Sensitivity of quinoa cv. ‘Titicaca’ to low salinity conditions

Environmental changes in the last decades have escalated to critical levels, presenting environmental risks to people, especially in terms of food supply, as it affects crop yield, production and quality (Alexandratos, 2005). Soil salinisation is one of the major environmental issues affecting crop production, especially in marginal landscapes or areas with limited resources (Rengasamy, 2010; Munns, 2011; Hussin et al., 2013). The intensive use of valuable natural resources such as land and water, along with high soil evapotranspiration and inefficient irrigation systems associated with poor water and soil management, inevitably accelerate secondary salinisation that usually results in the loss of productive areas (Munns, 2005; Hussin et al., 2013). In fact, soil and water salinity are wide-spread and currently, one-fifth of irrigated lands are salt-affected and 1.5 million hectares of lands are becoming unsuitable for agricultural production every year because of high salinity levels (Hossain, 2019).

In this sense, the Mediter ranean region is characterised by an extremely variable climate with dry-hot summers and wet-cold winters (Ceccarelli et al., 2007). Crop production in the southern Mediterranean region is restricted by limited water resources, drought and salinity. The adaptation of agriculture to changing climatic conditions includes the use of suitable crops, for example, species or genotypes with improved tolerance to abiotic stresses, such as cold, drought or saline soils (Rodríguez-Hernández et al., 2013; Fernández-García et al., 2014; Scheben et al., 2016; Cordero et al., 2019). One such crop, and perhaps the most promising, is quinoa. Quinoa (Chenopodium quinoa Willd.) is a native grain from the Andean highlands in South America that belongs to the Amaranthaceae family. It could be an excellent alternative crop in many regions of the world as is one of the few crops, if not the only one, that is capable of being successfully cultivated in the most extreme environmental conditions (Jacobsen et al., 2003). According to Adolf et al. (2013), quinoa is a halophyte that has the potential to become an important crop in arid regions and saline habitats and has also expanded in many parts of the world to satisfy new market niches for gluten-free foods. In general, quinoa combines a high inherent tolerance to salinity (Pulvento et al., 2012) and to other several environmental stress factors (Jacobsen et al., 2003; Razzaghi et al., 2011) together with a high nutritional quality (Repo-Carrasco et al., 2003). As a consequence, the demand for quinoa seeds has significantly increased over the last decade (Bazile et al., 2016). However, its production in the traditional areas of cultivation is not adequate to satisfy the increasing global demand, and thus it could be cultivated outside the equatorial Andes region of South America (Jacobsen, 2017).

Quinoa seeds are commonly known for their high nutritional value, mainly due to the amount of dietary fiber (7–10%), their high protein content and a wide range of minerals (Ca, Fe, Zn, Cu and Mn) and vitamins (B, C, and E), as well as health-promoting compounds, such as polyphenols (Ruales and Nair, 1993; Repo-Carrasco et al., 2003; Ruiz et al., 2014). On the other hand, quinoa leaves are considered nutritious vegetables, and based on their dry weight (DW), they present a better profile than grains. The protein content in their fresh leaves (14.77%) is greater than that in spinach (2.86%), chard (1.82%) and broccoli (2.98%) (Vazquez-Luna et al., 2019). Consequent to this large agricultural and nutritional potential, interest in quinoa has increased in recent years. Quinoa is traditionally classified into five ecotypes (highland, inter-Andean valley, salares, yungas and coastal lowlands), which are associated with the dispersion cores located in southern Peru and Bolivia close to Titicaca lake (Tapia, 2015).

During the last two decades, numerous articles in the literature have addressed salt and drought tolerance in quinoa (Jensen et al., 2000; Bhargava et al., 2006; Ruffino et al., 2010; Ruiz et al., 2014; Choukr-Allah et al., 2016). However, little is known about the growth of the ‘Titicaca’ cultivar (highland ecotype) in the Mediterranean region. ‘Titicaca’ cultivar represents one of the nine registered European quinoa cultivars (Jacobsen, 2017). In addition, it has properties that make it interesting as a result of its high antioxidant activity and its short time to flowering and to maturity compared to other cultivars (Reguera et al., 2018). According to Bazile et al. (2016), ‘Titicaca’ cultivar has a very good yield compared to other varieties; however, it presents a very heterogeneous yield and needs to be tested in more diverse conditions, considering its good yield production. On the other hand, there are an inadequacy in the number of studies that analyse the behaviour of this cultivar under greenhouse conditions and hydroponics, focusing on leaf production and nutritional content. In this sense, ‘Titicaca’ cultivar could be appropriate to leaf production as a product with high nutritional value. For this reason, this work aims to conduct more research into this cultivar of quinoa by analysing its behaviour against different levels of salinity, but this time under hydroponic and greenhouse conditions, and the impact of such salinity-levels on leaf biomass and mineral concentration. Thus, a broad study encompassing the biomass production parameters, as well as the water status, gas-exchange and quality parameters of this cultivar was assessed.

