Institute. At nightfall of 1 day before the measurement, potted materials were dark-adapted overnight (room temperature about 25 °C). On the next day, fully expanded younger leaves were selected for measurements. First, the measurement of for 10 s ,35. The  0.5 × α  . The light-response curve of photosynthetic rate was fitted by sigmoidal or hyperbolic equations. Data were analyzed by linear or curve–linear regression. All statistical analyses involved the use of Sigma Plot v10.0.


Older adults are considered one of the most vulnerable groups to COVID-19. However, previous studies on emotion and aging have found that older adults report better wellbeing than younger adults in global surveys and daily reports. To better understand older adults’ wellbeing during the COVID-19 outbreak, we examined age differences in daily affective experiences in this study.Two hundred and thirty-one participants from mainland China aged 18 to 85 were recruited to participate in the 14-day daily diary study, after a pretest. Their trait affect and demographic information were measured in the pretest. Their daily affect and stress levels were measured in the daily assessments.I found that older adults reported lower perceived stress related to COVID-19 in daily life, compared to younger adults. The negative relationship between daily perceived stress and high arousal positive affect and the positive relationship between daily perceived stress and high arousal negative affect was weaker in older than younger adults.These results provide initial evidence of daily affective wellbeing across different age groups in adulthood during the COVID-19 outbreak. Such information is important for developing interventions to promote better wellbeing during the COVID-19 outbreak. The Coronavirus Disease 2019 (COVID-19) epidemic started in December 2019 and quickly spread to more than 215 countries by mid-April 2020. Due to its serious effects on human health, it can also damage mental health by increasing levels of anxiety, stress, and worry among health professionals, and the general public . AlthougIn the literature on aging and emotion, older adults generally report a higher level of positive affect  and a lower level of negative affect  than younger adults in global surveys and daily diary studies . These rBased on the two-dimensional valence-arousal model , I examiMage = 44.74 years, SDage = 17.54 years; 69% women; 70% had a college degree; 41% had a job; 17% had a religion) participated in the study in February 2020. Thirty-three percent of the sample were aged between 18 to 35 years, 51.5% were aged between 36–60 years, and 15% were aged between 61 years and above. They were recruited through mass mailing through a university email system. All participants were born and raised in China, and lived in mainland China during the 14-day daily diary period. They came from 23 of the 32 provinces, cities, and autonomous regions in mainland China. Two participants came from Hubei province. We included their data in the analysis because the results did not change when deleting their data. However, four participants were excluded from the data analysis due to missing data in their daily diaries. Among the remaining 227 participants, 192 completed 14 daily questionnaires . One of them was identified as a confirmed or suspected COVID-19 case during the questionnaire period, but the deletion of the data did not change the pattern of the results. Descriptive information about the sample is presented in Two hundred and thirty-one Chinese participants aged 18 to 85  after completing the study. This study was approved by the Human Research Ethics Committee of the Education University of Hong Kong.The study was conducted during the peak period of COVID-19 spread in China. All questionnaires were completed online using the never” to 5 “always.” Based on the two-dimensional valence-arousal model, HAP was measured by the aggregate score of “enthusiastic,” “excited,” and “elated” (Cronbach’s α = 0.68); LAP was measured by the aggregate score of “calm,” “relaxed,” “peaceful,” and “serene” (Cronbach’s α = 0.76); HAN was measured by the aggregate score of “fear,” “hostile,” and “nervous” (Cronbach’s α = 0.71); and LAN was measured by the aggregate score of “dull,” “sleepy,” and “sluggish” (Cronbach’s α = 0.71).We used the Affect Valuation Index AVI;  to measuThe participants were asked to indicate their gender , partner status , education , religion , and overall subjective health (1 = very poor to 6 = perfect).not at all” to 5 “extremely.” In particular, HAP was measured by “enthusiastic” (intraclass correlation coefficients (ICC) = 53%); LAP was measured by “calm” (ICC = 44%); HAN was measured by “anxious” (ICC = 52%); LAN was measured by “dull” (ICC = 55%); positive affect was measured by “happy” (ICC = 51%); and negative affect was measured by “sad” (ICC = 49%).The daily version of the AVI  was usednever” to 5 “always”  + B2∗ + rLevel-2 model∗(age) + G02∗ + G03∗(socioeconomic status) + G04∗ (education) + G05∗(religion) + u0B0 = G00 + G01B1 = G10 + u1B2 = G20 + u2Level-1 modelAffective experiences = B0 + B1∗(day) + B2∗ + B3∗(daily perceived stress) + rLevel-2 model∗(gender) + G02∗(age) + G03∗ + G04∗(socioeconomic status) + G05∗(education) + G06∗(religion) + u0B0 = G00 + G01B1 = G10 + u1B2 = G20 + u2B3 = G30 + u32 = 1020.62, p < 0.001). Age was associated with daily lesser perceived stress . Ordinal day was associated with less positive affect, , suggesting that participants reported less stress in the later days of the daily diary period. Detailed results are reported in With perceived stress as the dependent variable, the variance components (VC) suggested that there was significant variance in the intercept to be explained across individuals , HAP , and LAP ; and was positively associated with daily negative affect , HAN , and LAN . The relationship between stress and HAP was moderated by age so that the negative association was weaker in older than younger adults . The relationship between stress and HAN was also weaker in older than younger adults . Age did not moderate the relationships between stress and other daily affective states. Detailed results are reported in Age differences in the relationship between perceived stress and daily affective experiences were then examined. Perceived stress was negatively associated with daily positive affect (Using a 14-day daily diary study, we examined people’s daily affective experiences across different age groups in adulthood during home quarantine when COVID-19 broke out in mainland China. We found that older adults reported a lower level of perceived stress related to COVID-19 than younger adults. In addition, we found that the negative association between daily perceived stress and daily high arousal positive affect was weaker in older than younger adults; and that the positive relationship between daily perceived health and daily high arousal negative affect was also weaker in older than younger adults.Consistent with the previous findings on age differences in perceived stress after stress induction , we founWe only found significant age differences in the relationship between daily stress and high arousal affect, regardless of valence. Such findings may be attributable to the fact that stress is a high arousal affective state. Therefore, the feeling of stress might be more relevant to high arousal positive and negative affect than the low arousal ones. It may also be possible that individuals regardless of age prefer low arousal affect states over high arousal affect states, when they see future time as increasingly limited . The sitDespite