Domestication influences morphological and physiological responses to salinity in Brassica oleracea seedlings

AoB PLANTS, 2019, 1–14 doi:10.1093/aobpla/plz046 Advance Access publication XXXX XX, 00 Studies AoB PLANTS, 2019, Vol. 11, No. 5 doi:10.1093/aobpla/plz046 Advance Access publication August 9, 2019 Studies Copyedited by: AS 1 Received: 21 December 2018; Accepted: 29 July 2019 © The Author(s) 2019. Published by Oxford University Press on behalf of the Annals of Botany Company. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Studies Domestication influences morphological and physiological responses to salinity in Brassica oleracea seedlings M. Lema1*, Md.Y. Ali2 and R. Retuerto1 1Department of Functional Biology, Faculty of Biology, University of Santiago de Compostela, Santiago de Compostela, 15782 Spain, 2Agrotechnology Discipline, Life Science School, Khulna University, Khulna, 9208 Bangladesh *Corresponding author’s e-mail address: mar[email protected] Associate Editor: BaorongLu Citation: Lema M, Ali MdY, Retuerto R.2019. Domestication influences morphological and physiological responses to salinity in Brassica oleracea seedlings. AoB PLANTS 11: plz046; doi: 10.1093/aobpla/plz046 Abstract Brassica oleracea cultivars include important vegetable and forage crops grown worldwide, whereas the wild counterpart occurs naturally on European sea cliffs. Domestication and selection processes have led to phenotypic and genetic divergence between domesticated plants and their wild ancestors that inhabit coastal areas and are exposed to saline conditions. Salinity is one of the most limiting factors for crop production. However, little is known about how salinity affects plants in relation to domestication of B.oleracea. The objective of this study was to determine the influence of domestication status (wild, landrace or cultivar) on the response of different B.oleracea crops to salinity, as measured by seed germination, plant growth, water content and mineral concentration parameters at the seedling stage. For this purpose, two independent pot experiments were conducted with six accessions of B.oleracea, including cabbage (group capitata) and kale (group acephala), in a growth chamber under controlled environmental conditions. In both taxonomic groups, differences in domestication status and salt stress significantly affected all major process such as germination, changes in dry matter, water relations and mineral uptake. In the acephala experiment, the domestication × salinity interaction significantly affected water content parameters and shoot Na+ allocation. At early stages of development, wild plants are more succulent than cultivated plants and have a higher capacity to maintain lower Na+ concentrations in their shoots in response to increasing levels of salinity. Different responses of domesticated and cultivated accessions in relation to these traits indicated a high level of natural variation in wild B.oleracea. Exclusion of Na+ from shoots and increasing succulence may enhance salt tolerance in B.oleracea exposed to extreme salinity in the long term. The wild germplasm can potentially be used to improve the salt tolerance of crops by the identification of useful genes and incorporation of these into salinity-sensitive cultivars. Keywords: Brassica crops; domesticated-wild; Na+ exclusion; salt stress; succulence. Introduction Exposure of plants to salt stress has become a threat to human civilization as it reduces the yield of most crops, which limits the goal of higher food production for the world’s ever-growing population. Also of concern is the fact that many areas are increasingly affected by salinization, mainly in intensively cropped and densely populated areas of the world. According Downloaded from https://academic.oup.com/aobpla/article-abstract/11/5/plz046/5545357 by Univ de Santiago de Composrela user on 14 April 2020 Copyedited by: AS 2 | AoB PLANTS, 2019, Vol. 11, No. 5 to the FAO (2017), ~800 million hectares of land are currently affected by salinization, and as a result 0.25–0.50 million hectares of agricultural land are lost every year. The main constraint to production is that very few crops are salt-tolerant, and cultivars of particular crop species vary greatly in relation to this trait (Greenway and Munns 1980; Ashraf and Sarwar 2002). Crop plants are generally glycophytic (i.e. sensitive to high salinity), although cabbage (Brassica oleracea var. capitata) is considered moderately sensitive to salinity (Bernstein etal. 1974). Kale (B. oleracea var. acephala) reaches acceptable growth rates and can tolerate saline irrigation during early stages of growth, and is therefore a potential candidate for use in drainage water reuse systems (Shannon etal. 2000). Wild B.oleracea grows in saline environments, inhabiting sea cliffs, where it cannot evade the effects of salt. The species is therefore expected to have developed at least some degree of salt resistance, using different mechanisms to avoid (salt regulation by exclusion, elimination, dilution and/ or compartmentalization) or tolerate the effects produced by the increased concentrations of ions. Significant morphological and physiological differences between domesticated and wild accessions have been found in maize (Chinchilla-Ramirez et al. 2017) and also in