The current work is part of a quinoa introduction and selection program initiated in 2000 in Morocco and involving Brigham Young University, the Institut Agronomique et Veterinaire Hassan II, and Copenhagen University of Denmark. Germplasm adapted to Moroccan production environments was to be identified in this long-term program through traditional breeding methodologies based upon the initial creation of highly diverse quinoa populations composed of the following: (1) in the first phase, introduction of internationally available cultivated quinoa varieties, landraces, and breeding lines; followed by, (2) introduction of breeding populations combining adaptive cultivated characteristics of lowland Chilean-origin × agronomically superior Andean Highland types; and then, (3) introduction of early-generation populations derived from disease-, pest-, and heat-resistant wild lowland × cultivated parent crosses. The primary objectives of this study, falling under phase 2 of the aforementioned project, were to (1) describe agro-morpho-phenological traits an F2:6 population of recombinant-inbred lines (RIL) developed from an NL-6 (female, lowland) × 0654 (male, highland) cross; (2) estimate the extent of genetic diversity present by the F6 generation in this population; and (3) estimate the discriminating potential and heritability of several of the evaluated traits.

### MATERIALS AND METHODS

### Plant Material

The quinoa population in this study was developed at BYU, Provo (USA), through a cross between the Dutch variety NL-6 (female) and Peruvian highland line 0654 (male) initially made by Alejandro Bonifacio, and was previously described in Maughan et al. (2012) as 'Pop39'. The NL-6 parent, which is derived from lowland Chilean germplasm, has yellow panicles, short stature, is early maturing, and is sufficiently heat-tolerant to allow for its cultivation at low elevations. Line 0654 is a highland Peruvian valley ecotype with red-purple panicles, late maturity, tall stature, and is heat susceptible. The population included in the current field evaluation consisted of 70 F2:6 lines plus the two parents.

### Field Characterization and Statistical Analyses

Characterization of Pop39 was performed on an experimental plot at the IAV Hassan II in Rabat, Morocco, near the laboratory to facilitate daily measurements of growth and phenology. The trial was sown indoors on January 29, transplanted to sandy loam soil in the field February 26–27, maintained through manual weeding, irrigated regularly as needed, and treated twice against aphids. No fungal control against mildew was applied. To protect against avian seed predation, an anti-sparrow net was used to cover the plots. The quinoa population was planted in two completely randomized blocks, with about 20 plants and five measurements per accession per block. Elementary plots were two rows of one-meter long and spaced 35 cm apart.

Sixteen quantitative and seven qualitative traits related to plant morphology, phenology and agronomic performance were measured. Quantitative measurements included seed diameter; plant height (at 60 days = PH60, 75 days = PH75, 90 days = PH90, and at maturity = PHM); panicle or inflorescence length (IL) and width (IW); sensitivity to downy mildew (SM); number of days to maturity (DM), and phenological stages and yield (GY) components, all of which were measured during the growing season and/or at harvest. Additional biomass data logged included main stem diameter (SD); root length (RL); aboveground fresh (FWA) and dry (DWA) weights; root fresh (FWR) and dry (DWR) weights; and overall biomass (BM). Qualitative traits used in the evaluation includes color of the seeds (ranged into a 1–10 scale by using classes of Photoshop color numerical codes), leaves (green/dark green) and stem (green/purple), and the panicle color (yellow/pink/orange/red/purple) and shape (globular versus amaranthiform).

The evaluation of downy mildew (Peronospora variabilis) resistance (Choi et al., 2010) was performed in the laboratory on young leaves according to the inoculation method of Mhada et al. (2015). Pathogen development was evaluated every 48 h on three leaves per accession using a 0–5 notation scale where score 0 corresponds to no lesion, 1 to small and disperse lesions with less than 1 mm diameter and no sporulation on the lower side of the leaf, 2 to clearly individualized lesions increasing in number and size with a diameter between 0.5 and 1 cm without showing any sporulation on the lower side of the leaf, 3 to brown lesions, covering less than 50% of the leaf surface with a beginning of the sporulation at the lower side, 4 to lesions of larger size, covering more than 50% of the leaf area and 5 to lesions covering more than 91% of the leaf area, with a high sporulation rating on the lower and upper area.