its interesting results, the study also has limitations. First, although we included multiple assessment points, this study was based on a correlational design. Thus, we were not able to examine the mechanism underlying the relationship. Second, the sample used may not be representative of the population of mainland China. In particular, the education level in this sample was higher than that of the general population. Although demographic information, such as education level, marital status, religion, and socioeconomic status, was controlled for in data analysis, a larger sample size is needed to validate findings of this initial study on daily affective experiences during the COVID-19 outbreak in future studies. Third and as aforementioned, we could not explain the non-significant interactions between age and fate control on daily affective experiences. Future studies should clarify this issue.In this 14-day daily diary study, we found that older adults reported a lower level of perceived stress related to COVID-19 than younger adults. The negative relationship between perceived stress and a high arousal positive affective state, and the positive relationship between perceived stress and a high arousal negative affective state were weaker in older than younger adults. These results provide initial evidence of the daily affective wellbeing of adults during the COVID-19 epidemic in China. These findings provide initial evidence on daily affect during home quarantine of individuals across different age groups in adulthood. Such information may be important for preparing different mental health services for people in different age groups.The raw data supporting the conclusions of this article will be available upon sending request to the corresponding author, without undue reservation.The studies involving human participants were reviewed and approved by the Human Research Ethics Committee of the Education University of Hong Kong. The patients/participants provided their written informed consent to participate in this study.The author contributes to theoretical framework, data analysis, and manuscript writing of the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We establish Chen inequality for the invariant  In 1985, Amari [Opozda, in , definedThe main Riemannian invariants are the curvature invariants, with many important applications, for example in physics. Among them, the most studied and well known are the sectional curvature, scalar curvature, and Ricci curvatures.In submanifold theory, beside the study of the geometric properties of submanifolds, establishing sharp relationships between intrinsic and extrinsic invariants is another topic of interest.n-dimensional Riemannian manifold K are the scalar and sectional curvatures of In 1993 [see also ,5). In tMoreover, rms see ,7)..δ initiators . Also, ure ; one; onestatThe dual connection of a torsion-free affine connection The curvature tensor fields with respect to the dual connections The curvature tensor of constant curvatureHessian structure.A statistical structure tureε∈R  if(3)R˜The curvature tensor fields  Hessian ).Hessian curvature tensor for On a Hessian manifold . One . One M˜mc if and only if Q˜ are(5)h and Since Furthermore, the Weingarten formulae are (9)∇˜Xξ=M, respectively. Then, the mean curvature vector fields are defined byLet The Gauss, Codazzi, and Ricci equations for statistical submanifolds, with respect to the dual connections, were established by Vos .Proposition 1. Let ∇˜ a∇ be the induced connection by where Proposition 2. Let ∇˜ awhere In , the preLemma 1.Let Moreover, the equality holds if and only if Proof. We shall prove this Lemma by mathematical induction.For Let us putd assumeP(n):Ln≤nBy using On the other hand, obviously one hasInequalities  and 20)20) implyquations  and (20)c. Then, it is flat with respect to the dual connections Let n-dimensional statistical submanifold of p. Consider orthonormal bases Let The sectional curvatures Using Equations (11) and (14), we getThe equation of Gauss for the Levi-Civita connection impliesAnalogously, we haveQ). Then, from Equations (11) and (14), we haveOn the other hand, let In addition, the equation of Gauss for the Levi-Civita connection impliesBy subtracting Equations  and (22)H and Let By summing the two above relations and substituting the result into Equation , we getIn summery, we may state our main result.Theorem 1.Let where Moreover, the equality holds if and only if for any An immediate consequence of Theorem 1 is the following.Theorem 2.Let then Theorem 1 represents a


Photobacterium, Alteromonas, Ruegeria, and Sulfitobacter. In all four species, we found that peptides induced bacterial extracellular aminopeptidase production. Amino acids led to cell growth with markedly lower aminopeptidase production by Photobacterium and Sulfitobacter, but not by Alteromonas and Ruegeria. These results suggest that the extracellular aminopeptidases of marine bacteria are primarily produced on demand in response to the presence of relevant substrates (peptides) in seawater. Peptidyl substances may be regulatory nutrients for marine bacterial growth in aquatic environments.Bacterial extracellular aminopeptidases are key enzymes in protein processing in oligotrophic seawater. To the best of our knowledge, the regulation of aminopeptidase production in microbes inhabiting seawater has not yet been reported. The present study attempted to experimentally clarify which organic materials affect bacterial extracellular aminopeptidase production by nutrient-rich and starved cells growing in artificial seawater using  The ocean contains a large carbon pool of dissolved organic matter (DOM), with the amount of carbon in DOM estimated to be similar to that in atmospheric CO2 . However2 . Some of2 . The upt. 600 Da . Heterot. 600 Da . Other tPseudomonas aeruginosa  under eutrophic conditions may easily access peptide substances; however, since marine bacteria in seawater are generally subjected to largely oligotrophic conditions, they need to expend energy in order to obtain organic nutrients. To reduce energy expenditure, bacteria in seawater may produce extracellular proteases in an “on-demand” manner, synthesizing enzymes only when the relevant substrates are present in the surrounding water. This hypothesis raises the question of whether organic matter other than peptide substrates also contributes to this proposed regulation of enzyme expression.We hypothesized that proteinaceous substrates control enzyme activity; bacteria under oligotrophic conditions may respond to organic matter substrates by activating efficient biochemical catabolism. Bacteria have generally developed advanced mechanisms for the regulation of catabolic pathways, as reported by In the present study, we attempted to clarify extracellular protease production by heterotrophic marine bacteria exposed to biodegradable DOM in aquatic environments. We considered the following (non-exclusive) possibilities: Proteases are induced by proteinaceous substances (substrates) and some or all of the amino acids that are generated by protein degradation. Additionally, other growth substances, such as polysaccharides, may promote enzyme activity. We also considered that the response observed may differ depending on the physiological conditions of bacterial cells upon encountering potential inducers. Therefore, the present study experimentally investigated the mechanisms by which protease activities are induced in bacteria growing in artificial seawater (ASW) under various physiological conditions. We used four species of heterotrophic bacteria isolated from