B. oleracea, among other crop species, under artificially imposed stress (Matesanz and Milla 2018). Genetic diversity in relation to salt tolerance and other traits originally present in wild B.oleracea has been reduced in modern cultivars due to human-mediated selection, which has caused several genetic bottlenecks. However, it can be assumed that some classes of alleles, in many cases with minor effects, may have been missed during domestication and/or crop improvement and remain unexploited in modern cultivars (Prada 2009). It is therefore important to explore how salt tolerance has changed between ancestors (wild forms) and current domesticate genotypes (landraces and modern cultivars). The main difference between landraces (local varieties) and cultivars is that landraces are grown in agricultural ecosystems, whereas cultivars arise as the result of formal breeding programmes. Most modern cultivars descend from a relatively small number of founder landraces (Prada 2009). Consequently, the genes controlling agronomic characters in current cultivars retain low levels of diversity relative to the entire gene pool (landraces and wild forms) from which they arise. For this reason, domestication status may be a determining factor in salt tolerance. Cultivated B.oleracea includes important vegetable and forage crops grown throughout the world, representing, together with cereals, the basis of world supplies (Cartea etal. 2011). Farmers have selected different forms of domesticated B. oleracea to produce crops with distinct uses and characteristics: broccoli, Brussels sprouts, cabbage, cauliflower and kale, among others. The diversification of B.oleracea, caused by selection processes, has led to differentiation of several botanical varieties or groups, such as italica, gemmifera, capitata, botrytis and acephala, corresponding, respectively, to the above-mentioned crops (Branca and Cartea 2011). Different B.oleracea crops have been demonstrated to display high phenotypic and genetic diversity (Cartea et al. 2003; Padilla et al. 2007; Branca 2008; Vilar et al. 2008), probably in relation to regional preferences, resulting in numerous choices of edible forms. Despite the relatively high variability in B.oleracea crops, the genetic variation could be further increased to maintain and extend the cultivation of Brassica species in suboptimal land affected by salt stress. According to Arzani and Ashraf (2016), the in situ genetic resources that have enabled the plants to adapt to survive and grow in saline habitats are crucial to increase the genetic variability in relation to salinity tolerance in crops. Investigation of whether such variation is found in wild Brassica and exploration of the underlying mechanisms of salinity tolerance are decisive for advancement in breeding programmes and increased production in saline environments. Domesticated (commercial or elite cultivars, germplasm and landraces) and wild forms could be crossed to broaden the genetic base and improve the salt tolerance of B. oleracea crops. Breeders should be aware of the high cost of recovering the genetic background of the receptor elite cultivar when wild species are used to enhance salt tolerance (Cuartero etal. 2006). Nevertheless, recovering the background of the elite cultivar from crosses between kale and wild B.oleracea may be feasible, taking in account that kale may represent an ‘evolutionary bridge’ for the other taxonomical groups included in B.oleracea (Branca and Cartea 2011). For most crop species, several evaluation and selection programmes have led to the establishment of germplasm collections. Conventional breeding programmes and other more innovative approaches, such as the use of in vitro selection, interspecific hybridization, marker-assisted selection, transformation, mutation and polyploidy, have been used to enhance salt tolerance in different crops (Flowers et al. 1997; Flowers 2004; Cuartero etal. 2006). However, salt-tolerant cultivars remain scarce, probably due to the quantitative nature of this trait, in which numerous genes have small effects. Several authors have demonstrated the existence of variations in salt tolerance among species belonging to the genus Brassica (Ashraf etal. 1999, 2001; Ashraf and McNeilly 2004; Agarwal etal. 2008; Kumar etal. 2009) and also within some Brassica species (Maggio etal. 2005; Ali etal. 2006) and crops (Sanoubar etal. 2016). For crop improvement, evaluation of salt sensitivity in all growing phases has been recommended to identify and prioritize efforts on the most saltsensitive developmental stage (Cuartero et al. 2006). The early stage of growth has been recognized as the most vulnerable life stage in plants, as weak seedlings do not reach the reproductive stage under stressful conditions (Chinchilla-Ramirez etal. 2017). The initial growth stage will be crucial for stress tolerance, as selective pressures will act on the most relevant seedling traits to overcome the restrictive conditions. Plants that are resistant/ tolerant to abiotic stress at initial growth stages will probably show enhanced resistance at later developmental stages. Research findings in diverse fields suggest that salt tolerance has changed during domestication and selection processes; however, no studies comparing Brassica genotypes with