In saline environments, plant vulnerability is determined by the concentration and the specific ions involved; therefore, adaptation to this environment is crucial for the establishment and development of the species (Koyro and Eisa, 2008). In this sense, the wide genetic variability in salinity tolerance in quinoa provides an excellent source for selection and breeding for improved tolerance (Ruiz-Carrasco et al., 2011; Zurita-Silva et al., 2014). According to Hinojosa et al. (2018), quinoa can tolerate high levels of salinity, ranging from a salt concentration of 150–750 mM of NaCl (∼15–75 dS · m⁻¹), which is greater than the salinity of seawater (>45 dS · m⁻¹). However, the optimal salinity conditions for quinoa growth are in the range of 100–200 mM NaCl (Eisa et al., 2017). Although quinoa has been classified as a highly salt-tolerant species (Jacobsen et al., 2003; Hariadi et al., 2011; Razzaghi et al., 2011; Eisa et al., 2012; Adolf et al., 2013), many quinoa cultivars show distinct variability in their germination and growth in response to salinity. Sun et al. (2014) revealed that ‘Titicaca’ cultivar is more sensitive to salinity than other quinoa varieties such as the Achachino variety. In our case, there was a significant decrease in shoot DW and root length of cv. ‘Titicaca’ from 100 mM of NaCl, and this decrease was more pronounced in the treatment with 200 mM (18.60 dS · m⁻¹). These results are in accordance with the previous work of Pacsi (2015), who observed that the INIA Salcedo variety did not show significant variations with low salinity concentrations, but that at concentrations of 200 mM the growth underwent a significant reduction.

Similarly, Gómez-Pando et al. (2010) found that some genotypes of quinoa showed a reduction in height when they were grown under salinity, as happened in our results with the ‘Titicaca’ cultivar, which exhibited a reduction in the length of shoots in plants grown with 200 mM NaCl and in the length of roots in plants grown with >50 mM NaCl. In addition, roots seemed to be more sensitive to salinity, as described by Panuccio et al. (2014), who demonstrated that quinoa is more susceptible to ion toxicity in roots than in above-ground organs. On the other hand, Ruiz-Carrasco et al. (2011), in studying ‘BO78’ quinoa variety, found a significant reduction in root length when the quinoa plants were subjected to 300 mM NaCl. In this work with ‘Titicaca’ cultivar, a significant decrease occurred with salinity levels over 50 mM, which indicates that this cultivar could be more sensitive to salinity. Furthermore, salinity caused a lower concentration of minerals such as K, Mg, Ca, P, Fe and Mn, which could be another factor that induced the decrease of the growth parameters (Amirjani, 2010). This plant biomass reduction, associated with saline conditions, can negatively affect seed yield of quinoa (Eisa et al., 2012; Talebnejad and Sepaskhah, 2015). Moreover, in accordance with Vazquez-Luna et al. (2019) and as stated above, quinoa leaves are considered nutritious vegetables. In this work, salinity conditions disminished mineral concentration of quinoa leaves, reducing its nutritional value.

A parameter widely used to characterise the plant's assimilation capacity is the leaf area (Hoogenboom et al., 1987). In this sense, as was seen in this work, ‘Titicaca’ cultivar showed a reduction in leaf area as salinity increased, a phenomenon that is similar to the one previously described by Hasegawa et al. (2000) and Koca et al. (2017). However, the leaf biomass was maintained with salinity levels below 200 mM, which could be related to the thickening of the leaves as a successful trait for plant species growing under saline conditions (Koca et al., 2017). Leaf thickening is considered as a mechanism to increase the water retention by mesophyll tissues to counteract salt toxicity (Naz et al., 2014).

Further, the reduction of the leaf area with salinity could indicate that salt excess has a negative impact on the quinoa's light absortion, and consequently, on its photosynthetic capacity. Salinity stress results in a decrease of photosynthesis in a wide variety of plant species (Sudhir and Murthy, 2004). However, many halophytes show a high rate of photosynthesis under elevated salinity conditions (Andersone et al., 2012), depending on the level of salt tolerance of the species and/or genotype (Brock et al., 2007). In this sense, Eisa et al. (2012) observed reduction of photosynthetic rate of quinoa with treatments above 100 mM NaCl, while in this work, cv. ‘Titicaca’ showed a slight but significant reduction even with 50 mM. Moreover, it is known that there is a positive correlation between the photosynthetic capacity and chlorophyll content (Morgan and LeCain, 1991). Our results, which were obtained in relation to the photosynthetic rate, agree with the levels of chlorophylls and carotenoids observed, showing a decreasing trend as the salinity concentration increases, similar to previous works (Lampkin, 1990; Adolf et al., 2012; Manaa et al., 2019). It is noteworthy to mention that salinity levels below 100 mM had similar pigment concentration than controls, unlike what was described by Eisa et al. (2012), where slight salinity levels (50 mM) stimulated pigment production.