seawater that were confirmed to produce extracellular proteases before the initiation of the study. The cells of each species were tested under two physiological states depending on their previous nutritional conditions. Specifically, each bacterium was cultured in the presence or absence of sufficient nutrients prior to the initiation of the experiments. Nutrient-rich cultured cells and starved cells were suspended in ASW, and their growth and extracellular protease activity were examined over time.Proteobacteria, the largest bacterial group in the marine environment . The resulting pellets were resuspended and washed once with ASW  at 4°C for 15 days. Cells were then collected by centrifugation. The resulting pellets were considered to be “starved cells”. The preparation processes for “MB-cultured cells” and “starved cells” are shown schematically in The four strains were precultured in 50-mL plastic centrifuge tubes (Sumitomo Bakelite), each containing 20 mL of Marine Broth medium , and incubated at 25°C overnight with shaking at 120 rpm. Cells in the log-growth phase were harvested by centrifugation at 5,400×with ASW , and wer6 cells mL–1) were distributed to 100-mL glass screw cap bottles. Separate suspensions were prepared using the MB-cultured cells and starved cells of each strain.Aliquots (70 mL each) of bacterial cell suspensions in ASW (at 1.0×10–1 (High-Peptone) or 12.5 mg L–1 (Low-Peptone). Amino acids were prepared as solutions of 20 types of proteinogenic amino acids , which were added to the experimental bottles to a final concentration of 100 μM each (High-Amino acids) or 10 μM each (Low-Amino acids). Carboxymethyl cellulose sodium salt  solution was added to the bottles (Cellulose) to a final concentration of 1.85 g L–1. D-(+)-glucose  solution was added to the bottles to a final concentration of 100 μM (High-Glucose) or 10 μM (Low-Glucose). The experimental concentrations of peptone and CMC were selected based on the expected average molecular weights of each commercial product: 1.25×103 g mol–1 for Proteose Peptone and 1.85×105 g mol–1 for CMC. Based on these conversions, High-Peptone and Low-Peptone were expected to contain approximately 100 μM and 10 μM peptone, respectively, while Cellulose was expected to contain approximately 10 μM CMC. Due to the lower solubility of polymeric CMC, cellulose supplementation was performed at a single concentration. As a control, a bottle without organic substances (Non) was also prepared. All prepared bottles  were incubated at 25°C without shaking. After 0, 5, 10, 24, 48, and 72 h, 4-mL samples were withdrawn from each bottle and subjected to bacterial counts and enzyme activity assays.To examine each bacterial growth and their extracellular protease activity in response to the organic matter added, four different organic substances were added to separate bottles : peptide–1 phosphate buffer stock was added to ASW to yield a final concentration of 2 μmol-P L–1, a level that is approximately equivalent to that of total phosphorus in natural seawater (As shown in –1) was calculated.Samples were subjected to serial 10-fold dilutions with ASW, and 100 μL of diluted samples was plated on each MB agar plate with replicates. After an incubation at 25°C for 1 or 3 days, colonies were counted and viable cell density (colony-forming units [CFU] mL2) per sample; the resulting values were used to calculate cell density (cells mL–1) in each sample.Samples were fixed with glutaraldehyde  at 4°C for 16 or 24 h. Fixed samples then were passed through a 0.2-μm Nuclepore filter  to collect bacterial cells on the filter, and cells were stained with 4,6-diamidino-2-phenylindole (DAPI) as previously described . Cell cot-butyloxycarbonyl-L-leucyl-L-seryl-L-threonyl-L-arginine MCA (Boc-Leu-Ser-Thr-Arg-MCA) is a trypsin substrate. Enzyme activities against these three substrates have been reported in seawater . The plate was incubated at 25°C for 1 h, during which time the fluorescence intensity of each well was measured five times  to monitor the time course of the change in fluorescence. The plate was shaken at 800 rpm for 60 s before each measurement. Measurements were performed using a microplate reader (Corona SH8100Lab) with excitation/emission wavelengths of 380/440 nm, respectively. Fluorescence changes were converted to increases in the concentration of a fluorophore , generated as a hydrolysis product of the MCA substrate. The same analysis was performed with autoclaved samples to assess non-enzymatically produced AMC. The enzymatic hydrolysis rates of substrates in the samples were estimated from changes in the concentration of AMC after the subtraction of non-enzymatically produced AMC. The cell-specific hydrolysis rate (fmol cell–1 h–1) was calculated by dividing activity by the total cell number at each culture’s sampling time point.Extracellular enzyme activities were measured according to the methods of seawater , and are® Excel® 2016 with a statistical chart for multiple comparisons. A difference was considered to be significant at P<0.01 (cell number) or P<0.05 (enzyme activity).Data are presented as an average±SD where appropriate. A two-tailed one-way ANOVA with post hoc Tukey-Kramer tests were performed to test the significance of differences among the experimental conditions examined. Calculations were performed using MicrosoftPhotobacterium and Sulfitobacter showed rapid increases in cell density at the early stage (<10 h) of culturing, whereas those of Alteromonas and Ruegeria increased after 10 h. The lag periods for all species before starting growth were longer with starved cells than with MB-cultured cells, indicating that the resumption of growth in ASW was delayed for starved cells. Growth in High-Peptone was significantly stronger (P<0.01) than that in Low-Peptone for all species tested.Photobacterium and Sulfitobacter were cultured in ASW supplemented with phosphorus, growth was observed under not only High- and Low-Peptones, but also Low-Amino acids  for Photobacterium and Sulfitobacter was weaker in media lacking supplemental phosphorus . Similar results were obtained for the acceleration of growth of starved cells by each organic substance; however, starved cells appeared to require more time to restart their growth in the presence of organic substances.Therefore, these experiments demonstrated that peptone-supplemented ASW is an efficient growth medium for the bacterial strains tested; ASW supplemented with amino acids also supported the growth of Gammaproteobacteria . The sole exception was the Leu-MCA hydrolyzing activity of Photobacterium, which continued to increase during the stationary phase. Alteromonas cultured in High-Peptone continued growing and exhibited increased enzyme activity at 72 h  Induction of Extracellular Aminopeptidase Production by Peptides in Some Marine Bacterial Species. https://doi.org/10.1264/jsme2.ME20150Supplementary Material


