Maternal salinity influences anatomical parameters, pectin content, biochemical and genetic modifications of two Salicornia europaea populations under salt stress

Salicornia europaea is among the most salt-tolerant of plants, and is widely distributed in non-tropical regions. Here, we investigated whether maternal habitats can influence different responses in physiology and anatomy depending on environmental conditions. We studied the influence of maternal habitat on S. europaea cell anatomy, pectin content, biochemical and enzymatic modifications under six different salinity treatments of a natural-high-saline habitat (~ 1000 mM) (Ciechocinek [Cie]) and an anthropogenic-lower-saline habitat (~ 550 mM) (Inowrocław [Inw]). The Inw population showed the highest cell area and roundness of stem water storing cells at high salinity and had the maximum proline, carotenoid, protein, catalase activity within salt treatments, and a maximum high and low methyl esterified homogalacturonan content. The Cie population had the highest hydrogen peroxide and peroxidase activity along with the salinity gradient. Gene expression analysis of SeSOS1 and SeNHX1 evidenced the differences between the studied populations and suggested the important role of Na+ sequestration into the vacuoles. Our results suggest that the higher salt tolerance of Inw may be derived from a less stressed maternal salinity that provides a better adaptive plasticity of S. europaea. Thus, the influence of the maternal environment may provide physiological and anatomical modifications of local populations.

Salt stress is one of the main environmental factors that limits growth of plants worldwide. An environment with high, medium or low salinity may impact plants' ability to tolerate high salinity, and this impact varies between and within species. In heterogeneous environments such as natural and anthropogenic sites, plants could develop multiple strategies through producing offspring that differ in their salt stress tolerance 1,2 . For instance, according to El-Keblawy et al. 3 , the halophyte Anabasis setifera with phenotypic plasticity is more able to survive in harsh environments conditions due to the maternal environment being able to produce progeny that fit specific habitats well. Maternal effects might influence adaptive plasticity between generations, which can be considered an adaptive evolution due to the advantage conferred to the offspring reflected by an increased survival 4,5 . Many studies have demonstrated that maternal habitats can cause plants' growth to respond differently depending on environmental conditions and, subsequently, this affects the next generation 3,5,6 . Some studies have reported contradictory results: for instance, El-Keblawy et al. 3 showed that the halophyte Anabasis setifera has greater salt tolerance when taken from non-saline habitat as compared to a population from a low saline habitat (17.5 mS cm −1 ), while Van Zandt and Mopper 4 reported that Iris hexagona seeds from maternal high salinity germinated earlier and in greater quantity than did seeds from low salinity plants. Currently, many

Results
Morphometrical parameters in salinity gradient. Overall www.nature.com/scientificreports/ increase of 128% and 246% was observed between the minimum and maximum A for Cie and Inw, respectively. Between populations, A showed the highest difference between Cie and Inw at 1000 mM NaCl, with an increase of 159% in Inw with respect to Cie. The degree of succulence (S) of the stems was also calculated, these results adequately show the change between salt treatments, the values are in accordance to Delf 17 report. The highest S is observed for Inw population at 1000 mM. Then, a maximum increase in the cell's diameter (Cdiam) and roundness (R) of water-storing cells was observed from 0 to 1000 mM NaCl, for Inw population, 75.5%, 11.3% respectively, while Cie population, has its maximum Cdiam 49.7% increase from 0 to 200 mM NaCl treatments, and R increases 11.5% from 0 to 600 mM NaCl (Fig. 2b,c). Therefore, a significant different behaviour from Cie and Inw population was detected as shown in 3D plot (Fig. 2d) which comprises the three morphological parameters of both populations through the 6 salinity treatments. Inw population showed a wider distribution suggesting a better adaptation during the experimental salinity stress with respect to Cie which presents a reduced distribution in the 3D plot.
Biochemical modification to salt stress. Proline (P) showed an increase with salinity gradient (Fig. 3a).
