Salt tolerance at seed germination is not consistent with that in the seedling stage
Seed germination often occurs on soil surface in saline environment, exposing the seeds and seedlings to higher salt levels than older plants. Some halophytes are even relatively more sensitive to salinity during the establishment stage [24, 25]. In our study, seed germination of five highland quinoa cultivars did not reduce at low salinity level (100 mM NaCl), but two of them showed significant decrease at high salinity level (400 mM NaCl) (Fig. 1). In a previous study with four lowland quinoa genotypes in Chile, only one (BO78) revealed significant reduction in seed germination rate at high salinity level (300 mM NaCl) ; while for a less salt-tolerant cultivar selected in Denmark (cv. 5206), inhibitory effect was obvious only at high salinity levels (> 400 mM NaCl) . Across our studied highland cultivars, tolerance at high salinity level during germination was not consistent with relative plant growth (biomass) reduction. Taken together, it can be said that, at germination, salt tolerance of quinoa largely depends on its genotype and/or ecotype. Since these two processes are governed by different mechanisms [26, 27]; salt tolerance between seed germination and seedling is not necessarily correlated.
Small plants are more tolerant to salt
Plant water content or leaf water content, though widely employed, is not a strong indicator of turgor in salt-stressed plants undergoing osmotic adjustment. This is due to the fact that, greater solute content of cells at higher salinity largely results from ion (e.g. Na+ and Cl-) and organic solutes accumulation but not from water loss, especially in halophytes [1, 2]. In response to the increased salinity levels, plant water content did not vary greatly, but plant height, leaf area, and root length decreased sharply (Fig. 2A-F) (c.f. ). Compared with the control (i.e. no salinity), plant height of some Peruvian quinoa accessions reduced under salinity, whereas some of them even indicated taller growth . Rather than developing deep and dense root system to ameliorate the negative effects of drought to “find” more water , highland quinoa decreased root growth and elongation (Fig. 2C,F), thus, avoiding excessive uptake of Na+ and Cl- and also preventing an escalation in salt concentration in soils . Increase, decrease and maintenance of root/shoot ratio were previously found to be a genotype-dependent response in quinoa . Decreased root/shoot ratio with increasing salinity levels (Fig. 2E) indicated stronger influence of salt on root biomass than shoot biomass [24, 26]. But the plant’s early morphological response via adjustment of root and shoot biomass did not play an important role in salt stress because of absence of any close relationship between root/shoot ratio and salt tolerance (Fig. 7B). Reduction in leaf area (Fig. 2D), the most obvious avoidance mechanism to cope up with salt stress for many crops including halophytes [2, 26], resulted in a consequent functional reduction in assimilatory unit of plants and decreased water use by the plant, thus conserving soil moisture.
We did not find significant change in plant biomass between the control and the lowest salinity level (100 mM NaCl) in each studied cultivar, although salinity stress progressively reduced shoot, root, and total biomass (Fig. 2E,F,H). This contrasted with previous results where, in some quinoa genotypes, optimal growth was achieved at intermediate salinity levels (i.e. 100-200 mM) [10, 12, 27]. Slower growth at initial stage under stress conditions might be an adaptive response of plants to survive that allows them to store resources, repair damaged structures, and restart physiological functions [15, 16]. A negative trend between plant biomass and salt tolerance within each salt level (Fig. 3) indicated that quinoa cultivars with smaller size (biomass) are more salt tolerant, especially with strong relationship existing at lower salinity levels (< 300 mM NaCl). Compared with the high salt levels, genetic variation in Na+ exclusion may contribute to greater tolerance at moderate salinity conditions where leaf Na+ content is below toxic level. Thus, our results provided some support for the presumed tradeoff between seedling’s growth potential and salt tolerance at the intraspecific level of highland quinoa, as stress adaptation is costly. This “trade-off” was previously observed in 2 quinoa cultivars having highly contrasting origin with respect to salinity: the salt-tolerant cultivar Utusaya belonging to Salares ecotype, and, the less salt-tolerant Danish-bred cultivar Titicaca . Even in adult plants, a trade-off between salt-stress adaptation and plant growth was found among two coast-lowland quinoa landraces (VI-1, Villarrica) and a salt-tolerant Salares cultivar (R49) . However, in response to drought, the relationship between plant's growth potential and drought tolerance across eight desert grasses may be somehow explained by differential response of plants on soil water content, rather than the inter-specific differences in drought tolerance .