Most mutations in human cancer are low-frequency missense mutations, whose functional status remains hard to predict. Here, we show that depending on the type of nucleotide change and the surrounding sequences, the tendency to generate each type of nucleotide mutations varies greatly, even by several hundred folds. Therefore, a cancer-promoting mutation may appear only in a small number of cancer cases, if the underlying nucleotide change is too difficult to generate. We propose a method that integrates both the original mutation counts and their relative mutational difficulty. Using this method, we can accurately predict the functionality of hundreds of low-frequency missense mutations in p53, PTEN, and INK4A. Many loss-of-function p53 mutations with dominant negative effects were identified, and the functional importance of several regions in p53 structure were highlighted by this analysis. Our study not only established relative mutational difficulties for different types of mutations in human cancer, but also showed that by incorporating such a parameter, we can bring new angles to understanding cancer formation. As a result, such mutations are enriched in cancer, and are found in numerous cancer samples. It is generally perceived that if a mutation occurs in higher number of cancer cases, it is more likely to be a driver mutation4. However, most mutations in cancer only occurs in very small number of cancer cases, and the functional impacts of these mutations are hard to predict.Gene mutation is a major cause of tumorigenesis. Certain mutations on important cancer genes such as KRAS and p53 drive cancer formationTo address this problem, it is necessary to consider that the chance of observing a mutation in cancer cases is influenced by at least two major aspects: (1) how difficult it is to generate the mutation; and (2) whether the mutation promotes cancer, therefore it will be selectively enriched in cancer cases. If different mutations are initially generated at significantly different rates, it will greatly impact the mutational distribution in cancer genome database such as Catalog of Somatic Mutations in Cancer (COSMIC). Certain cancer-driving, but too-hard-to generate mutations may appear exceedingly rare in cancer database, yet certain passenger-type mutations may pile up in greater numbers, if the underlying mutations are too easy to occur.9. Second, the abilities to recognize, repair, and tolerate different types of mutations are also different11. Third, although difficult to predict, different nucleotide sequences surrounding the mutation site may cause local variances, which may physically or chemically affect the chance of mutagenesis. In addition, certain sequences are also more prone to be edited by enzymes such as APOBEC13. Therefore, different flanking nucleotide sequences can also affect mutation rate15.At nucleotide level, there are 12 routes of interchanges between A/G/T/C for single nucleotide substitutions, which underly most cancer mutations. The chances of generating each kind of mutations are certainly not equal. Many factors contribute to such phenomenon. First, different endogenous and exogenous mutagenic events lead to different types of nucleotide substitutionsTaken together, the probability to generate different types of nucleotide change may vary greatly. If two mutations both change the functional status of an important gene and promote cancer, they should be found in multiple cancer samples. However, if one of such mutation is too difficult to generate at nucleotide level, the number of cancer cases carrying that mutation will decrease significantly. Considering this, if we can define the relative difficulty to generate each type of nucleotide mutations in cancer, we will be able to better estimate the functional importance of cancer mutations.16, it is difficult to predict what percentage of cancers are influenced by each signature, and to what extent. Moreover, some of the major contributors to cancer, including nitrosamine, have not been assigned a Sanger mutation signature. Therefore, the relative difficulty to generate different types of mutation in cancer has not been adequately established. In this report, through analysis of mutational data from 26,000 cancer genomes, we established the relative mutational difficulty for different types of cancer mutations and showed that it can help accurately interpret functional importance of cancer mutations.Although mutational signatures for ageing, UV, APOBEC, smoking, and other cancer causes have been establishedGiven the complexity of mutagenesis in cancer, it is very difficult to construct a mathematical model that could weight in all relevant factors to forwardly predict how much more difficult it is to generate one type of mutation versus the other. However, such differences do factually exist, and they collectively determined the mutation distributions in human cancer. Based on this notion, we argue that by analyzing large human cancer genome dataset, we can reversely derive the relative difficulties for each type of mutation , which is about 200-folds more than the probability of A→C mutation on an ACATC sequence . Notably, the revised count for this mutation is comparable to that of the hotspot R282W mutation , suggesting M133R is also a deleterious mutation despite its low frequency in cancer database.To more precisely assess these p53 mutations, we also took into consideration that the same type of mutations is generated at different rates in different cancer types Fig. . TherefoThe global view of p53 missense mutations is provided in Fig. To establish a cut-off value that could help identify p53 mutants that still retain wild-type function, we compiled revised count values for all p53 synonymous mutations and found them to be mostly below 700  retains wild-type function Fig. . Interes18. Quantitative polymerase chain reaction (qPCR) analysis showed that these p53 mutants were expressed at similar levels  was achieved in this cell line via retroviral vectors. Hotspot p53 mutant exerts dominant negative effects over endogenous wild-type p53, and cells could not efficiently elicit cell death when treated with DNA damaging drugs. As a result, the percentage of GFP-positive, hotspot p53 mutant-expressing cells will increase after drug treatment , which host several hotspot mutations and are responsible for DNA-interaction24 Fig. . Other h 25 Fig. . Colony  25 Fig. . Such mu 25 Fig. . Taken t27. We complied revised counts for these five conserved domains  methodlap Fig. .Fig. 5Co32. Such a dataset (PHANTM) provides direct experimental readout of p53 mutations. Our predictions of the functionality of p53 mutations are highly consistent with their experimental results, with an 88% overlap . For each nucleotide mutation, −2, −1, +1, +2 nucleotides were extracted from the corresponding coding sequence.4. Therefore, we collated all mutations into 12 × 44 = 3072 groups.At the central position, there are 12 routes of interchange between A/G/T/C. The permutations at −2, −1, +1, +2 nucleotides amount to 4−5, which is the highest amongst all 3072 combinations. We set the “difficulty” score for such a mutation as 1. The mutational tendency of A→C mutation in a CGATG sequence is 0.93 × 10−7, and its relative difficulty score is calculated as 1.85 × 10−5/0.93 × 10−7 = 200. Difficulty scores for all other combinations were generated using the similar method.For the aforementioned 3072 groups, we first counted how many mutations from the 26,154 cancer genomes belong to each group. Next, we counted how many times each penta-nucleotide sequence appears in coding sequences of the 19,940 genes. For example, there are 10,389 C→T mutations on TTCGT sequences in 26,154 coding genomes. There are 21446 TTCGT sequences per coding genome. Therefore, the mutational tendency of C→T on TTCGT is approximately 10389/(21446 × 26154) = 1.85 × 10Of note, the numbers of different penta-nucleotides in the coding genome vary greatly. For example, in the coding genome there are 3001 TAGCG sequences and more than 100,000 TGGAG sequences. Therefore, it is necessary to divide the number of mutations by the number of available sites to accurately understand the relative mutational difficulty.In the above analysis, we aim to estimate the mutational tendency for each type of mutation in human cancer. Certain