The results show that P was significantly higher in Inw in comparison to the Cie population under salt stress, mainly at 400, 800 and 1000 mM. Meanwhile, hydrogen peroxide (HP) was significantly higher in Cie with respect to Inw population through 0, 200, 600, 800 and 1000 Mm NaCl treatments. A significant increase was observed at 800 and 1000 mM NaCl in Cie and very slightly at 1000 mM NaCl in Inw population. Regarding the enzyme activity analysed, peroxidase (POD) activity increased markedly at 800 and 1000 mM NaCl in Cie with respect to Inw, which maintains a homogeneous low POD activity through all the treatments. These results correlate with the HP analysis (Fig. 3b,c). Then, the lowest catalase (CAT) activity for Inw was found at medium salinity treatments 200, 400, 600 mM, while the highest CAT activity was found at the extremes (0, 800 and 1000 mM), in contrast, for Cie population the CAT activity decreased along with the salinity gradient, both populations showed significant difference between them in all the treatments (Fig. 3d).
Chlorophyll a (Cha), b (Chb) and carotenoid (Carot), content show a remarkable decrease in both populations under NaCl stress ( Table 1). The chlorophyll content among Inw and Cie was significantly different in Cha at 200 mM and in Chb at 0 and 200 mM. No significant differences between the two populations were found in total chlorophyll content, but in carotenoid, the highest content was found only at 0 mM for Cie and at 0 and 200 mM in Inw. However, comparing both populations, significant differences were observed through all treatments. Interestingly the total soluble protein content was higher for Cie at 0 mM treatment and it progressively decreases along with salinity gradient, while it increases with salinity in Inw.
High and low methylesterified HGs content and distribution under salt stress. Immunofluorescence analysis of the location of high and low methylesterified HGs (HM-HGs and LM-HGs) showed variances in the total levels of methylesterified HGs as well as in their distribution through the semi-thin cross-section of the fleshy tissue among the stem, epidermis, palisade tissue, cortex, vascular bundles and vascular cylinder. An increase in the total intensity level of HM-HGs when subjected to salt stress was identified with JIM7 antibody in the stem cross-section for Inw, whereas for Cie the highest total intensity levels of HM-HGs were observed only at 200 mM NaCl, then a gradual decrease occurred along with the salinity gradient (Fig. 4a). LM-HGs homogalacturonans distribution identified with LM19 antibody show significantly higher levels of total intensity for Inw with respect to Cie in all salt treatments, with exception of treatment at 200 mM (Fig. 4b).
Also, the HM-HGs and LM-HGs quantity varied between treatments and populations as observed in Fig. 5a-f (Ciechocinek) vs.  Table 2. In particular, epidermis tissue showed Figure 1. Stem-cortex cell's area changes of S. europaea after 2 months in Cie (a-f) and Inw (g-l) populations grown under different NaCl concentrations. Scale bar 150 μm. n = 300 ± 50 cells, 12 individuals per treatment. S: correspond to the degree of succulence in the stems 17,24 . The F value of S that corresponds to the 2-way ANOVA for the interaction salt treatment × population is F 5,48 = 5.5; p < 0.001.  Table 2). For LM-HGs in Inw, non-significant differences were found between treatments ( Fig. 6h-l arrowheads, respectively; Table 2). In the palisade tissue a significant increase in LM-HGs is identifiable at 1000 mM NaCl for the Inw population (Fig. 6l, Table 2).

Scientific
Evaluation of the differences between S. europaea populations. All the variables were evaluated in each population using principal component analysis (PCA) (Fig. 7a); both populations show a similar tendency at the low salt treatments. Figure 7a shows the PC1 and PC2 axes, which accurately describe the variance of the samples (75.43%). This plot shows which plants are the most tolerant with regard to salt stress and how they correlate with the active variables that describe the low or high stress. It also shows that the Inw population seems to cope better with salinity. The biplot demonstrates that I1000 mM correlates well with the cell area variable, which is the morphometric trait that suggests Inw is less affected under stress salinity; this agrees with the image growth analysis reported in a previous salinity tolerance study for the same populations 25 . Variables  www.nature.com/scientificreports/ related to high stress, such as HP and POD, correlate better with high salinity treatments in Cie (Fig. 7a). This biplot also shows how the individuals move through the two-dimensional space of the main components, from the positive to the negative quadrant of PC1 as salinity increases. The results were also grouped on a 3D plot ( Fig. 7b) according to their similarities through the three principal component scores (PC1, PC2 and PC3) that describe the variance of the samples (84.87%), which shows that Cie plants are more susceptible to salt stress. Factorial scores from the PCA of each sample were used to calculate the distance between the two points under the same treatment P1 = (x 1 , y 1 , z 1 ) and P2 = (x 2 , y 2 , z 2 ) in the 3D space of the PCA (Fig. 7b). The comparisons of C0 vs. I0 (3.30) against C1000 vs. I1000 (8.39) were created in the 3D cartesian axis (x = PC1, y = PC2, z = PC3), with distance results indicating that the greater the stress, the greater the separation. In addition, the shortest distance C200 vs. I200 (2.12) is observed at the optimum salinity for S. europaea-growth, at between 200 and 400 mM NaCl.