Chlorophyll content generally increased in the most salt-tolerant quinoa cultivar (i.e. B2), but decreased in others (Fig. 5F). It was also reported that chlorophyll content significantly decreased in a less salt-tolerant cultivar (Titicaca) but increased in a salt-tolerant cultivar (Utusaya) . Positive relationship between chlorophyll content and salt tolerance (Fig. 7C) may be considered as a compensatory mechanism aimed to protect quinoa for their inability to efficiently exclude Na+ from uptake into leaves and enhance CO2 fixation, with less growth penalty.
The measured antioxidant enzymes may not be a key element for salt tolerance
Increased activities of antioxidant enzymes (like SOD, CAT, POD) might mitigate effects of oxidative damage that often characterize plant responses to stress . SOD dismutates O2·– into H2O2; whereas, presenting in the peroxisomes, CAT mainly catalyzes decomposition of H2O2 into water and oxygen, and POD mainly scavenge H2O2 in chloroplasts. As one of the first line of defense against oxidative stress, SOD activity had no positive or negative correlation with salinity tolerance in glycophytes [29, 30]. But halophytes may show an exceptional ability to utilize the immediate stress-induced SOD production, thus sending stress signals through H2O2 formation to protect themselves from adverse conditions [31, 32]. It was found that, the enhanced antioxidant enzyme activities in quinoa cultivar Titicaca could be one of the factors responsible for salt tolerance, although having lower activity than its counterpart in cultivar Utusaya [24, 33]. Previously, a controversial increase and decrease in CAT and POD activity under salt and drought stress in Pichaman genotype of quinoa was found; that might be attributed to both genetic variation and variances in kinetics of stress development . At the lowest salt level, antioxidant enzymes counteracted the adverse effects of O2– as well as H2O2, since the activities of antioxidant enzymes (SOD, POD and CAT) increased but MDA did not accumulate greatly in leaves (Fig. 4). Consequently, a degree of oxidative damage at cellular level was mitigated. Antioxidant system works in a coordinated manner, SOD activity producing cytotoxic H2O2, while itself being neutralized by superoxide . Whereas at higher salt levels (>100 mM NaCl), SOD activities continued to increase; POD and CAT activities first increased and then decreased, and, MDA also accumulated significantly (Fig. 4). Significant increase in leaf MDA content at higher salt levels in all cultivars can be evidence of increased damage to membranes due to generation of more reactive oxygen species (ROS). The plausible answer in our study might be the important role played by H2O2 as a second messenger, triggering cascades of adaptive responses at both physiological and genetic levels, where rapid conversion of O2– to H2O2 by SOD is essential for generating early defense signal in halophytes [2, 31]. Having enough SOD ‘in stock’, quinoa plants rapidly induce H2O2 levels, rather than detoxifying by SOD, which give them a certain adaptive advantage. Compared with Arabidopsis thaliana (a glycophyte), it was clear that Cakile maritime (a halophyte) could quickly send stress signals through H2O2 and had an efficient antioxidant mechanism to scavenge it upon completion of signaling . It was also reported that high salt-induced reduction of CAT activity might be explained by the requirement to increase H2O2 levels to be used in stress signal transduction . In addition, H2O2 played an important role in the regulation of K+/Na+ homeostasis and increased resistance to salt stress in callus tissue of salt-tolerant Populus euphratica . Thus, it will be interesting to evaluate the actual role (signaling or scavenging) of CAT and POD in halophytes like quinoa.
Salinity tolerance was not closely related to antioxidant enzyme activities (SOD, POD, or CAT) across different salt levels and quinoa cultivars (Fig. 7G-J), similar to the genotypes of barley  and some halophytes (e.g. Atriplex lentiformis) . The antioxidant enzymes, therefore, may not be a key element for salt tolerance in highland quinoa. As a halophyte, quinoa possesses efficient means to handle salt load (e.g. Na+ exclusion from the cytosol) without the requirement of a high level of antioxidant activity, as they simply prevent excessive ROS formation in salt stress conditions [10, 32]. Genetic differences in salinity tolerance are not necessarily owe to differences in the ability to detoxify ROS. Additionally, although halophytes may use the antioxidant machinery more efficiently than glycophytes, non-enzymatic antioxidants, e.g., glutathione reductase and glutathione, and compatible solutes (mannitol, myo-inositol, proline and glycine-betaine, etc.) present in quinoa can play a crucial role [3, 27].