contrasting domestication status have been carried out. We aimed to determine whether traits leading to salt tolerance in wild B.oleracea that survives on sea cliffs have been lost during domestication and/or improvement processes. Although the effects of soil salinity on the performance of Brassica species is well known and widely documented (see above-mentioned references), studies involving accessions belonging to different botanical varieties or groups are scarce. The objective of this study was to determine the influence of domestication status (wild, landrace or cultivar) on the response of B.oleracea, including the taxonomic varieties acephala (kale) and capitata (cabbage), to increasing levels of salinity, as measured by germination, plant growth and allocation traits, and water content and mineral concentration parameters. Materials and Methods Plant material and growth conditions For this research, two independent experiments were carried out with the capitata (cabbage) and acephala (kale) groups of Downloaded from https://academic.oup.com/aobpla/article-abstract/11/5/plz046/5545357 by Univ de Santiago de Composrela user on 14 April 2020 Copyedited by: AS Lema etal. – Domestication influences salinity in Brassica oleracea | 3 B.oleracea. Experiment 1 (‘capitata experiment’) was conducted between October 2013 and January 2014 and included cabbage accessions with different domestication status: the commercial and extensively used cultivar ‘Corazon de Buey Grande’ (capitata cultivar, Rocalba S.A.), the landrace MBG-BRS535 (capitata landrace—a local variety collected from Galegos, Lugo, Spain at 42.9385°N, −7.5685°W) and the wild accession EXT-BRS0151 (wild-capitata—collected from sea cliffs in Foz, Lugo, Spain at 43.6279°N, −7.3340°W; Fagundez et al. 2015). Experiment 2 (‘acephala experiment’) was conducted between September and December 2014 and included kale accessions with different domestication status: the commercial and extensively used cultivar ‘Gigante Caballar’ (acephala cultivar, Rocalba S.A.), the landrace MBG-BRS0510 (acephala landrace—a local variety collected from Baleira, Lugo, Spain at 43.0285°N, −7.1756°W) and the wild accession EXT-BRS0217 (wild-acephala—collected from sea cliffs in Cudillero, Asturias, Spain at 43.5664°N, −6.1478°W). The six accessions were provided by the Brassica Genebank held in the Misión Biológica de Galicia (MBG-CSIC),Spain. In both experiments, plants were maintained under controlled environmental conditions in a plant growth chamber (temperature 24± 2°C, 50–60% relative humidity, photoperiod of 16 h light and 8 h dark and 500–550 μmol photons m−2 s−1 of photosynthetically active radiation) belonging to the Department of Functional Biology, University of Santiago de Compostela, Spain. Salt treatments The experiments included four treatments with different concentrations of NaCl in the irrigation water: 0, 50, 100 and 150 mM NaCl. The electrical conductivity (EC) values for 0 (control), 50, 100 and 150mM NaCl solutions were, respectively, 1.45, 5.15, 9.48 and 13.28 dS m−1. Ten-day-old seedlings grown under non-saline conditions were transferred in 1-L plastic pots with perforated bottom filled with organic substrate (‘Sondermischung’, Gramoflor GmbH & Co. KG). One month after the seedlings were transplanted, sodium chloride was added to the irrigation water. The four treatments were arranged in a completely randomized design. Plants belonging to both groups (acephala and capitata) were exposed to salt treatments for 3months. Three times a week, the pots were placed in a tray filled with water with the respective amount of salt, and the soil was allowed to reach 100% water holding capacity by being soaked for 4h in the saline solution. The pots were then returned to the culture shelves in the growth chamber. Germination Three replications of 20 seeds per accession were germinated in Petri dishes on filter paper soaked with sodium chloride solution (50, 100 and 150mM NaCl) or non-saline water (control—0mM NaCl). Seed germination was evaluated daily, and those seeds in which the radicle had emerged through the seed coat were considered to have germinated. The germination percentage was recorded after 10 days. The mean germination time (MGT=−∑Dn/∑n, where ‘n’ is number of seeds emerged on day, and ‘D’ is the number of days from sowing to the emergence) was calculated according to Abbas etal. (2013). In the capitata experiment, after 10days none of the seeds of the wild accession were able to germinate at 150mM. Plant growth and water content parameters At the end of the experiments, all plants (n = 240, 10 plants by 4 treatments and 6 accessions) were de-potted along with substrate. The roots were then carefully washed with flowing tap water to remove the soil. The plants were separated into roots and shoots (leaves and stems), and the fresh weight was measured with a digital balance (Mettler Toledo mod. ML203/01, 220± 0.001g, Columbus, OH, USA). The plant parts were then oven-dried separately at 40 °C for at least 7 days to constant weight, and the root and shoot dry weights were measured. The values of root mass ratio (RMR = root mass/plant mass), shoot mass ratio (SMR = shoot mass/plant mass) and total water content [TWC = (leaf fresh mass − leaf dry mass)/leaf fresh mass] were calculated. Salinity