Expression patterns of SeNHX1 and SeSOS1 genes involved in Na + segregation of S. europaea stem. The expression patterns of NHX1 and SOS1 in S. europaea stems under saline treatments were analysed with real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). These genes NHX1 and SOS1 encode a tonoplast Na + /H + antiporter and apoplast antiporter, respectively. SeNHX1 and SeSOS1 expression does not show a significant difference within treatments of the same population, but a significant difference in gene expression is visible between populations. SeNHX1 and SeSOS1 were equally expressed in the Inw population, while the Cie population showed the highest expression for SeSOS1 but very low SeNHX1 expression, as shown in Fig. 8. This confirms differences between these two populations. Different letters indicate significant differences between treatments within population and * indicates significant difference between populations within treatment (p < 0.05), n = 3.

Discussion
According to the S. europaea anatomical cells results obtained through image analysis, cell's area has a similar result under 0 mM NaCl in both populations, but significant differences were observed when populations were subjected to salt stress. The Inw population has the highest values for all cell parameters tested. The highest value observed was in Inw at 1000 NaCl. Our results are in accordance with Akcin et al. 26 , who demonstrated that Salicornia freitagii stem anatomical characters such as thickness, length and width of water-storing tissue significantly increased when the halophyte grows under high salinity. While the roundness of the cells analysed in this study show that at higher salinities, cells lose their natural hexagonal shape, therefore, this parameter was useful to determine that cells turn round probably due to the high-water storage within them. The highest roundness was observed for Cie at 400 and 600 mM and for Inw population at 800 and 1000 mM suggesting that these rounded cells store higher amount of water. These parameters, area of cells and roundness, can be associated with an increase in (S) succulence as a way to aid in storing additional water by increasing vacuolar volume for   [26][27][28][29] , who also showed that succulence is an adaptation mechanism in salt-tolerant cultivars subjected to saline stress. Results of succulence in the present work, adequately show the change between salt treatments which are associated to the area of the cells, large cells can be linked to high turgidity and hence to the S of the plant. In the same line, proline, which allows additional water to be reserved in the water storage cells from the environment, positively correlates with the anatomical analysis. The 22% and 40% higher results for P in the Inw population at 800 and 1000 mM NaCl, respectively, relative to the Cie population can be linked to the increased cell area and roundness in the Inw population. These features may allow cell water potentials to decrease 30,31 . Kumar et al. 31 demonstrated that between two cultivars of Morus alba L. subjected to salt stress, the proline metabolism was significantly altered and the extent of alteration varied between both cultivars, where proline accumulation was higher in the salt tolerant cultivar than in the salt sensitive one because higher content of proline leads to the maintenance of turgor by preventing the loss of water and ion toxicity, supporting its salt tolerance. Also, our results are in accordance with studies carried out by Aghaleh et al. and Akcin and Yalcin 30, 32 for S. europaea. Moreover, the Cie population showed the highest HP and POD values, especially at the highest salinity, with percentage differences of 285% and 219%, respectively, with respect to Inw; in the Fig. 3b,c we can determine which population is more salt-tolerant. According to Kong and Seo 33 , salt-tolerant cultivars showed less HP content compared to salt-sensitive cultivars, due to the effect of salinity induction of reactive oxygen species (ROS), such as HP, which severely reduced overall plant growth in sensitive species. In the present study, the results indicate that the Cie population is more salt-sensitive than is the Inw population. Aghaleh et al. 32 tested the effects of salt stress on the activities of antioxidative enzymes in two Salicornia species at NaCl concentrations (0, 100, 200, and 300 mM), finding that the salinity progressively enhanced the POD activity, whereas the CAT activity was only registered at the low salinity. POD and CAT play a key role in removing ROS produced in plant cells under abiotic