Collected data were subjected to descriptive analyses, analysis of variance (ANCOVA) with two factors, the accessions as a fixed factor and the blocks as a random factor, and means comparisons using the Fisher test with XLSTAT software (XLSTAT-Pro 7.5). PAST software (v. 2.16, Hammer et al., 2001) was used to perform multivariate analyses to subtract the principal components which account for much of the variance, to compute the correlations matrix and to set up the dendrogram that gathers the F2:6 accessions into clusters using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) procedure (Sokal and Michener, 1958).

## RESULTS

### Qualitative Traits

The 72 studied lines represented a wide range of morphological variability that was reflected in seed and panicle coloration, plant and panicle size and shape, seed diameter, grain yield, maturity, and resistance to downy mildew (**Figure 1**). The population exhibited segregation for seed color, with 46% of the RIL's having white seeds and 22% having red seeds – the two main coloration patterns. The remaining lines were yellow (15%), white-speckled red (3%), yellow speckled with red (7%), and light-yellow or orange (3% each).

Forty-six percent of the RIL's in the population had green stems and light green leaves (NL-6 parental type), 43% had red stems and dark green leaves (0654-type), and the rest of the lines (8%) were heterogeneous, having green stems streaked with red and light green leaves or fully red stems and light green leaves.

With respect to panicle variation, 35% of the RIL's had yellow panicles like P1, 25% were purple like P2, 19% had red panicles, and 3% had pink panicles. Several RIL's exhibited panicle color segregation or heterogeneity, including yellow/red, yellow/purple, orange/ red, yellow/purple, and orange/purple. In addition to panicle color, the amaranth panicle shape (amaranthiform) was present in 60% of the RIL's, with the remaining 39% exhibiting globular (glomerulate) panicles.

Branching pattern of the main stem was another discriminating trait. Sixty-nine percent of the RIL's did not

exhibit branching. Since branching is a wild-type trait and neither parent normally has a branched stem, the presence of significant stem branching in ∼30% of the lines indicates the two parents may have complementary genes suppressing stem branching.

### Quantitative Traits

fpls-07-01222 August 13, 2016 Time: 15:7 # 4

All 16 quantitative traits showed very highly significant differences among the RIL's for practically all the studied characters, providing strong evidence for the important diversity generated within the Pop39 and highlighting the relevant discriminating power of the variables used. Seed diameter, for example, varied from 1.71 to 2.11 mm and there were highly significant differences among lines, with 28% of the RIL's having larger seeds than the 0654 parent.

Sorting RIL's by plant height at maturity differentiated the population into five classes or groups, producing a distribution skewed slightly toward shorter plant height. The three intermediate classes – classes 2, 3, and 4 –encompassed more than 80% of the lines (**Table 1**). At maturity, the NL-6 parent was 63.90 cm tall and thus fell into in class 1; the 0654 parent at 214.88 cm height was at the upper end of class 5. Consequently, transgressive segregants for plant height were not detected in the population.

Descriptive and variance analyses of biomass and its components highlighted the important diversity present among the F2:6 lines. The main stem diameter varied between 0.54 and 1.89 cm (**Table 2**). The NL-6 parent had a diameter of 0.71 cm and for parent 0654 it was 1.15 cm. The main root length of the whole population averaged 10.15 cm and varied among lines between 8.50 and 12.80 cm. The panicle length also presented a wide variation among lines; it was three times greater in line L70 (120.70 cm) compared to L26 (36.90 cm). The panicle width also varied, ranging from 3.61 to 17.50 cm in lines L26 and L90, respectively (**Table 2**).

Lines could be classified into five biomass groups, with the NL-6 parent falling into class 2 (13.16–20.10 g) and 0654 grouping with class 5 (50.52–79.39 g). The aboveground and root biomasses had values for the 0654 parent of 196.85 and 12.94 g, respectively (**Table 2**). The 0654 parent also presented the highest stem dry weight (70.79 g). Total plant dry biomass varied between 6.19 and 79.39 g for lines L26 and 0654, respectively. Standard deviations were variable among characters, being very high for the fresh weight of the aboveground plant and the length of the panicle. In contrast, standard deviations were minimal for stem diameter, plant size, and root dry weight (**Table 2**).

Five seed yield classes were identified, with >80% of the lines falling into the three lowest classes and yielding less than 12 g. The 0654 parent grouped in class 1 and yielded only 2.20 g, while NL-6 yielded 12.18 g (class 4). Five of the RIL's in class 5 produced 17–23 g of seeds per plant, indicating the potential for positive transgressive segregation for yield in Pop39. The grain yield measured per individual ranged between 0.13 and 22.42 g; the average yield of the whole F6 population was of 8.70 g/plant (**Table 1**). Thus, variation for seed production was highly significant in the population.