cancer-promoting mutations on genes such as KRAS and BRAF are strongly selected for during cancer formation. The number of such mutations are significantly increased in the dataset, not because they are easy to generate, but because they are strongly enriched by the tumorigenesis process. Therefore, their presence in the dataset may skew our estimation of the natural mutational tendency for each type of mutation. Considering this, in the above calculation, we excluded mutations that occur in more than five cancer sample, in order to achieve a closer estimate of mutational tendency. Of note, about 2% all of mutations in the 26,000 cancer genomes Fig.  occur inSupplementary Fig. −4 in endometrial cancer. Since in previous calculation we set the mutational difficulty score as 1 for a mutation rate of 1.85 × 10−5, we can calculate the relative mutational difficulty for C→T on TTCGT as 0.1 ( = 1.85 × 10−5/1.88 × 10−4) in endometrial cancer.To generate cancer type-specific mutational difficulty scores, mutations were first grouped by cancer types, from which mutation rates were calculated using similar methods. For example, we observed 1195 C→T mutations on TTCGT sequences in 296 endometrial cancer samples, and the mutational tendency of C→T on TTCGT is approximately 1.88 × 10Mutational data for p53, PTEN, and INK4A was last acquired from COSMIC on January 2018. At the time, p53 mutation data were from 130,448 cancer samples, PTEN from 72,199 samples and INK4A from 72,566 samples. Current numbers in COSMIC database have slightly increased due to website updates.40. In addition, U251 cells, which deleted the CDKN2A locus, could tolerate ARF expression, but not INK4A expression. Therefore, for later experimental validation, we cloned and analyzed INK4A mutants in this study.Of note, the CDKN2A locus contains two genes, INK4A and ARF. Previous studies showed that recurrent mutations on the CDKN2A locus do not change the function of the ARF geneFor each mutation, we first extracted the penta-nucleotide sequence surrounding the mutation site and matched it with relative mutational difficulty scores. For mutational sites that are adjacent to intron–exon junctions, the genomic sequence was used to extract the nucleotide sequences surrounding the mutational site.Next, we calculated the revised mutational count based on original mutation count and cancer type-specific relative mutational difficulty. For example, if a p53 mutation occurs in 10 colorectal cancers and 5 lung cancers, and the relative mutational difficulties for the mutation is 1 in colorectal and 3 in lung cancer, the revised count for such a mutation can be calculated as 10 × 1 + 5 × 3 = 25. Revised counts calculated using this method were used to predict the functional impact of p53, PTEN, and INK4A mutations in our study.41. The revised count for T125T is 1353 and is predicted to be a loss of function mutation by our method.Supplementary Fig. Supplementary Table In the COSMIC database, the S149F mutation on p53 is caused by single nucleotide substitution in 5 samples, and the revised count is lower than 700. However, in one additional cancer sample, a CC to TT nucleotide change also caused the S149F mutation. Because of the rarity of such double mutations, we did not assign relative mutational difficulty score to such double mutations. Therefore, we cannot make functional prediction for this mutant, and an “*” is marked in the “revised count” column for S149F. Such phenomenon also occurred for S166L, V218M, and R158C, and these mutations are labeled similarly with an “*” in Supplementary Table 42. Briefly, Eμ-Myc p19Arf−/− cells are infected with retrovirus that express GFP and mutant p53, such that 20–50% of cells are GFP positive. Cells are treated with DNA damage drug at doses that would kill 80–90% of uninfected Eμ-Myc p19Arf−/− cells. In this assay, if p53 mutant exerts dominant negative effects on endogenous wild-type p53, after DNA damage drug treatment the GFP positive, p53 mutant-expressing cells will be relatively more resistant than GFP-negative cells that only express wild-type p53. At 72 h, treated and untreated cells are analyzed by flow cytometry. GFP percentages of live (PI-negative) cells are recorded and used to calculate relative resistance index (RI).The experiment was carried out with a protocol modified from43.The value of relative RI can be calculated as RI = (G2 − G1 × G2)/(G1 − G1 × G2). G1 means how many percentages of cells are GFP positive before drug treatment. G2 means how many percentages of cells are GFP positive after drug treatment. The explanation for such calculation was provided in ref. Relative RI larger than 1 means the corresponding p53 mutant displayed dominant negative effect, protected cells from DNA damage, and the rate of GFP+ cells in surviving cells increased after drug treatment. Relative RI smaller than 1 means the corresponding p53 mutant displayed wild-type function, sensitized cells to DNA damage, and the rate of GFP+ cells in surviving cells decreased.l-glutamate, and 5 μM β-mercaptoenthanol). Phoenix, HCT116 p53−/−, Saos-2, U251, A549, 293T, and 293A were cultured in Dulbecco’s modified Eagle’s medium supplemented with glutamate and 10% (v/v) FBS. 786-O cell was cultured in RPMI medium supplemented with glutamate and 10% (v/v) FBS.Eμ-Myc p19Arf−/− cell was cultured in B-cell medium , Saos-2, HCT116 p53−/−, U251, 786-O cells were obtained from the Cell Bank, China Academy of Sciences . Doxorubicin was purchased from Selleck.Antibodies against Phospho-Akt (Thr308) (D25E6) , Akt (pan) (C67E7) , Phospho-GSK-3β (Ser9) (D84E12) , Phospho-PDK1 (Ser241) (C49H2) , and PTEN (pan) (Y184)  were used for Western blot analysis.EcoRI and XhoI sites. The PCR product was cloned into the EcoRI/XhoI sites of the pMSCV-IRES-GFP vector. cDNAs with missense mutations were constructed by overlap extension PCR. All mutation constructs were sequenced to confirm that the appropriate mutations had been incorporated and that no additional mutations were generated.Wild-type p53, INK4a, and PTEN expression vectors were constructed as follows. The full-length open reading frame of p53, INK4a, and PTEN cDNAs were amplified by PCR using KOD plus neo DNA polymerase (Code No. KOD-401 Lot No. 646300) and a pair of primers with All p53, INK4a, and PTEN mutants tested in this study are listed in Supplementary Table To test the functional status of p53 mutants, retrovirus that expresses p53 mutants, puromycin resistance gene and GFP was used to infect Saos-2 cells or HCT116 p53−/− cells. Cells are infected with similar virus MOI such that 30–50% of cells are GFP positive for all experimental groups. We were able to determine the expression level of p53 mutants in HCT116 p53−/− cells since they can tolerate p53 mutants that retain wild-type gene function. The results showed that under such infection protocol, expression levels of different p53 mutants were comparable  for three times and dried. Images were obtained by a digital camera. Similar protocols were used to test INK4a mutants in U251 cells.21. Two microlitre of plasmid solutions were injected into tail vein of a mouse in about 7 s. This creates immediate high pressure at the liver portal vein, and plasmids will enter liver cells under such conditions. In this experiment, sgRNA targeting PTEN was used to inactivate PTEN to facilitate liver cancer development, similar to Yang et al.22. cDNAs of c-Myc and different p53 mutants were cloned into a sleeping beauty system, so that upon entering liver cells, they will be stably integrated into host cell chromosomes. In this experiment, p53 mutants were not generated by CRISPR, but