stresses. In this study, the Cie population showed higher levels of POD activity under high salinity, probably due to the remarkably higher content of HP that this population has under high salinity with respect to Inw. Meanwhile, the decrease in photosynthesis activity when plants are subjected to salinity is reflected in the reduction of chlorophyll and CO 2 fixation due to the lower stomatal conductance 27,32 . Some plants grown under high salinity have a lower stomatal conductance as a strategy to conserve water 34,35 . Consequently, CO 2 fixation is reduced and photosynthetic rate decreases. The chlorophyll content of both populations was significantly different at 200 mM NaCl (Table 1), with no difference at high salinity. In this line, it is important to note that Chb type is an adaptive feature of adapted chloroplasts, because high Chb content produces an increase in the range of wavelengths absorbed by the chloroplasts, which is attributed as a mode of adaptation when plants  www.nature.com/scientificreports/ are subjected to some abiotic stressor 36 . In the present study, Inw showed a statistically significant higher Chb content compared to Cie under 0 and 200 mM treatments, while Inw was the one with higher Carot content as a sign of a better adaptability to salt stress. The lower content of protein in the Cie population under salt stress and higher content in Inw population, suggests the possible connection of protein with an osmotic adjustment that confers higher salt tolerance. The importance of protein for abiotic and biotic stress adaptation was thoughtfully reviewed by Sasidharan et al. 37 , who stated that the regulation of cell wall protein activity results in growth modulation during stress, and that this can be mediated by the regulation of wall modifying proteins that alter cell wall structure and allow it to yield to turgor, thus driving a cellular expansion, which was corroborated with the cell area analysed in this study for each population. According to Zagorchev et al. 12 , around 30 kDa proteins are involved in the cell wall rigidity, which plays a crucial role in plant growth and development during stress adaptation. With regard to high and low methylesterified HGs, levels and distribution were noticeably different in each population. As pectin is important for the cell wall structure and could be modified in response to different signals such as salt stress, analysis of pectins received major attention in the present study. Overall, it is already known that a large majority of the genes encoding proteins modifying cell wall structure, are down-or up-regulated under salt treatment. In the case of pectin, Fan et al. 38 reported that genes encoding methylesterases  www.nature.com/scientificreports/ inhibitor family proteins are up-regulated under saline conditions, which decreases the level of methyl esterification of pectins and affects their normal function by inhibiting pectin methylesterase activity. This behaviour was reflected in the present study for Cie, the less salt-tolerant population. However, differences in the content of HM-HGs may occur between salt-tolerant populations. For instance, Uddin et al. 39 indicate that under stress conditions the concentration of methylated pectic epitopes tends to drop, especially for those species that are less tolerant to salt stress. Meanwhile, Liu et al. 40 indicate that the degree and pattern of the methyl-esterification of pectin to some extent determines the stiffness of cell walls and, with this, the tolerance to salinity. In the same study it is stated that the overexpression of the gene (AtPMEI13) that causes a decrease in pectin methylesterified enzyme activity in Arabidopsis enhances the total levels of methyl-esterification pectins, which was reflected in an improvement in seed germination and survival growth rate under salt stress. Also, Le Gall et al. 11 reported that in salt-sensitive species, the high salinity triggers the de-esterification of loosely bound pectins that impede the swelling of cells, affecting the plant more than those with higher tolerance. According to Peaucelle et al. 15 , the de-esterification of homogalacturonans can lead to cell wall stiffening through the creation of "egg boxes", and to enzymatic degradation of pectin, which indicates a denser and less extensible cell wall. On the other hand, HM-HGs can be involved in remodelling the cell wall structure andmechanical properties, which under salt stress helps to regulate the cell wall elongation and cell shape for better water accumulation, which translates into higher resistance