TABLE 1 | Main traits class's interval, average, and lines' percentage in quinoa F2:6 RIL Pop39.

PHM, Plant height at maturity; GY, grain yield; DM, days to maturity; YPV, resistance to peronospora variabilis. RPV scores: 1 = very resistance, 2 = resistance, 3 = slightly resistance, 4 = slightly susceptible, 5 = susceptible, 6 = very susceptible.

TABLE 2 | Biomass and its components in F2:6 quinoa Pop39.

SD, main stem diameter; RL, root length; IL, inflorescence length; IW, inflorescence width; FWA, above ground fresh weight; FWR, root fresh weight; DWA, above ground dry weight; DWR, root dry weight; BM, biomass. SD, standard deviation.

The 70 F6 quinoa lines grouped into five precocity classes when sorted by their number of days to maturity (**Table 1**), with a population DM average of 148 days and a standard deviation of 16.78 days. About 65% of the F2:6 lines required less than 5 months to mature (<150 days). The remaining 35% of lines required >150 days to mature and grouped in classes 4 and 5. The ANOVA for DM, as with the other characters, had a highly significant p-value. The NL-6 parent was earlier than any of the RIL's, having reached maturity after only 124 days (**Table 1**). The 0654 parent, L48, and L90 were the latest-maturing genotypes at 192 days.

### Downy Mildew Resistance

Analysis of variance for resistance to mildew showed significant differences among lines from the second day of the inoculation (**Table 3**). Perenospora variabilis symptoms did not show up right after the inoculation on all the RIL's – indicating that initial reaction to the pathogen depends on the genotype. The 70 F6 lines were classified in six groups (**Table 1**), with the most resistant ones in class 1 (L55, L7, L27, L41, and L89) and the most susceptible in class 6 (L37, L12, L18, L92, L44, and L95). The NL-6 parent was slightly susceptible to mildew while 0654 was among the most resistant. Fifty percent of the accessions (36/72) were classified between the two parents, indicating that transgressive segregation for resistance was substantial in Pop39. The resistant 0654 parent did not show any sporulation at day 12 from the inoculation; this behavior was also the case for other lines of the three first classes. Spores appeared on the 10th day on NL-6, which was also the case in lines falling into the other three susceptible classes. The most highly susceptible lines developed their first symptoms well before 10 days after inoculation.

Collected data showed that the pathogen infected every single RIL; however, the most susceptible genotypes rapidly developed symptoms and allowed for sporulation, whereas resistant lines delayed the progression and spread of disease, preventing spore growth and dissemination.

### Heritability

Broad-sense heritability and variances for morphological characters are presented in **Table 4**. Phenotypic variances were greatest for PH (1154.7) and FWA (1066.97). The highest value of genotypic variance was also noted for PH (838.53); SD presented the lowest genotypic variance (0.017). Highest heritability values were observed for DM (89%) and PH (73%). Heritabilities were intermediate for DWA (55%), IL (46%), and BM (46%). Root length had the lowest heritability at 10%. Grain yield heritability was measured at 42% in this population and environment.

### Principal Component Analysis

Multivariate analysis was undertaken to highlight genotype groups with similar traits and significant correlations among characters. The principal components analysis (PCA) was computed to assess the contributions of individual variables to the global variance. Fifteen quantitative traits were used and data analysis showed that the four first components explained 96% of the variance. These 15 traits' contributions are presented in **Table 5** for each of the first four axes. The first principal component (PC1) explained 74% of the total variance. The variables positively correlated to PC1 were PH (0.92); IL and IW (0.85 and 0.64, respectively); SD (0.69); BM (0.94); and DM (0.65). Resistance to mildew was negatively correlated to PC1 (−0.46). Grain yield was correlated to PC2 (0.6), to which DM was negatively correlated (−0.47).

According to the correlation matrix in **Table 6**, the most significant relationships among traits were GY with IL (0.52) and BM (0.52); DM with PHM (0.71), DWA (0.72), DWR (0.64), and BM (0.54). Susceptibility to mildew (SM) was negatively correlated to PHM (0.54) and DM (0.47).