The effects of the photosynthetic rate's decrease can be caused by diffusion limitations through stomatal closure and/or biochemical limitations, mainly to Rubisco, that affect the CO₂ assimilation rate (Flexas et al., 2016). The correlation between photosynthetic rate and g_s in different varieties of quinoa was demonstrated by González et al. (2014) and Gámez et al. (2019). Causes of the decreased photosynthesis in this work with quinoa cv. ‘Titicaca’ due to salinity could be diffusional, because its stomata closed from 50 mM NaCl treatment. Furthermore, it is known that one of the mechanisms of response to salinity in quinoa is to reduce water loss through fast stomatal closure (Eisa et al., 2012; Ruiz et al., 2016). ABA regulation seems to be one of the mechanisms utilised by quinoa, when facing salinity-inducing decrease of turgor of stomata guard cells and thus stomatal closure (Zang et al., 2006; Jacobsen et al., 2009). In this work, a decrease in transpiration rate was also observed. This is evident when an increase in the concentration of NaCl causes higher values of WUE (Cocozza et al., 2013; Sun et al., 2014; Miranda-Apodaca et al., 2018). In this sense, one aspect that deserves further comment is that in our work, the reduction in the transpiration rate (73.62%) induced by high concentration of salts was proportionally greater than the one of the photosynthetic rate (32.40%), which could improve WUE according to Eisa et al. (2012). Thus, low levels of salinity could be beneficial in terms of water consumption without negative effect in plant aboveground biomass.

It is known that salinity causes a reduction in leaf water potential (Razzaghi et al., 2011; Eisa et al., 2012), as this work demonstrates. Moreover, as observed by Munns (2009), root Ψ also suffered a significant decrease in saline treatment of 200 mM of NaCl, although in this work with cv. ‘Titicaca’, reductions were significant at 50 mM of NaCl. According to Adolf et al. (2013), decline of Ψ is due to an osmotic adjustment carried out by quinoa under saline stress due to an accumulation of Na in vacuoles. In this sense, our results showed that the amount of Na was higher as the salinity increased, while the amount of K, Mg and Ca decreased with salinity consequent to being antagonistic to Na (Niu et al., 1995; Dodd et al., 2010). In addition, in terms of leaf water content, it was observed that quinoa remains unchanged until the treatment of 200 mM NaCl. Similar results were obtained by Riccardi et al. (2014), who observed that quinoa grown with 200 mM NaCl undergoes a significant reduction in water content due to a decrease in quinoa water absorption.

Saline glands seem to be an efficient mechanism to resist drought and salinity (Agarie et al., 2007; Ben-Hassine et al., 2009). Salt-secreting structures, namely salt bladders and salt glands, are the unique structures that directly secrete salt ions out of the plant (Shabala et al., 2014; Yuan et al., 2015), with most studies concentrating on two plants, C. quinoa and Limonium bicolor. Quinoa is a typical halophyte that possesses salt bladders, and its salt secretion mechanism and salt transport pathway were illustrated in detail by Shabala et al. (2014). Conversely to the results of Orsini et al. (2011) and Ruiz et al. (2016), we observed that salt exposure caused a significant enhancement in the number of saline glands in comparison with plants which were not exposed to salt treatment. On the other hand, there were no significant differences in the salt gland density among the saline treatments tested. In our cv. ‘Titicaca’ plants grown under hydroponic conditions, the exclusion mechanism based on saline glands could have reached saturation at low salinity levels. Orsini et al. (2011), working with the variety ‘B078’ of quinoa, found a saturation of this exclusion mechanism at 150 mM NaCl.

To conclude, quinoa cultivar ‘Titicaca’ is not as effective in tolerating salinity as expected. In fact, a salt condition of 100 mM that persisted for a week was severe enough to reduce the photosynthetic rate and the shoot biomass of plants. Despite that fact that the quinoa plant has been classified as a halophyte, genotype variations and understanding of the mechanisms of adaptation and tolerance against salinity are fundamental aspects to be taken into account in future research. In this sense, this study could serve as a foundational model for future studies on this peculiar species, the seeds and leaves of which are of high nutritional value.