to abiotic factors such as salinity 11 , as confirmed in the present study. Herein, high methylesterified pectin was detected at high levels mainly in epidermis (ep) and in vascular bundles (vb) ( Table 2). This vb tissue corresponds to the collenchyma cells, which are elongated cells composed of cellulose and pectin, with irregularly thick cell walls that provide support and structure. These cells are often found under the epidermis and associated with vascular bundles. A study carried out on three maize hybrids with contrasting salt tolerances showed an accumulation of highly methylated pectin in the salt-tolerant maize genotype, which favoured their cells' elongation 39 . Another study reported by Muszyńska et al. 2 showed that high methylated pectin (identified by immunolabelling with JIM7 antibody) increased within the cell wall of Populus tremula under saline conditions. This increase was linked with a rise in the modulus of elasticity and a decrease in cell wall plasticity in order to keep the turgor pressure necessary for plant growth. According to these authors, under salt stress, cell walls of salt sensitive cultivars can became more rigid (less flexible) while turgor pressure is maintained. Thus, maintaining good cell wall flexibility might be part of the mechanism by which salt-tolerant cultivars adapt to environmental stresses. Pectin polysaccharides are also believed to play an important role in cell adhesion and tissue cohesion 41 , which would be very important for adaptation to stress in plants that live in saline environments. For instance, in the halophyte Sonneratia alba a decrease in calcium content was detected, which may be a strategy by this halophyte to reduce cell rigidity 42 . Meanwhile, Le Gall et al. 11 reported that, in Salicornia europaea, the genes encoding the cell wall proteins of the primary cell wall (including UDP-l-rhamnose synthase and cellulose synthases) decrease under saline conditions, while other genes that encode pectin methylesterase inhibitor proteins increase. Byrt et al. 43 reported that there are associations between higher cell wall pectin content and increased tolerance to salinity.
According to Rasouli et al. 44 , salinity altered the physical properties of epidermic cells, specifically in the guard cell wall. In their study they demonstrated that the cell-wall-modifying enzymes such as acetyl-and methyl-esterifications esterases of pectin were upregulated in the epidermic cells of the halophyte Chenopodium quinoa. They concluded that the methyl-esterifications of pectins at epidermis are critical for salt tolerance by increasing the mechanical strength in the guard cells that are exposed to salinity. So, pectin methyl-esterification is essential for plant responses to environment stresses, which was also observed in the present study through the quantification of the fluorescence of the high methyl esterified pectin. The Inw population had a higher level of HM-HGs in the epidermis cells than did Cie, through all the salt treatments. This finding may also be associated with the fact that pectins in guard cell walls provide strength and flexibility in order to accommodate the turgor-pressure-driven changes in size and shape that underlie the opening and closing of stomatal pores during abiotic stress factors 45 . Moreover, for the case of vascular bundles, Fan et al. 38 reported that under salinity the S. europaea genes involved in cell wall metabolism are well linked with the vessel differentiation increment in xylem. In this sense, highly methyl-esterified pectin together with lignin in xylem are the main passage for assimilation of water and mineral elements 46 . So, the accumulation of large amounts of salt in S. europaea shoots under salinity requires a more rigid support transport system from root to shoot, which may be an important strategy for this halophyte when adapting to salinity. This may explain our results with regard to the higher levels of HM-HGs in the vb for Inw population. The results of the correlation between investigated parameters are of great interest and some have not been reported before, especially the positive correlation between proline and cell area (0.728) ( Table 3); this result confirms that the higher the cell's area, the higher the proline content, which promotes plant succulence. Roundness has similar correlation tendency with proline (0.672) due to cell turgidity, while the plants under 0 mM were mainly described by higher total content of chlorophyll (Fig. 7a), and these two variables (R and TC) have a high significant negative correlation (0.781).