**Figure 1** presents the PCA plot of Pop39 F2:6 RIL's for PC1 and PC2. On the positive side of axis 1 are grouped 15 entries – 0654 (P2), L70, L41, L61, L29, L27, L55, L57, L72, L53, L68, L13, L8, L85, L48 – that shared the characteristics of tall plant height, long and wide panicles, large stalk diameter, large biomass, and relatively long growth cycle. On the negative side of axis 1, RIL's L60, L67, L21, L22, L80, L31, NL-6 (P1), and L26 presented lower values of the aforementioned characters. Lines on the positive side of axis 2 (L70, L16, L36, and NL-6) are characterized by higher grain yield and earlier maturity; on the negative side of axis 2 are RIL's that matured later and had lower seed yield, notably lines L13, L14, L48, L27, and L66.

### Dendrogram

A dendrogram was computed based on UPGMA using PAST v. 2.16 (Hammer et al., 2001). The F2:6 quinoa RIL Pop39 separated into three clusters (**Figure 2**). Group A included short-stature lines (61–140 cm) with small panicles (36.9–69.4 cm) and low biomass (6.19–29.56 242 g). With the exception of lines L9 and L89, they were also early maturing and susceptible to P. variabilis. Cluster A also included the NL-6 parent (P1).

Cluster B included six lines (0654 or P2, L29, L41, L72, L61, L70), all of tall size (140–215 cm) and having both long (90.2– 120.7 cm) and wide (12.63–16.14 cm) panicles. They were also of high biomass (50.52–7.39 g) and high grain yield (12–23 g). Parent 0654 was an exception because it yielded only 2.20 g/pl. However, 0654 and line L41 were very resistant to mildew, L70 and L29 had intermediate levels of resistance, while L61 was very susceptible.

Cluster C (**Figure 2**) consisted of 24 lines of somewhat large size (115–170 cm), long panicles (57.8–120.7 cm), high biomass (20.77–48.02 g), and late maturity (140–192 d). These RIL's were mostly resistant to downy mildew, except for highly susceptible lines L30, L12, and L18.

### DISCUSSION

The presented cross operates between two contrasting lines, NL-6 adapted to European lowlands and of short size (63.90 cm), and 0654 from the Peruvian valleys and taller (214.88 cm). There is a large genetic distance between F2:6 accessions, and much variability within the population. Several authors reported that the valley ecotype is generally taller than highland ecotype

(Gandarillas, 1979; Cáceres, 1993; Mujica and Jacobsen, 1999; Carmen, 2008).

The F2:6 quinoa RIL's presented a large genetic variability, which is translated to qualitative traits of stem, leaf, inflorescence and seed color diversity, and also by inflorescence shape and size variation. Seed color varies from black, brown, red, pink, yellow, orange to white. The inflorescence presents a very wide range of colors from white, yellow, pink, red to dark red, brown, and purple. Within the F2:6 accessions, the leaf color was light green in 47% RIL's or dark green in 45%; the stems were red (43%) or green (46%) and the inflorescence were mainly yellow (35%) or red (25%), but also orange, pink, purple.

Several authors have investigated the genetic control of a number of qualitative characters. A single gene with several alleles controls leaf color, red color is dominant over purple, dominating over green. The red color is dominant at leaves axils (Tapia et al., 1979; Izquierdo Fernández et al., 2001; Carmen, 2008). Concerning the panicle shape, a 3:1 segregation of glomerulate upon amaranth forms was reported (Carmen et al., 2008). Male sterility identified in quinoa is nucleocytoplasmic, with three fertile plants for one sterile (Izquierdo Fernández et al., 2001; Carmen et al., 2008).

There is a wide range of seed colors being white, yellow, pink, dark red, brown, and purple (Cusack, 1984; Risi and Galwey, 1989). The genetic determination of this trait requires studying several cross descendants' between homozygote lines with contrasted seed colors. The tetraploid status of the species has to be taken into account while analyzing the offspring.

The UPGMA clustering and PCA showed that the highlandphenotype, relatively unadapted lines in Rabat's coastal climate formed a distinct group far apart from the other clusters. Highland-phenotype accessions– those most like the Peruvian 0654 parent – are of tall stature and have a more indeterminate growth pattern when cultivated in the mild and humid coastal environment. Moreover, since 0654 is susceptible to severe decreases in seed yield when exposed to elevated temperatures during flowering (personal observations), lowyielding lines may have inherited this characteristic from 0654.