were introduced by cDNA to avoid uncertainty with CRISPR mutagenesis.The mouse liver cancer model was performed using published protocol as published in Chen et al.21. Such plasmids were mixed with sgPTEN–Cas9 plasmid22, together with Sleeping Beauty transposase-expressing plasmid in PBS. Gene mixture was delivered to mouse by tail vain hydrodynamic injection. Concentrations of Sleeping Beauty transposase and Myc-expressing plasmids were at 0.5 and 1.25 μg/ml, respectively. Other plasmids or corresponding empty vectors were used at 5 μg/ml. The experiments were all done in female FVB mice at 7 weeks of age. The mice were hosted in SPF housing condition. The experimental was approved by the institutional animal care and use committee.Specifically, Myc cDNA and p53 mutants were cloned into a transposon system using the PT3 vectorU251 cells expressing wild type or mutant forms of INK4A were analyzed. When grew to proper density (about 70–80%), cells were collected and fixed overnight in 70% ethanol. Cells were then treated with 0.2% Triton X-100, 50 μg/ml propidium iodide and 100 μg/ml RNase A for 40 min, then analyzed by FACS.RNA was purified using GeneJET RNA Purification Kit (thermo scientific) and qPCR was performed on a StepOne real-time PCR machine (BIO-RAD) using SYBR Green PCR master mix (Promega). mRNA level of actin was used as control. Primers used for qPCR analysis are listed in Supplementary Table t test. p Values < 0.01 were marked as *** in figures, p values < 0.05 were marked as ** in figures.Differences of event frequency between two groups were analyzed using Student’s unpaired two-tailed Supplementary InformationSupplementary TABLE.S1Supplementary TABLE.S2Supplementary TABLE.S3Supplementary TABLE.S4Supplementary TABLE.S5Supplementary TABLE.S6Supplementary TABLE.S7Supplementary TABLE.S8


There was no interaction between salinity and K. Salinity decreased germination percent (GP), not always significantly, and drastically reduced seedling biomass. ‘Raccoon’ significantly increased GP at 60 mmolc L−1 while ‘Gazelle’ maintained GP up to 60 or 120 mmolc L−1. After 50 days under saline irrigation, shoot biomass increased significantly at 30 and 60 mmolc L−1 at the lowest K dose but, in general, neither salinity nor K dose affected shoot biomass, suggesting that salinity supported plant growth at the most K-deficient dose. Salinity did not affect shoot N, P, or K but significantly reduced Ca, Mg, and S, although plants had no symptoms of salt toxicity or mineral deficiency. Although spinach seedlings are more sensitive to salt stress, plants adjusted to salinity with time. Potassium requirement for spinach growth was less than the current crop recommendation, allowing its cultivation with waters of moderate to high salinity without considerable reduction in yield, appearance, or mineral composition.Information is scarce on the interaction of mineral deficiency and salinity. We evaluated two salt-tolerant spinach cultivars under potassium (K) doses (0.07, 0.15, 0.3, and 3.0 mmol Salinity treatments were applied on 10 December 2018 and continued until 28 January 2019. Before treatments were applied, all pots were flushed four times with deionized water to remove any remaining fertilizer (including K) from the sand. After that, treatments combining Na (as NaCl) and K (substituting K for Na) began and continued for 7 weeks (50 days) with harvest taking place from 29 January to 1 February 2019. All solutions  contained all macronutrients and micronutrients needed for spinach growth, as previously determined [−1). Then, shoots and roots were washed with tap water, twice with deionized water to remove remaining sand (roots) and mineral fertilizers from tissue surface (shoots and roots) before drying in a forced-air oven (70 °C) for at least 48 h to obtain constant shoot and root dry weight  in g pot−1 before grinding for mineral analysis.For Experiment 2 (vegetative growth), 7 seeds were sown directly into pots at 1.3 cm depth. Seeds were sown on 19 October 2018 and harvested on 28 January 2019 (101 days). Pots had the same dimensions and growth medium as described above. After germination, plants were thinned to the most homogeneous three plants per pot. Plants were watered with 1/8-strength Hoagland’s nutrition solution  made witIn experiment 1 (germination and seedling establishment) plants were too small for mineral analysis and only the fresh weight of shoots was recorded. For Experiment 2, plants were harvested, separated into shoots and roots, washed with tap water, followed by deionized water and blotted dry with paper towels and bagged to be dried in a forced-air oven at 70 °C until stable dry weight. Dry weight was recorded for shoots and roots. Samples were then ground in a Wiley mill to pass a 20-mesh (0.84 mm) screen. Tissue mineral concentration was based on shoot or root dry weight. Chloride was determined from nitric/acetic acid extracts by amperometric titration. The concentrations of tissue Na, P, K, Ca, Mg, and total-S, and of the micronutrients Fe, Cu, Mn, Zn, and Mo were determined from nitric acid digestions  of the dried, ground, plant material by Inductively Coupled Plasma Optical Emission Spectrometry . Nitrogen was determined by combustion in a Rapid N Exceed® analyzer .−1) and 4 concentration of K  prepared with deionized water (ECw = 0.05 dS m−1). NaCl and K  were added to a basic, modified (no K), ½-strength Hoagland’s solution (Control) containing the same concentrations of macro- and micronutrients and 0.5-3.5 mmol L−1 of Na+ 2·4H2O, NaNO3, KNO3, KCl, NaH2PO4, MgSO4·7H2O (3BO3 (0.023 mM), MnSO4·H2O (0.005 mM), ZnSO4.7H2O (0.0004 mM), CuSO4·5H2O (0.0003 mM), and (NH4)6Mo7O24·4H2O (0.0001 mM) were added to every solution to meet plant micronutrient requirements. The concentrations of K+ were achieved by substituting varying amounts of KNO3 for NaNO3 to achieve target K+ levels. This resulted in slight decreases in Na+ with increasing K+ that are insignificant, relative to the amounts of total Na+ applied, with real values given in c L−1.The following salts were used to obtain target salinities in irrigation solution: NaCl, Ca(NOSO4·7H2O . NaFe-EDp < 0.05). Statistical analysis was performed using SAEG version 9.1 [www.systatsoftware.com) software. Interactions were analyzed regardless of the significance of the F test. All interactions between Na and K doses were analyzed statistically independently of the significance of the F test. Analysis was also done for individual effects of either Na or K doses for all the parameters related to mineral tissue accumulation and biomass accumulation, except for germination percentage, shoot fresh weight, N, P, Ca, Mg, and S in roots and shoots of both cultivars. These parameters were analyzed taking the F test into consideration, also for individual effects of either NaCl or K doses. Because there was no significant effect of K dose, but there was of Na dose , the results are presented for salinity inside of each K dose.Data for each cultivar was used for analyses of variance. Means were compared using Fisher’s LSD test  at 60 mmolc L−1, both at 22 and 32 days after seeding, while ‘Gazelle’ maintained GP up to 60 mmolc L−1 at 13 days and up to 120 mmolc L−1 at 32 days after germination  and in the coefficient of velocity of germination (CVG) with increased NaCl-induced salinity for both cultivars, regardless of the K dose. Thus, results were presented in terms of nominal NaCl doses , callingmination . The CVGSeedling shoot fresh weight decreased for both cultivars as salinity increased , regardl−1, approximately) and 5.6-fold for Cl . In shoots, from control to the highest salinity treatment, the accumulation of Na and Cl in ‘Gazelle’  used in the germination study. From here on, all the results are related to this second experiment. The effects of increased NaCl on tissue ionic composition were evaluated through the tissue accumulation of Na and Cl in root and shoot tissues of ‘Raccoon’ and ‘Gazelle’ . In geneGazelle’  was simi K doses .c L−1) to sufficient (3.0 mmolc L−1). Although the decrease lessened at higher NaCl doses, it was still significant for shoots of ‘Raccoon’ and for roots and shoots of ‘Gazelle’ (c L−1 NaCl) the decrease in Na:K ratio in shoots was seen stepwise, mainly for ‘Gazelle’, despite the low increase in K concentration for deficient doses. At higher salinities, this decrease in Na:K ratio was only observed when K dose increased from deficient to 3.0 mmolc L−1  (−1 for both cultivars), even when K was applied at deficient doses   and of Nnt doses . Howevernt doses . Interesnt doses .