response models were constructed and salt-tolerance index (ST-index) was calculated for each accession, according to Steppuhn etal. (2005a, b). For this purpose, relative yield (Yr) was calculated by dividing the absolute yield (Y, total plant dry mass) by dry mass under control conditions (0mM NaCl), when salinity has no influence on mass production (Ym). To test the response to salinity, different models were chosen for each accession on the basis of the best-fit line and highest r2 (Steppuhn etal. 2005b). The ST-index, proposed by Steppuhn et al. (2005a) as an indicator of the inherent tolerance of agricultural crops to salt stress, was calculated as ST-index=ECe50 (1+ b), where ECe50 is the electrical conductivity of the root zone that reduces the relative yield to 50% of the absolute yield (ECe at Yr=0.5) and ‘b’ is a regression constant. ECe was derived from the electrical conductivity of the irrigation water solutions (ECi) by the relationship ECe=0.5 ECi. Before the plants were harvested, two leaf discs of 1.8 cm diameter were punched from the uppermost fully expanded leaf on each plant and were immediately weighed to determine the fresh mass. The discs were then placed in a 5-cm diameter Petri dish containing distilled water, for 24h at 4°C in darkness, to obtain the saturated mass. Once the fully turgid leaf discs were weighed, they were oven-dried and again weighed to determine the dry mass. The succulence degree [SD=(turgid mass − dry mass)/leaf area, in g dm−2], relative water content [RWC=(fresh mass − dry mass)/(turgid mass − dry mass) * 100], specific leaf area (SLA=leaf area/dry mass, in dm2 g−1) and saturated water content [SWC=(turgid mass − dry mass)/dry mass] were determined according to Ogburn and Edwards (2012). Mineral concentration The roots and shoots of all harvested plants were dried in oven at 40°C for 7days, before being ground to a fine powder in a ball mill (Retsch® model MM400, Verder Holding B.V., Vleuten, GG, The Netherlands), digested with nitric acid and extracted by an ultrasound method. Samples (10 mg) were placed in 10mL of HNO3 (0.2N; 0.5% Tritin X-100) for ultrasonic-assisted pseudo-digestion with an ultrasonic processor (SONICS VibraCell™ model VCX 130, Sonics & Materials, Inc., Newtown, CT, USA), operated during 60s at 80% power. The concentrations of Na+ and K+ were determined in liquid phase by flame atomic absorption spectrometry (Perkin Elmer mod. 2100, Norwalk, CT, USA), and the corresponding concentration in the plant tissue was calculated (expressed as percentage dry mass). For analytical quality control, duplicate samples were analysed once every nine samples, and analytical blanks were used to control for possible contamination. Certified reference material (‘poplar leaves: GBW07604’) was also analysed once every nine samples. Data on germination and plant growth and allocation traits were obtained in both experiments (six accessions, including cabbage and kale). Plant water content and mineral concentration were also determined in kale accessions. The acephala group (Experiment 2) was selected for the abovementioned analyses because the growth habit and organ morphology are similar to those of wild types. Furthermore, this Downloaded from https://academic.oup.com/aobpla/article-abstract/11/5/plz046/5545357 by Univ de Santiago de Composrela user on 14 April 2020 Copyedited by: AS 4 | AoB PLANTS, 2019, Vol. 11, No. 5 group represents an ‘evolutionary bridge’ between wild types and the other taxonomic groups in B.oleracea (Branca and Cartea 2011). The greatest differences relative to other taxonomical groups were expected to be observed in water content and mineral concentration. Statistical analysis All data used in this study are included as Supporting Information (see File S1 to S4). Data from acephala and capitata experiments were analysed separately. Two-way analysis of variance (ANOVA) was used to evaluate the effects of salinity (0, 50, 100 and 150mM NaCl) and domestication status (cultivar, landrace and wild), and their interaction, all of which were included in the model as fixed-effect factors. For each trait, Fisher’s protected least significant difference (LSD) was used to compare mean values, at a significance level of 0.05 (Steel etal. 1997). Regression analysis was used to model different response functions relating relative yield of B. oleracea accessions to root-zone salinity. Phenotypic correlation coefficients between traits associated with mineral content, plant growth and water content parameters were calculated. All analyses were performed using SAS® 9.4 software and JMP® 12.1.0 (SAS Institute Inc., Cary, NC, USA). Results The ANOVA revealed significant effects of domestication status (P ≤ 0.046) on germination, growth and allocation traits and TWC in both experiments (Table 1). Salinity significantly affected germination traits in both experiments, as well as root dry mass (P=0.044) and TWC (P<0.0001) in acephala and SMR and RMR in capitata (Table 1). Similar responses to salinity were observed in domesticated and wild accessions as the domestication status × salinity interaction was not significant for any of the abovementioned variables, except germination traits (Table 1). In the acephala experiment, the ANOVA revealed that domestication status significantly affected all