Moreover, the inverse correlation (− 0.688) between HM-HGs and HP is also an interesting finding, suggesting that when the plant is under salt stress, the chemical cell wall composition is restructured; this was observed in a reduction in pectin content along the salinity gradient. However, the more salt-tolerant population Inw increases its HM-HGs content, suggesting that this component is related to better salt resistance; furthermore, a significant positive correlation was detected between HM-HGs vs. A and Cdiam (0.605 and 0.639 respectively). Meanwhile, HP positively correlated with POD, as expected (0.863). The results in Fig. 7a illustrate population salt tolerance and how the two populations move through the biplot showing the active variables that correlate with the studied individuals depending on their salt tolerance. In the Inw population, individual I1000 correlates well with cell area and proline content, while in Cie, individuals C1000, C800 and C600 correlate well with HP and POD. Then, the C0 and I0 individuals correlate well with TC, Cha, Chb and Carot. Additionally, factorial www.nature.com/scientificreports/ scores (Fig. 7b) were useful in demonstrating that the highest separation between Inw and Cie parameters was found at the highest salinity, indicating also that Cie has more stress-modifications at this salinity level. The present gene expression results confirmed that Inw can be considered a more salt-tolerant plant in comparison to the Cie population. This is because, according to Lv et al. 47 , NHX1 is one of the Na + /H + antiporters in tonoplast responsible for Na + transport from cytosol to vacuole, and it plays a central role in salinity tolerance. In the present study, SeNHX1 was highly expressed only in the Inw population, suggesting the important role of Na + /H + in the Na + influx to vacuole in plant cells. For instance, Hayatsu et al. 42 reported that in the halophyte Sonneratia alba, the Na + content in the vacuole was higher than in glycophyte Oryza sativa, concluding that halophilic cells gain salt tolerance by transporting Na + into their vacuoles. Meanwhile, the SeSOS1 gene was highly expressed in the salt-sensitive population (Cie), suggesting that excreting Na + in apoplast is the main mechanism by which this population copes with salinity. Jha et al. 48,49 stated that the transcript of a Na + /H + antiporter gene from Salicornia brachiata (SbNHX1) increased under different NaCl concentrations. However, in the present study, no significant differences were found between salt treatments. This phenomenon may be attributed to the different plant species, salt treatments and experiment duration. Fan et al. 38 found that more than half of the differentially expressed transcription factors in Salicornia are directly or indirectly involved in the salt response but also in the growth and development process of S. europaea roots and shoots. So, only a small fraction participated exclusively in the stress response, which means that salinity efficiently induces the growth of S. europaea and that most of these genes can be activated through all the development growth process, independently of the salt content.
All the discussed results confirm our hypothesis that different maternal salinity populations of the same S. europaea species adapt differently to salt stress at the anatomical, pectin, biochemical and gene level, which can be important in the context of Salicornia europaea species as future crop 50 , especially considering seed sources.

Materials and methods
Plant materials, growth conditions and salt treatments. Soil samples were performed as in previous experiments with S. europaea 25 , seeds were collected at two maternal sites, the first of which represents natural salinity related to inland salt springs at the health resort of Ciechocinek (Cie) (52°53′N, 18°47′E) characterised by a high soil salinity of ca 100 dS m −1 (~ 1000 mM NaCl), and the second of which is associated with soda factory waste that affects the local environment in Inowrocław-Mątwy (Inw) (52°48′N, 18°15′E) and with a lower salinity of ca 55 dS m −1 (~ 550 mM NaCl). The complete soil description is reported in Piernik et al. 51 and Szymanska et al. 52,53 . Populations are isolated by a distance of ca 40 km without any saline environment between them, however, they were somehow connected due to the presence of salt springs in the nineteenth century. The seeds came from one generation and were collected in early November 2018. The seeds were germinated and grown according to the same steps reported in Cárdenas-Pérez et al. 25 with a slight modification in the number of salt treatments at 0, 200, 400, 600, 800 and 1000 mM NaCl. In total, 144 plants were cultivated, and, therefore, a complete randomised factorial design 2 6 was used, which included (12 plants × 6 treatments × 2 populations) with 14 response variables. After 2 months of development, anatomical analysis such as cell area (A), roundness (R) and maximum cell diameter (Cdiam) were estimated in 12 samples, whereas high and low methyl esterified pectins (HM-HGs and LM-HGs), proline (P), hydrogen peroxide (HP), total soluble protein (Prot), catalase activity (CAT), peroxidase activity (POD), chlorophyll a, b and total (Cha, Chb and TC), carotenoid (Carot) contents, as well as SeNHX1 and SeSOS1 gene expression, were all determined per triplicate (plants were randomly selected). The collection of plant material, comply with relevant institutional, national, and international guidelines and legislation, IUCN Policy Statement on Research Involving Species at Risk of Extinction and Convention on the Trade in Endangered Species of Wild Fauna and Flora. The voucher specimen of the plant material has been deposited in a publicly available herbarium of the Nicolaus Copernicus University in Toruń (Index Table 3. Pearson correlation matrix of the anatomical, biochemical and pectin content parameters. Values in bold are different from 0 with a significance level alpha = 0.05. www.nature.com/scientificreports/ Herbarium code TRN), deposition number not available (dr. hab. Agnieszka Piernik, prof. NCU undertook the formal identification of plant species, and permission to work with the seeds was provided by the Regional Director of Environmental Protection in Bydgoszcz, WOP.6400.12.2020.JC).