−1 , followed by Mn, which ranged from 254  to 304 mg kg−1 , with the remaining micronutrients  ranging from 19 to 29.7 mg kg−1. Salinity increase led to a significant reduction of Cu (35%), Fe (39%), and Mn (16.5%) but had no effect of the concentrations of B (18–24 mg kg−1) or Zn (22 to 29.7 mg kg−1) , Fe (44%), Mn (43%), and Zn (24%) (.)Regarding root micronutrients, ‘Raccoon’ Fe was the highest, ranging from 820  to 1350 mg kgmg kg−1) . In ‘GazZn (24%) .−1 (when K was provided at 3.0 mmolc L−1) for ‘Raccoon’ and from 30.5 (deficient K) to 73.5 g kg−1 (3.0 mmolc L−1 K) for ‘Gazelle’, while N ranged from 43.4 to 49.3 g kg−1 for ‘Raccoon’ . The data showed a similar increase for ‘Raccoon’ at the same NaCl dose  as if a small increase in salinity had favored root development  NaCl but decreased significantly at 100 mM NaCl, and further at 200 mM NaCl [−1 (60 mmolc L−1) would not affect either GP or seedling growth of Gazelle (c L−1 NaCl (7.0 dS m−1). It has been previously shown that the lack of response to K fertilization at high levels of NaCl could be explained, at least in part, by the reduction in plant growth caused by NaCl ion toxicity, which limits the absorption of water and nutrients from the soil [c L−1 may reflect the fact that ‘Gazelle’s seedlings had more time to adjust to salinity.While there was no effect of K dose on germination, ‘Raccoon’ had its germination improved by salinity while ‘Gazelle’ was more tolerant to salinity than ‘Raccoon’ during germination and seedling establishment. Different researchers cited in the introduction demonstrated that crop germination decreased with increased salinity of irrigation water. The salinity threshold of approximately 120 mmolGazelle’  was high Gazelle  if irrig Gazelle  clearly the soil . Althougc L−1 of NaCl [c L−1 in this study resulted in Na shoot accumulation of approximately 50–60 g kg−1, smaller than the 60–70 g kg−1 of shoot Na reported previously [c L−1 of Na. The same can be said for Cl. Our data indicate that spinach plants have control mechanisms triggered by salinity that allows plants to control salt tissue accumulation once Na and Cl reach a certain level. However, the salinity threshold beyond which the plant will no longer be able to control its tissue accumulation of Na and Cl has not been determined. It is noteworthy to mention that in naturally-saline waters, the concentration of Cl− can be as high to twice as high as the concentration of Na+.The significant increase in both Na and Cl in roots and shoots of both cultivars was expected with increasing NaCl concentrations. The steady and stepwise increase in tissue Cl, which surpassed the increase in tissue Na , indicat of NaCl . Intereseviously  for the c L−1 in K was enough to show that K tissue accumulation was antagonistic to Na tissue accumulation. These results agree with a previous report that concluded that a 20-fold increase in K from 0.25 to 5.0 mmolc L−1 decreased Na tissue accumulation [The ratio of Na:K is considered an important measure of tolerance of a species or cultivar to salinity. However, in the case of these two spinach cultivars, this ratio only increased with salinity because Na tissue levels increased significantly at every step while K tissue concentrations remained constant . Howevermulation .42− to compensate for a constant concentration of NO3− in the leaf , and in response to a steady increase of Na+ in saline irrigation waters, as suggested by others [Plants maintained adequate concentrations of N, P, and K in roots, regardless of salinity or K dose. Although Ca, Mg, and S decreased significantly with salinity, regardless of K dose, plants maintained a minimum concentration of these macronutrients needed for growth ,47. Rooty others . Similary others  or when y others .Regarding root micronutrients, Fe and Mn were the highest among other micronutrients tested. Although salinity significantly reduced Fe, Cu, and Mn (and Zn in ‘Gazelle’), these micronutrients still were present at sufficient concentrations for root development. In a study evaluating the effect of combined salinity with N doses in spinach, the authors reported a significant decrease in Fe with increased salinity, in agreement with our results but, contrary to our results, they reported small, but significant, increases in Zn and Cu as salinity increased , which c42− ions under an increased concentration of Na+, as previously discussed under root mineral composition, and as suggested by others [+ retention in roots [+ in both roots and shoots, regardless salinity increase in irrigation waters or the low availability of K in irrigation waters. This K homeostasis against its availability in soil or water was discussed at the molecular level elsewhere [Our shoot macronutrient data for both ‘Raccoon’ and Gazelle clearly established that concentrations of N, P, and K were maintained in shoots across salinity levels . The incy others . Similary others . It has in roots . The higlsewhere .Interestingly, shoot micronutrients, in general, showed no significant differences in either cultivar as salinity increased. The concentrations of Fe and Mn were also the highest, as they were in roots, but at much lower magnitudes .c L−1), also under elevated irrigation-water salinity [Homeostasis of N, P, and K was maintained in both spinach cultivars, regardless of the significant increase in Na and Cl and regardless the fact that three of the K doses were from 10 to 43 times lower than what was considered sufficient for spinach growth in previous works ,40. The salinity .c L−1 [c L−1 at the lowest K dose suggests that plants of both cultivars benefited from NaCl when K was deficient  at 30 and 60 mmolc L−1 . The SDWeficient . Althougc L−1) showed a significant decrease in dry shoot biomass for ‘Raccoon’ after 30 mmolc L−1 when K was 5.0 mmolc L−1, while ‘Raccoon’ significantly increased SDW with 30–60 mmolc L−1 NaCl when K was 0.25 mmolc L−1 [c L−1 was also observed for both ‘Raccoon’ and ‘Gazelle’ when K dose was 0.07 mmolc L−1 in this experiment  of 8.9 to 10.4 dS m−1, respectively, at the end of the experiment. These soil–paste salinities correspond to irrigation-water salinities (ECiw) of 19.6 and 22.9 dS m−1, respectively, thus explaining the significant decrease in shoot biomass at these two salinities. In this study, plants were cultivated in an all-sand medium to reduce K to deficient levels for seven weeks under the salinity treatments, and a leaching fraction of 0.30 was applied. Thus, the low effect of salinity on SDW, even at 160 mmolc L−1 (ECiw = 17 dS m−1), can be partly explained by the fact that the sandy medium allowed a more efficient leaching of NaCl, preventing salinity build-up