water content parameters and mineral concentrations, except for shoot K+ concentrations and shoot K+/Na+ ratio (Table 2). In addition, salinity significantly affected all traits except shoot K+ concentrations. Domestication groups differed widely in their response to salinity as regards RWC (P<0.0001), SLA (P=0.0001) and SD (P=0.0098) (Table 2). Germination The effect of salinity on seed germination percentage depended on domestication status in the acephala (P<0.001) and capitata (P<0.001) experiments (Table 1). While in the different landraces and cultivars, the germination percentage was not affected by salinity treatment (except in the acephala landrace), in the wild accessions seed germination percentage decreased significantly as the salinity increased (Fig. 1A and B). Mean germination time varied significantly in relation to domestication status (Table 1), with the highest values corresponding to wild accessions, indicating a positive effect (i.e. reduction in MGT) of domestication on this character (Fig. 1C and D). Salinity increased the MGT (Table 1), although in the acephala experiment this effect depended on the domestication status, and only the wild accession was significantly affected by salinity (Fig. 1C). Plant growth and allocationtraits Domestication significantly affected total, shoot and root dry mass (P always ≤ 0.049; Table 1; Fig. 2A). Although salt stress reduced total, shoot and root dry mass in both experiments, significant differences were only observed for root dry mass (P=0.044; Table 1) in the acephala group (Fig. 2B). The reduction in root dry mass was greater for exposure to 150mM NaCl, but the values were statistically similar to those of plants irrigated with 50 and 100mM NaCl. For all dry mass measurements, all domestication groups responded similarly to salt stress, as revealed by the absence of a significant domestication status × salinity interaction. In both experiments, accessions differed significantly in their RMR (P always < 0.025) and SMR (P always < 0.025) (Table 1). According to LSD comparisons, landraces dedicated a higher proportion of total biomass to roots than the wild accessions and cultivars (Fig. 2C), although in capitata the values were similar for the wild accession and landrace (17 and 18%, respectively). Shoot mass ratio increased gradually with increasing saline stress in both experiments. However, the salinity increased Table 1. Result of the two-way ANOVA assessing the effect of salinity on germination, growth and allocation traits and TWC of six accessions of Brassica oleracea with different domestication status (cultivar, landrace and wild) and representing acephala (kale) and capitata (cabbage) taxonomic groups. ns, not significant. *Significant at P<0.05; **P<0.01; ***P<0.001. The numbers of degrees of freedom (df) for germination and growth/allocation traits were, respectively, 36 and 112. Domestication status (df=2) Salinity (df=3) Domestication status × salinity (df=6) Brassica oleracea acephala Seed germination (%) 485.0*** 25.7*** 6.22*** MGT (days) 74.1*** 36.7*** 18.2*** Total plant dry mass (g) 8.15*** 1.88ns 0.49ns Shoot dry mass (g) 8.66*** 1.70ns 0.61ns Root dry mass (g) 8.13*** 2.79* 0.40ns SMR 18.9*** 1.45ns 1.78ns RMR 18.9*** 1.46ns 1.78ns TWC 7.32*** 21.97*** 0.38ns Brassica oleracea capitata Seed germination (%) 269.6*** 15.2*** 5.19** MGT (days) 78.0*** 10.4** 2.59ns Total plant dry mass (g) 6.27** 1.10ns 0.55ns Shoot dry mass (g) 7.26** 1.11ns 0.65ns Root dry mass (g) 3.09* 1.68ns 0.35ns SMR 3.83* 3.01* 2.08ns RMR 3.83* 3.01* 2.08ns TWC 3.29* 0.95ns 1.07ns Downloaded from https://academic.oup.com/aobpla/article-abstract/11/5/plz046/5545357 by Univ de Santiago de Composrela user on 14 April 2020 Copyedited by: AS Lema etal. – Domestication influences salinity in Brassica oleracea | 5 significantly the above-ground dry matter (P=0.034), and the RMR consequently decreased (P=0.034), only in capitata (Table 1; Fig. 2D). The TWC was lowest in the landraces, which differed significantly from cultivars in both experiments and also from the wild accessions in acephala (P < 0.041; Table 1; Fig. Table 2. Result of the two-way ANOVA assessing the effect of salinity on water content parameters and mineral concentration of three accessions of Brassica oleracea with different domestication status (cultivar, landrace and wild) and representing the acephala (kale) taxonomic group. ns, not significant. *Significant at P<0.05; **P<0.01; ***P<0.001. The number of degrees of freedom (df) for water content parameters and mineral concentration traits were 239 and 59, respectively. Domestication status (df=2) Salinity (df=3) Domestication status × salinity (df=6) Water content parameters RWC 13.4*** 86.3*** 7.84*** SWC 10.8*** 4.75** 1.82ns SLA (dm2 g−1) 24.7*** 6.91*** 4.77*** SD (g dm−2) 39.6*** 41.1*** 2.89** Mineral concentration Root K+3.89* 13.9*** 0.80ns Root Na+8.62*** 13.1*** 1.46ns Root K+/Na+ ratio 3.72* 9.11*** 0.31ns Shoot K+0.29ns 1.54ns 2.03ns Shoot Na+6.30** 52.5*** 2.36* Shoot K+/Na+ ratio 2.18ns 15.4*** 1.3ns Figure 1. Effect of increasing salt concentrations (0, 50, 100 and 150mM NaCl) on percentage of germination and MGT in Brassica oleracea acephala (A, C) and B.oleracea capitata (B, D) experiments including accessions with different domestication status (cultivar, landrace and wild). Data are means of three measurements per domestication status and salinity level (error bars=SE). Significant salinity × domestication status interactions are indicated by solid lines; non-significant interactions are indicated by dashed lines (P > 5%, LSD test). Means indicated by equal letters are not significantly different (P > 5%, LSD test). Downloaded from https://academic.oup.com/aobpla/article-abstract/11/5/plz046/5545357 by Univ de Santiago de Composrela user on 14 April 2020 Copyedited by: AS 6 | AoB PLANTS, 2019, Vol. 11, No. 5 2E). In addition, in plants irrigated with saline water, the TWC was significantly lower than in control plants in the acephala experiment (P<0.0001; Fig. 2F). Asimilar trend was observed in capitata, but the differences in response to the different salinities were not significant (P=0.421; Fig. 2F). Additional information about intra-taxonomic group variation can be obtained by analysing the relative yield of all B.oleracea accessions as a function of salinity in domesticated (Fig. 3A) and non-domesticated (wild) accessions (Fig. 3B). The quadratic model [Yr=a + (b * ECe) + (c * ECe 2)] and the exponential model [Yr = a * exp(b * ECe)] provided the best-fit lines and highest R2 values for domesticated accessions (cultivars and landraces) and wild accessions, respectively (Table 3). The values of EC in the root zone that reduced the yield to 50% of the maximum (ECe50) and the ST-index are also shown in Fig. 3. In general, the relative yield decreased with increasing salt concentration, except for those cultivars in which an initial stimulatory effect was induced by salt (Fig. 3A). The highest ST-index values corresponded to the capitata cultivar (14.93), the wild-acephala (14.01) and the acephala landrace (13.00). By contrast, the lowest values of this index were obtained for the capitata landrace, acephala cultivar and wild-capitata (7.42, 8.52 and 9.59, respectively). Water content parameters Highly significant differences (P < 0.001; Table 2) between domestication status, salt treatments and their interaction were detected for RWC, SLA and SD in the acephala experiment. The interaction was not significant (P = 0.096; Table 2) for SWC. Increasing concentrations of NaCl in the irrigation water Figure 2. Effect of domestication status (A, C, E) and increasing salinity (B, D, F) on growth, allocation and TWC of domesticated (landraces and cultivars) and nondomesticated (wild) Brassica oleracea accessions belonging to the acephala and capitata taxonomic groups. Data are means of 40 and 30 measurements per domestication status and salinity, respectively (error bars=SE). Significant effect of salinity on growth, allocation and TWC are indicated by solid lines; non-significant effects are indicated by dashed lines (P > 5%, LSD test). Means indicated by equal letters are not significantly different (P > 5%, LSD test). Downloaded from https://academic.oup.com/aobpla/article-abstract/11/5/plz046/5545357 by Univ de Santiago de Composrela user on 14 April 2020 Copyedited by: AS Lema etal. – Domestication influences salinity in Brassica oleracea | 7 caused a significant reduction (P< 0.001) in RWC in all three domestication groups (Table 2). In the highest salt treatment, the RWC value was highest in the acephala landrace, differing from acephala cultivar and wild plants (Table 4; Fig. 4A). The highest SWC was recorded in the acephala cultivar (13.94), relative to the wild plants (13.09) and the acephala landrace (12.37; LSD=0.66). Saturated water content tended to increase with salinity (from 12.33 for control conditions to 13.74 for 150mM NaCl treatment; LSD = 0.77). Salt stress significantly decreased the SLA of all accessions, although the difference between accessions Figure 3. Yield response to increasing root-zone salinity of (A) domesticated (cultivars and landraces) and (B) non-domesticated (wild) Brassica oleracea accessions. Landraces and cultivars are categorized into acephala (kale) and capitata (cabbage) taxonomic groups. Wild accessions are named according to the experiment where they were included. Each point represents the mean total dry mass of 10 plants of each accession. Table 3. Coefficient of determination (R2) and root-mean-square error (RMSE) and parameters (a=constant that reflects the shape of the curve, and b=defines the intensity of the model) from regression analyses relating relative yield and root-zone salinity in six accessions of Brassica oleracea with different domestication status (cultivar, landrace and wild) and representing the acephala and capitata taxonomic groups. Accession R2RMSE a b Brassica oleracea acephala acephala cultivar (kale) 0.8689 0.1377 0.8445 0.1917 acephala landrace (kale) 0.9922 0.0134 1.0150 0.0243 wild-acephala 0.9509 0.0334 1.0087 0.0520 Brassica oleracea capitata capitata cultivar (cabbage) 0.9857 0.0349 0.8676 0.1828 capitata landrace (cabbage) 0.8951 0.1047 0.9623 0.0252 wild-capitata 0.6135 0.1495 1.0110 0.0790 Downloaded from https://academic.oup.com/aobpla/article-abstract/11/5/plz046/5545357 by Univ de Santiago de Composrela user on 14 April 2020 Copyedited by: AS 8 | AoB PLANTS, 2019, Vol. 11, No. 5 was greatest for the control (0 mM) and at maximum salt concentration (150mM), with the acephala cultivar showing the lowest values across