Anatomical image analysis. From the middle primary branch (fleshy segment shoot) of S. europaea plant treatments (0, 200, 400, 600, 800 and 1000 mM NaCl), slices of fresh tissue were obtained by cutting them with a sharp bi-shave blade. The thinner slices of approximately 0.5 mm were selected and used in the microstructure analysis. The size and shape of the stem-cortex cells from the fresh water-storing tissue were characterised by a light microscope (Olympus BX51, USA) connected to a digital camera (DP72 digital microscope camera) and digital acquisition software (DP2-BSW). The microscope images were captured at a magnification of 10 ×/0.30 in RGB scale and stored in TIFF format at 1280 × 1024 pixels. A total of 300 ± 50 cells from five individuals per treatment were analysed. Finally, the shape and size of the cells were obtained from the captured images. Cell image analysis (IA) was performed in ImageJ v. 1.47 (National Institutes of Health, Bethesda, MD, USA). The following anatomical parameters were obtained. Firstly, the cell area (A) was estimated as the number of pixels within the boundary. Secondly, the maximum cell's diameter (Cdiam) was determined by the distance between the two points separated by the largest coordinates in different orientations, and the cell roundness (R) was obtained through the equation R = (4 A)/(π (Cdiam) 2 )-where a perfectly round cell has R = 1.0, while elongated cells will show an R → 0. Finally, the degree of succulence (S) in stem was calculated according to 24 with slight change S = (Fresh Weight-Dry Weight)/stem Area, where the Area of the stem (As) was calculated as: As = π × r 2 , the diameter of the stems was obtained according to Cárdenas-Pérez et al. 25 .
Immunolocalisation experiments. The samples dissected from the middle segment of the shoot (3 individuals per treatment) were prepared for embedding in BMM resin (butyl methacrylate, methyl methacrylate, 0.5% benzoyl ethyl ether (Sigma) with 10 mM DDT (Thermo Fisher Scientific) according to Niedojadło et al. 54 .
Next, specimens were cut on a Leica UCT ultramicrotome into serial semi-thin cross sections (1.5 µm) that were collected on Thermo Scientific Polysine adhesion microscope slides. Before immunocytochemical reaction, the resin was removed with two changes of acetone and washed in distilled water and PBS pH 7. Fluorescence quantitative evaluation. For the quantitative measurement, each experiment was performed using consistent temperatures, incubation times and concentrations of antibodies. The aforementioned ImageJ (1.47v) software was used for image processing and analysis. The fluorescence intensity was measured for five semi-thin sections for each experimental population (Inowrocław and Ciechocinek) at the same magnification (100 ×) and the constant exposure time to ensure comparable results. The threshold fluorescence in the sample was established based on the autofluorescence of the control reaction. The level of signal intensity was expressed in arbitrary units (a.u.) as the mean intensity per μm 2 according to Niedojadło et al. 54 .