Accumulation of organic osmolytes may be adaptive
The contents of organic solutes in leaves (i.e. soluble sugar, protein and proline) generally increased with increasing salt levels for each studied cultivar (Fig. 5A-C), which was also widely reported in some quinoa genotypes in response to drought and salt stress [23, 36]. Accumulation of soluble sugars and other compatible solutes (e.g. proline) not only allow plants to decrease osmotic potential and maintain the cellular turgidity necessary for cell expansion under salinity stress conditions (osmotic adjustment) but also act as osmoprotectants, helping the cells to protect and maintain membrane integrity [4, 30]. The enhanced production of total soluble sugars in quinoa seedlings was presumed to adjust osmotically to saline environment . As an osmolyte that is frequently found in plants subjected to drought and salinity conditions [32, 37], the increased sugar content in quinoa might be due to salinity stress, which was further supported by high activities of soluble acid invertase and sucrose–phosphate synthase in salt-stressed quinoa seedlings . Excessive high content of sugar may, however, inhibit photosynthesis by a feedback mechanism, causing a reduction of leaf development and hence plant growth; similar to the negative correlation observed here between sugar contents and salt tolerance (Fig. 7F). Carbohydrates act as an active carbon sink prior to plant growth; an increase in carbon storage and decrease in plant growth could be a plastic or evolutionary response to carbon-limiting conditions [16, 37].
Apart from osmotic adjustment, proline is considered to perform multiple antioxidant functions, thereby ensuring membrane stabilization, and protection of photosynthetic machinery against oxidative stress in developing leaves . However, there were inconsistent results about whether proline accumulation serves as a mechanism to tolerate salinity or it was a negative consequence of salinity [38, 39]. For instance, in rice, some authors suggested that salt-stress induced proline accumulation was related to the degree of salt tolerance . Whereas others argued that proline accumulation in salt-sensitive rice was a symptom of salt stress injury reflecting poor performance and greater damage, resulting from the increased ornithine d-aminotransferase activity and the endogenous pool of its precursor glutamate . Proline enhancement occurred at the onset of the lower salinity level in quinoa (Fig. 5C), supporting the hypothesis that this accumulation is initially a reaction to salt stress damage [4, 38]. However, the relationship between the ability of proline accumulation on stress imposition and specie’s stress tolerance is not very clear, although plant species differed greatly in the amount of proline responded to stress. For instance, cell elongation in roots in drought-stressed maize was maintained in cells that accumulate proline; hence, proline accumulation was not associated with reduced growth . The inverse relationship between proline content and salt tolerance in highland quinoas (Fig. 7E) may be related to the fact that the synthesis of proline consumed high nitrogen source and energy, at the cost of plant growth. This result in our studied highland quinoas contrasted partly with that in four lowland quinoa genotypes, where the most salt tolerant genotype showed the highest increment in proline content upon salt conditions , but consistent with salt-sensitive barley  and sorghum . Even at the cellular and, ultimately, organismal level, it is clear that the level of proline accumulation and the amount of growth are inversely correlated under salt conditions; being a part of adaptation process, proline may act as a signaling molecule capable of activating multiple responses . Being demonstrated in a range of halophytic species, the beneficial effect of adaptive proline accumulation is that it served as an osmolyte and protection for quinoa plants under salt-stress conditions, rather than enhancing plant growth (salt tolerance) . Whereas, more work are needed to understand the relationship between proline accumulation, stress adaptation, and control of plant growth and development in quinoa, especially in the field.
K+/Na+ ratio is positively correlated to salt tolerance
Leaves had much lower Na+ content, but higher K+ content than roots (Fig. 6A-D). As a whole-body response in adaptation to salt, plants preserve Na+ in the roots, due to their relatively higher tolerance to ion toxicity than leaves, and restrict Na+ flux to the shoot and leaves. Compared with roots, much lower leaf Na+ content suggested that these quinoa cultivars are “Na+ excluders” . Na+ exclusion from cells and compartmentalization and safe lock of excessive Na+ in the vacuole of leaves in quinoa are important protective ways in response to salt-induced ion toxicity at the cellular level . Moreover, quinoa plants tolerate saline conditions by dumping excess salt into specialised epidermal bladder cells on the leaves, which constitutively sequester and excrete them actively from metabolically active cells [10, 43]. It is now becoming clear that the inward-rectifier high-affinity K+ transporters (HKT1.2) is the underlying one-way accumulation system playing a key role for Na+ load into bladder cells in quinoa . This surplus salt, mainly Na+, compartmentalize from the leaf blades into the bladder hairs located on the leaf surfaces, from where it can be washed off by rain.