all salt treatments (Fig. 4B). The SD values were higher in wild plants than in domesticated accessions under all experimental conditions (Fig. 4C). However, although the SD of wild plants increased gradually and significantly with increasing salinity, no significant differences were observed between SD for 100 and 150 mM NaCl in the domesticated accessions. Mineral concentration In the acephala experiment, wild plants contained significantly less Na+ in shoots (P=0.0038) and roots (P=0.0007) than the domesticated accessions (Table 2; Fig. 5A). In addition, the root K+ content was significantly higher in the wild accession than in the acephala cultivar, while the difference relative to the acephala landrace was not significant (P=0.027; Fig. 5A). Under saline and non-saline conditions, the mean concentrations of Na+ and K+ were higher in shoots than in roots in all accessions (Table 4). Differences between accessions were also observed in the root K+/Na+ ratio: the values were highest in the wild accession but not significantly different from those in the acephala landrace (Fig. 5A). The concentrations of K+ decreased and those of Na+ increased in roots as the salinity increased, independently of domestication status (Fig. 5B). In addition, the salt treatment significantly decreased the K+/Na+ ratio in the shoots and roots of all accessions (Fig. 5B). A significant domestication status × salt treatment interaction (P=0.045; Table 2) was observed for shoot Na+, indicating that the wild and cultivated accessions responded differently to increasing concentrations of NaCl in the irrigation water (Table 2; Fig. 5C). Although the behaviour of the accessions was similar at 0 and 50mM, the wild plants contained significantly less Na+ in shoots than both domesticated accessions at higher NaCl concentrations (100 and 150mM NaCl). Notably, the shoot Na+ was slightly higher in the wild accession at above 50mM NaCl, whereas in the acephala landrace and acephala cultivar, the shoot Na+ increased considerably with increasing salt concentrations (Fig. 5C). Correlations between traits, based on domestication status across salt treatments, were calculated. Germination percentage was significantly and positively correlated with shoot (r=0.624; P=0.030) dry mass. In addition, succulence was significantly associated with SLA (r = −0.795; P = 0.002), RWC (r=−0.736; P=0.007) and total plant (r=−0.624; P=0.030) and shoot (r = −0.654; P = 0.021) dry mass. Regarding the mineral concentrations, the RWC was positively correlated with root K+ (r=0.859; P=0.0003) and shoot K+/Na+ ratio (r=0.627; P=0.029) and negatively correlated with Na+ in shoots (r = −0.8168; P=0.0012) and roots (r = −0.796; P= 0.002). The shoot K+/Na+ ratio was more strongly influenced by Na+ (r=−0.796; P=0.0019) than by K+ (r=0.285; P=0.3686), but the root K+/Na+ ratio was influenced to a similar degree by both cations (r = 0.919 and P<0.0001 for K+; r=−0.868 and P=0.0003 for Na+). Discussion In the present study, the growth and allocation traits in seedlings of domesticated and non-domesticated (wild) accessions exhibited similar responses to varying saline conditions. However, differences in the relative yield between cultivars and landraces within domestication groups were marked, although expected, as accessions from two different taxonomical groups were compared. In addition, the effects of domestication (transition from wild plants to landraces) and modern breeding (transition from landraces to cultivars) were variable and depended on the trait considered. Domestication has evidently mediated the seed germination and leaf succulence and shoot Na+ of seedlings, while modern breeding, along with domestication, may also have influenced the RWC andSLA. Although we could not reveal how domestication has led to the loss of salt tolerance of an elite cultivar because the phylogenetic relationships among the wild types, landraces and elite cultivars used in this study are unknown, we provide strong evidences that wild types have potential as sources of salt tolerance. Germination We found that increasing salinity significantly affected the time to emergence as well as the final germination percentage. However, the extent of the response varied between domesticated and wild accessions in relation to percentage (in both groups) and time to germination (only in the acephala Table 4. Mean values of water content parameters and K+ and Na+ concentrations (in shoots and roots) of three accessions of Brassica oleracea with different domestication status (cultivar, landrace and wild) and representing the acephala (kale) taxonomic group grown under different salinities (0, 50, 100 and 150mM NaCl). Mean values indicated by different letters are significantly different (P<0.05) according to LSD test. Mean values shown in italics are not significantly different. RWC SWC SLA (dm2 g−1) SD (g dm−2) Root Shoot K+Na+K+/Na+ ratio K+Na+K+/Na+ ratio (%dry mass) (%dry mass) Domestication status Cultivar 62.7c 13.94a 5.31a 2.68b 1.59b 1.00a 2.02b 3.38 3.50a 1.35 Landrace 67.7a 13.09b 4.85b 2.60b 1.78ab 0.82b 2.75ab 3.08 3.80a 1.15 Wild 65.4b 12.37c 4.30c 3.10a 2.10a 0.68b 3.90a 3.25 2.79b 1.95 LSD 1.9 0.66 0.28 0.12 0.38 0.15 1.60 – 0.58 – Salinity 0 mM 73.5a 12.33b 5.13a 2.42d 2.44a 0.52c 5.83a 3.41 0.96c 3.89a 50 mM 69.3b 