Maughan et al. (2012) reported that single-nucleotide polymorphism (SNP) markers identified in this same population (Pop39) displayed 95% normal (non-distorted) segregation, with most of the skewed markers favoring the NL-6 alleles. They further speculated that this pattern of segregation distortion favoring SNP alleles from the NL-6 parent might be due to heat-induced sterility coming from the 0654 parent, even though the population was advanced to homozygosity each generation under relatively mild (25◦C) temperatures in the greenhouse. Consequently, it would be interesting in a future study to address the question of whether or not these NL-6 skewed regions carry alleles for heat tolerance.

This highland RIL's are, however, highly resistant to downy mildew; they develop small lesions and have a long latent period when inoculated with oospores of P. variabilis under controlled conditions. The pathogen affected most genotypes at different intensities. 0654 parent was ranked among the most resistant F2:6 RIL's group, probably carrying the resistance

#### TABLE 6 | Correlation matrix among variables measured in F2:6 RIL quinoa Pop39.

factor. This assumption is in agreement with Kitz (2008) from the same population. The highest resistance was that of RIL L55, which confirms our earlier greenhouse observations (unpublished).

According to Alfano and Collmer (1997), Campbell et al. (1999), and Eulgem et al. (2004), the valley ecotype is usually more resistant to downy mildew than early and short size lowland ecotype, corresponding to group A of the dendrogram. These results are in concordance with Bonifacio and Saravia (1999).

The data analyses confirmed the significant diversity of the quantitative traits. Seed yield was positively correlated to panicle length (0.52) and total biomass (0.52). Bhargava et al. (2007) state good correlation between grain yield and plant biomass while Carmen (2008) reports positive correlations with plant size, panicle width, total dry matter and crop earliness.

Mujica (1988) puts up two genetic indexes that are based on high inheritability traits that are highly correlated to grain

yield; those are central glomeruli diameter (Index I1) and stem and panicle diameters (Index I2). The efficiency of the two indexes reached 144.48 and 148.32%, respectively; they represent relevant parameters for germplasm evaluation and selection studies (Mujica, 1988).

Bertero et al. (2004) reported striking disparities for quinoa character correlations between cold/highland and lowland/tropical environments. For example, they reported grain yield versus biomass correlations of 0.92 and 0.48 in cold/highland versus warm/lowland test sites, respectively. Although Rabat is on the coastal plain and at mid-latitude, its proximity to the ocean moderates the temperature; nonetheless, the observed correlation of yield versus biomass of 0.52 in this study follows the heat-stress pattern detected by Bertero et al. (2004). Biomass was still a close second behind (IL, 0.521) for magnitude of correlation with grain yield in this study.

Bertero et al. (2004) also reported a disparity for quinoa grain yield-maturity correlations, having observed values of 0.87 and 0.37 in cold/highland and warm/lowland environments, respectively. In the present study, we found that grain yield and days to maturity were negatively correlated at −0.31. This is most likely due to later-maturing lines encountering debilitating late spring-summer heat stress, especially for lines carrying alleles from the heat-susceptible parent 0654.

Bertero et al. (1999) reported strong effects on seed size and maturation from high temperatures and post-anthesis exposure to long days in Altiplano (variety 'Kanckolla') and Valley (variety 'Blanca de Junin') quinoa genotypes. Since our parental cultivar '0654' is of similar intermediate-late maturity and environmental adaptation as 'Blanca de Junin', it is not surprising that plant height, inflorescence size, biomass, seed diameter, and maturation all contributed heavily to Axis 1 of the PCA (**Table 5**). With a February sowing date at Rabat (∼34◦ N latitude), lines having greater allelic contributions for daylength sensitivity from '0654' would be expected to have delayed maturation, higher biomass, etc., whereas daylength-neutral alleles from the coastal genetic background of 'NL-6' would have contributed to markedly earlier maturation and relatively reduced biomass parameters under the same lengthening-day conditions – and without transgressive segregation for maturation from 'NL-6' contributing to a longer growth cycle.

Clearly, salient breeding objectives in the mild coastal climate of Rabat should include higher seed yield, shorter growing cycle and downy mildew resistance – though given the correlation

### REFERENCES

between more lengthy growth cycle and higher mildew resistance, and in light of Rabat's lack of severe summer heat or winter freeze constraints, mildew resistance probably should not be sacrificed for earlier maturation. This is based on the presumption that the humid climate in Rabat is conducive to mildew spore exposure and growth year-round. Nevertheless, evaluations of larger numbers of segregating progeny from lowland × highland quinoa populations like Pop39 should facilitate uncoupling of traits like heat tolerance/early maturity + mildew susceptibility.