Biochemical analysis. Proline content (P) was measured according to Ábrahám et al. 55 . Five hundred milligrams of fresh stem material was minced on ice and homogenised with 3% aqueous sulfosalicylic acid solution (5 μl mg −1 fresh plant material), centrifuged at 18,000×g, 10 min at 4 °C, and the supernatant was collected. The reaction mixture: 100 μl of 3% sulphosalicylic acid, 200 μl of glacial acetic acid, 200 μl of acidic ninhydrin reagent and 100 μl of supernatant. Acidic ninhydrin reagent was prepared according to Bates et al. 56 . The standard curve for proline in the concentration range of 0 to 40 μg ml −1 . The standard curve equation was y = 0.0467x − 0.0734, R 2 = 0.963. P was expressed in mg of proline per gram of fresh weight. Hydrogen peroxide (HP) levels were determined according to the methods described by Velikova et al. 57 , and 500 mg of stem tissues were homogenised with 5 ml trichloroacetic acid 0.1% (w:v) in an ice bath. The homogenate was centrifuged (12,000×g, 4 °C, 15 min) and 0.5 ml of the supernatant was added to potassium phosphate buffer (0.5 ml) (10 mM, pH 7.0) and 2 ml of 1 M KI. The absorbance was read at 390 nm, and the HP content was given on a standard curve from 0 to 40 mM. The standard curve equation was y = 0.0188x + 0.046, R 2 = 0.987. HP concentrations were expressed in nM per gram of fresh weight. Chlorophylls (Cha and Chb) and carotenoids were extracted from fresh plant stems (100 mg) using 80% acetone for 6 h in darkness, and then centrifuged at 10,000 rpm, 10 min. Supernatants were quantified spectrophotometrically. Absorbance was determined at 646, 663 and 470 nm and calculations were performed according to Lichtenthaler and Wellburn 58 , when 80% of acetone is used as dissolvent. Total chlorophyll content was calculated as the sum of chlorophyll a and b contents. Total CAT activity was determined spectrophotometrically by following the decline in A 240 as H 2 O 2 (ε = 39.9 M −1 cm −1 ) was catabolised, according to the method of Beers and Sizer 59 . Decrease in absorbance of the reaction at 240 nm was recorded after every 20 s. One unit CAT was defined as an absorbance change of www.nature.com/scientificreports/ signal was recorded at the end of the extension step in each cycle. The specificity of the assay was confirmed by the melt curve analysis i.e., increasing the temperature from 55 to 95 °C at a ramp rate 0.11 °C/s. The fold-change in gene expression was calculated using LightCycler 480 Software release 1.5.1.62 (Roche, Penzberg, Germany).
Statistical and multivariate analysis. In order to determine the projection of the effect of salt treatment in plants we followed Cárdenas-Pérez et al. 25  The data was fit with a modified three parameter exponential decay using SigmaPlot version 11.0 66 . The relationships between variables were performed using a Pearson analysis, while a significance test (Kaisere Meyere Olkin) was performed in order to determine which variables had a significant correlation with each other (α = 0.05). Then, a 3D plot was developed using the three principal component factors according to the Kaiser criterion which stated that the factors below the unit are irrelevant. The three main factorial scores of the PCA from each sample were used to calculate the distance (D) between the two points (populations) under the same treatment P1 = (x 1 , y 1 , z 1 ) and P2 = (x 2 , y 2 , z 2 ) in 3D space of the PCA (Eq. 1).
where x, y, and z are the three main factorial scores in the PCA corresponding to the evaluated treatment in Inw and in Cie. Distances were used to evaluate and determine in which salt treatment the greatest differences between the populations were recorded.

Conclusions
This work shows that cell's image analysis was efficient at evaluating the salinity-anatomical modification response of S. europaea and can be used to identify differences between populations coming from different maternal salinities. By analysing the cell parameters of area and roundness, we can conclude that these parameters are a good indicator of both succulence and plant salinity tolerance. The biochemical analysis proved that anatomical parameters that confer higher salinity tolerance strongly correlate with the cell's modifications, as confirmed by the Pearson correlation, which highlighted the relationships between anatomical and biochemical parameters. PCA provided evidence that the plants from the anthropogenic saline (Inw) with lower maternal soil salinity (~ 550 mM) habitat are more tolerant to saline stress at laboratory conditions than are those from the natural site with high maternal soil salinity (~ 1000 mM). Our results suggest that the higher salt tolerance of the Inw population may be derived from the maternal salinity being less stressful, and from the better adaptive plasticity of S. europaea. Based on our analysis as a whole, it is clear that our applied methods are able to demonstrate that the two S. europaea populations from different maternal habitats do indeed have different mechanisms of salt adaptation at a cellular and biochemical level at high salinities, as well as a positive salt-tolerance effect under lower salinities. The gene expression analysis suggested the important role of Na + sequestration into the vacuoles and confirmed that the Inw population can be considered the most salt-tolerant in comparison to Cie. Therefore, these results can be used in the future for the selection of resistant plants. The present correlation results between anatomical and biochemical modifications vs. maternal soil salinity are novel in the study of salt-resistant plants, meaning that researchers can apply this correlation analysis straightforward, for future experiments related to plant salinity-development responses. Although further studies are required, these preliminary results support the idea that maternal effects influence offspring physiology under stress environments. However, future studies are required to consider the ecological context in which plasticity and maternal effects are expressed, such as studies of the patterns of natural populations in term of their environmental heterogeneity.