13.09ab 5.02a 2.70c 2.13a 0.72b 3.22b 3.09 2.93b 1.06b 100 mM 59.7c 13.38a 4.65b 2.93b 1.59b 1.02a 1.98bc 3.66 4.31a 0.85b 150 mM 58.6c 13.74a 4.47b 3.14a 1.15c 1.01a 1.24c 2.79 4.95a 0.62b LSD 2.2 0.77 0.33 0.13 0.43 0.18 1.86 –0.67 1.05 Downloaded from https://academic.oup.com/aobpla/article-abstract/11/5/plz046/5545357 by Univ de Santiago de Composrela user on 14 April 2020 Copyedited by: AS Lema etal. – Domestication influences salinity in Brassica oleracea | 9 group). The slight reduction in germination for landraces and cultivars contrasted with the abrupt decrease in final germination in wild accessions. Seed mortality at salinity below 150 mM NaCl was not confirmed; however, the pronounced decrease and/or suppression of seed germination at higher salinity levels in wild accessions probably corresponds to an adaptive mechanism described in species surviving in littoral habitats and which is decisive in the ability of such species to colonize saline environments (Ishikawa and Kachi 2000). In a comparison of wild species from contrasting coastal habitats, Mariko etal. (1992) found that even under favourable conditions sea cliff species germinate slowly. This probably guarantees germination at the ‘safe site’ and the subsequent establishment of seedlings in these environments. Salt-adapted Brassicaceae species also show high sensitivity to NaCl during germination (Orsini etal. 2010). Delayed and decreased germination in wild accessions is not associated with salt tolerance at advanced growing stages, as reported for other crops (Foolad etal. 1999; Flowers 2004; Cuartero etal. 2006). In the present study, the salt tolerance during seed germination shown by the domesticated accessions, in terms of higher germination percentage and lower time to germination, may be related to human-mediated selection processes (Fuller 2007). While domestication and selection processes lead to high germination percentage and rapid germination in cultivated genotypes, wild genotypes have evolved adaptations to saline environments, such as increased succulence of leaves. During domestication, wild B.oleracea plants were removed from hostile saline habitats, where non-saline water supplies were limited and occasional, to benign farming environments with less restrictive conditions regarding the availability of fresh water. Under the latter conditions, plants were artificially selected for early emergence and high rates of germination over several generations, leading to changes in seed characteristics that resulted in seeds showing loss of dormancy (Fuller and Allaby 2009) (i.e. with a more permeable and thinner seed coat that enables rapid germination even under saline conditions, at least at concentrations reaching 150mM NaCl). This trait may be undesirable in hostile conditions (i.e. sea cliffs inhabited by the wild type) because it increases the vulnerability of plants to further adverse conditions, but is intentionally selected in plants growing in agricultural environments (i.e. landraces and cultivars grown as crops) (Fuller and Allaby 2009). Plant growth and allocationtraits The study findings did not indicate any clear relationship between salinity tolerance and domestication status of the accessions. Contrary to our expectations, the value of the ST-index was lower in the wild-capitata accession than in the wild-acephala and similar to those of the domesticated gene pool. This finding may be explained by hybridization between cultivated and wild accessions, which is possible as there are some domestic gardens close to sea cliffs in this area. Branca and Cartea (2011) reported evidence of crosses between wild populations of several Brassica species growing near villages and B.oleracea landraces in Sicily. In the present study, the wild and cultivated accessions belong to the same species, and crosspollination may be frequent. The wild-capitata could survive in its saline natural habitat due to remaining pre-domestication traits such as dormancy (i.e. strong inhibition of germination, especially under adverse conditions). In addition, higher levels of salt tolerance are expected to occur in wild accessions at advanced stages of development. Salt particularly influences growth processes, so that growth rates and biomass production are reliable criteria for assessing the degree of salt stress in a plant and its salt-tolerance capacity (Flowers and Flowers 2005). In addition, changes in biomass allocation may be determinant in salt tolerance (Byrt and Munns 2008), and shoot growth is generally more salt-sensitive than root growth (Munns 2002; Benincasa etal. 2013). It can be assumed that a reduction in leaf area relative to root growth will decrease water uptake, thus conserving soil moisture and Figure 4. Changes in (A) RWC, (B) SLA and (C) SD with increasing saline content of the irrigation water (0, 50, 100 and 150mM NaCl) in leaves of three accessions of Brassica oleracea acephala with different domestication status (cultivar, landrace and wild). Data are means of 10 measurements per domestication status and salinity (error bars=SE). Means indicated by equal letters are not significantly different (P > 5%, LSD test). Downloaded from https://academic.oup.com/aobpla/article-abstract/11/5/plz046/5545357 by Univ de Santiago de Composrela user on 14 April 2020