in the pot, and partly because spinach plants may have adapted to NaCl during the seven weeks (50 days) of cultivation, while in a previous experiment with ‘Raccoon’, plants were only allowed to grow under salinity for only 28 days [Previous results with the same cultivars grown in soil under lower NaCl concentrations  [c L−1 used  was from 23 to 50 times lower than recommended for spinach fertilization [c L−1 of K combined to a soil salinity of ECe = 6.0 dS m−1 [iw of 13.2 dS m−1 in this study. However, our results disagree with a previous study that reported that spinach plants benefitted from extra K when salinity increased from 50 to 250 mM NaCl [c L−1, plants grew well up to 80 mmolc L−1 NaCl (ECiw = 9.3–9.8 dS m−1), regardless of the K doses and had no significant decrease in plant dry weight [−1, without salinity, reported that spinach shoot dry weight showed no difference among all the K doses tested [−1. In our experiment, spinach shoot biomass was not generally affected by salinity up to 160 mmolc L−1, despite significant shoot reductions in Ca, Mg, and S, and regardless of the very low doses of K provided. Thus, spinach plants behaved unlike what was expected for glycophytic plants irrigated with water electrical conductivity (ECiw) up to 17 dS m−1 (160 mmolc L−1 NaCl) combined with deficient K in the growth medium.Our results clearly indicate that K requirements of spinach are much lower relative to the ones previously recommended for the crop (63 to 138 kg haolc L−1) . Despitelization , plants  mM NaCl . In a sas tested . Howevere of 2.0 dS m−1 (estimated ECiw = 4.4 dS m−1) [iw of 17 dS m−1 tested here, we can estimate an ECe = 7.7 dS m−1 for ‘Raccoon’ and Gazelle, or 3.85-fold higher than the ECe provided for spinach (no cultivar mentioned) by the previous authors.Our results show clear differences in shoot biomass between seedlings and older spinach plants in response to the same water-salinity treatments. Plants exposed to salinity at the 6–8 true-leaf stage and allowed to grow for seven weeks into adult plants were more salt-tolerant than seedlings. Eggplant varieties subjected to increasing salinity during germination and seedling stages also responded differently depending on plant growth stage, with salinity tolerance of all cultivars increasing at the later growth stages . Spinach dS m−1) ,55,56. Bc L−1 for Gazelle, indicating that Gazelle was more tolerant to salinity than ‘Raccoon’ during germination and seedling establishment, the most salt-susceptible stage. Interestingly, germination % of ‘Raccoon’ improved significantly at 60 mmolc L−1 NaCl, suggesting that moderate salinity can improve the germination of this cultivar. Our results also indicate that the responses of spinach to salinity depends on the plant growth stage and on the cultivar. At the later growth stage, ‘Raccoon’ and Gazelle were both tolerant to irrigation waters of moderate to high salinity.In general, spinach did not respond to K doses either during germination or vegetative growth and there was no interaction between salinity and K. However, salinity decreased germination parameters of Gazelle at the highest NaCl dose, and drastically reduced seedling biomass at every increase in NaCl for ‘Raccoon’ but only at 120 and 160 mmolc L−1, equivalent to a range of 23- to 50-fold less K than recommended for the crop), adult plants maintained K concentrations over the minimum required (20 g kg−1 SDW) for plant growth. Therefore, mature plants are more salt tolerant than young plants and saline waters would be better adapted to the production of “freezer spinach” (vegetatively mature plants) instead of “baby spinach” (young plants).Mature plants of both cultivars accumulated large concentrations of Na and Cl in both roots and shoots, and these ions could have contributed to osmotic balance to maintain cell turgor without causing symptoms of ion toxicity. Both cultivars maintained their macro and micronutrient at adequate, or close to adequate, tissue levels for growth. Even under severe K deficiency (0.07 mmolc L−1 NaCl). This demonstrates that the improvement in the nutritional status of the plant does not overcome the osmotic effects associated with the increase in the total concentration of salts in the root zone.The increase in K from deficient to sufficient reduced the Na:K ratio in the shoots, regardless of the level of salinity but the beneficial effects of K on RDW and SDW were observed only at lowest level of salinity , as an osmoticum to maintain cell turgor but not as a substitute of K+ in its physiological function, salinity treatments above control levels had similar concentrations of Cl−. Regarding mineral tissue accumulation, Cl tissue accumulation was as substantial as that of Na. Interestingly, Cl is recognized to be essential to plant growth only at micronutrient levels (mg kg−1), while Na is a needed nutrient for the growth of halophytes, but not of glycophytes, such as spinach. The spinach cultivars studied here have demonstrated to be salt tolerant and our results for Na:K ratio confirmed the results of a previous study with the same cultivars that reported that sufficient K decreases Na tissue accumulation.Plants from both cultivars seemed to benefit from salinity at the lowest K dose of 0.07 mmolOf course, these results cannot be directly extrapolated to field cultivation of spinach in general because salinity tolerance depends on the cultivar, type of soil, season, and irrigation frequency applied. What is clear is that K fertilization can be significantly decreased to increase farmer profits. These results also raise the question: Is K the only mineral that can be drastically reduced in spinach cultivation or would similar shoot biomass be achieved with N reduction? At a time that more food is needed to feed a growing world population and freshwater becomes scarcer and more expensive, or is unavailable, for irrigation in arid and semiarid areas of the planet, we must revisit mineral needs for crops cultivated under saline irrigation as a way to maintain food production, increase farmer’s profits, while decreasing agriculture footprint and slowing down the salinization of our soils and water resources.Salicornia spp., Sarcocornia spp., Suaeda spp., Chenopodium spp., and Atriplex spp., which have all adapted to saline and arid environments. The fact that germination percent improved with time and that plants showed no significant decrease in biomass at the adult stage, regardless of K dose and after 50 days of irrigation with moderate to high-salinity waters lead us to conclude that spinach can be successfully cultivated with saline recycled waters in arid and semiarid regions and requires much less K fertilization than previously recommended.Spinach seems to be a salt-tolerant glycophyte in a family (Amaranthaceae) that has halophytes such as


Scientific Reports 10.1038/s41598-020-70911-4, published online 19 August 2020Correction to: This original version of this Article contained errors.In the original version, the author Galit Levi Dunietz was incorrectly indexed.In Figure 1, the description for “Laboratory evaluation” was incomplete.“Blood samples for fasting glucose, insulin, HbA1c, and CRP were drawn on the morning following the overnight sleep study”now reads:"Blood samples for fasting glucose, insulin, HbA1c, lipid panel and CRP were drawn on the morning following the overnight sleep study"In Figure 2C, the colour of the lines used to identify “offspring of mothers with mild SDB” and “offspring of mothers without SDB” was erroneously inverted.These errors have now been corrected in the PDF and HTML versions of the Article.