Salt increased K+ and Na+ contents in both leaves and root (Fig. 6A-D); the accumulated inorganic ions in tissues thereby enable quinoa plants to maintain cell turgor and to reduce transpiration under salt stressed conditions, via adjustment of water potential [2, 12]. Potassium is released from roots to xylem for transport to the leaves; increased leaf K+ content could be attributed to an exchange between Na+ and K+ in the proximal part of roots . Our results are consistent with the previous works that accumulation of inorganic ion and organic solutes occurred in salt-stressed quinoa plants [5, 12, 25], although inorganic osmoregulation had the strongest contribution to osmotic adjustment (ca. 90%) . Higher K+ content with increasing salinity levels indicated that K+ uptake was enhanced by Na+ supply. This seems to be counterintuitive, as the two ions competing for major binding sites in the important metabolic processes in cytoplasm and K deficiency always occurred when imposed by salinity . In other work on barley  and some halophytes, e.g. quinoa [5, 10, 12], K+ accumulated in some tissues and contributed more efficiently in osmotic adjustment in cells of leaves under high salinity conditions. In the former work, it was interpreted that a higher demand is for “free” K+, not “structural” K+, in order to osmotically adjust and support leaf expansion. We only measured total ion content but not fluxes themselves, therefore, it is not possible to confirm such possibilities from our study. Essential for a range of physiological processes in response to salt stress, leaf K+ loss may activate an amount of caspase-like proteases triggering programmed cell death, and thus enhance leaf senescence . In this context, the quinoa plants' ability to increase K+ uptake and retention in plant tissues (especially in leaf) is a part of its extraordinary salinity tolerance (c.f. ).
Salinity tolerance was significantly negatively correlated with K+ and Na+ contents in leaves or roots, except for root K+ (Fig. 8A,B). Compared with the organic osmolytes (i.e. protein, sugars, and proline) in leaves, inorganic ions showed higher correlations with salinity tolerance (Figs. 7,8), indicating that they probably made larger contribution in osmoregulation. Relative to organic osmolytes with high energy cost of de novo synthesis , it is much more advantageous and metabolically cheaper for plants to use inorganic solutes for osmotic adjustment, assuming it will not interfere with cell metabolism. But strikingly, in 11 genotypes of “Na+ includer”, a positive correlation was observed between the accumulated Na+ amount and plant’s salinity tolerance . An inverse relationship between leaf Na+ accumulation and salinity tolerance often occurred when different genotypes within a species are compared, but this is not the case in inter-specific comparison, such as in wheat and barley . On the other hand, the general assumption of increased levels of K+ to mitigate salt stress is probably oversimplified. In NaCl-treated Arabidopsis plants, over-accumulation of Na+ and K+ triggered growth reduction, through stomatal regulation or systemic stress responses, rather than Na+ toxicity and water deficit . The negative correlation between leaf Na+ content and plant salinity tolerance suggested that the major mechanism contributing to salinity tolerance was to exclude salt from their leaves, rather than vacuolar Na+ sequestration. At the whole-plant level, protecting young leaves from excessive Na+ amounts has long been considered as a key attribute of Na+ compartmentalization in many species [1, 43]. Quinoa is no exception.
In addition, maintenance of ion (especially K+) homeostasis is essential for ionic and pH homeostasis, enzyme activities, and cytosolic K+ attributed to the plant adaptive responses to a broad range of abiotic stresses [1, 44]. Quinoa plants accumulated more Na+ than K+ under salinity stress, as the K+/Na+ ratios in both leaves and roots decreased with increasing salinity levels (Fig. 6E,F). Being one of the most important cations for plant growth, K+ is required as an enzyme cofactor and as a vacuolar osmoticum. The catalytic sites normally bind the essential K+ and maintain a high cytosolic K+/Na+ ratio to enhance salt tolerance . Similarities between Na+ and K+ lead to competition during transport in these sites. The K+/Na+ ratio in leaves or roots was positively correlated with salinity tolerance (Fig. 8C), which was also found in drought resistant quinoa  and, commonly, in glycophytes . Thus, maintenance of ion homeostasis is critical for salt tolerance of our studied highland quinoa plants. As an index of salinity tolerance, K+/Na+ ratio in the vegetative tissues (i.e. leaf or root), therefore, can be used as a convenient selection criterion in the breeding of highland quinoa cultivars.