The extensive morphological variation present in just 70 F2:6 lines of the NL-6 × 0654 RIL population, combined with the pattern of relatively moderate linkage distortion detected in this population using molecular markers (Maughan et al., 2012), bodes well for the continued use of lowland coastal × Andean highland quinoa breeding populations. The detection of positive transgressive segregants for seed size and yield, in particular, combined with an observed heritability for yield of approximately 40%, bodes well for future selection to develop higher-yielding quinoa lines for the Moroccan coastal environment.

### AUTHOR CONTRIBUTIONS

OB and NB designed the study. PM and EJ prepared the population. NB and OB collected the data. NB, OB, and SJ analyzed the data. OB, SJ, PM, and EJ obtained funding to support this project. NB, OB, and EJ took primary responsibility for writing the manuscript.

### FUNDING

Funding for advancement of the population to the F2:6 generation was provided by Brigham Young University and the Holmes Family Foundation through grants in support of undergraduate research.

### ACKNOWLEDGMENTS

The SWUP-Med project of the European Union provided principal funding for this study. The original cross for this population was made by Alejandro Bonifacio and we are grateful for his contribution of the F1 seed.

Cusack, D. (1984). Quinoa: grain of the Incas. Ecologist 14, 21–31.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Benlhabib, Boujartani, Maughan, Jacobsen and Jellen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Quinoa Seed Quality Response to Sodium Chloride and Sodium Sulfate Salinity

Geyang Wu1 , Adam J. Peterson2 , Craig F. Morris3 and Kevin M. Murphy2 \*

1 School of Food Science, Washington State University, Pullman, WA, USA, 2 Department of Crop and Soil Sciences, Washington State University, Pullman, WA, USA, 3 United States Department of Agriculture/Agricultural Research Service-Western Wheat Quality Laboratory, Washington State University, Pullman, WA, USA

Quinoa (Chenopodium quinoa Willd.) is an Andean crop with an edible seed that both contains high protein content and provides high quality protein with a balanced amino acid profile in embryonic tissues. Quinoa is a halophyte adapted to harsh environments with highly saline soil. In this study, four quinoa varieties were grown under six salinity treatments and two levels of fertilization, and then evaluated for quinoa seed quality characteristics, including protein content, seed hardness, and seed density. Concentrations of 8, 16, and 32 dS m−1 of NaCl and Na2SO4, were applied to the soil medium across low (1 g N, 0.29 g P, 0.29 g K per pot) and high (3 g N, 0.85 g P, 0.86 g K per pot) fertilizer treatments. Seed protein content differed across soil salinity treatments, varieties, and fertilization levels. Protein content of quinoa grown under salinized soil ranged from 13.0 to 16.7%, comparable to that from non-saline conditions. NaCl and Na2SO4 exhibited different impacts on protein content. Whereas the different concentrations of NaCl did not show differential effects on protein content, the seed from 32 dS m−1 Na2SO4 contained the highest protein content. Seed hardness differed among varieties, and was moderately influenced by salinity level (P = 0.09). Seed density was affected significantly by variety and Na2SO4 concentration, but was unaffected by NaCl concentration. The samples from 8 dS m−1 Na2SO4 soil had lower density (0.66 g/cm3 ) than those from 16 dS m−1 and 32 dS m−1 Na SO , 0.74 and 0.72g/cm3 2 4 , respectively. This paper identifies changes in critical seed quality traits of quinoa as influenced by soil salinity and fertility, and offers insights into variety response and choice across different abiotic stresses in the field environment.

Keywords: quinoa, soil salinity, protein content, hardness, density

## INTRODUCTION

Quinoa (Chenopodium quinoa Willd.) has garnered much attention in recent years because it is an excellent source of plant-based protein and is highly tolerance of soil salinity. Because soil salinity affects between 20 and 50% of irrigated arable land worldwide (Pitman and Läuchli, 2002), the question of how salinity affects seed quality in a halophytic crop like quinoa needs to be addressed. Protein content in most quinoa accessions has been reported to range from 12 to 17%, depending on variety, environment, and inputs (Rojas et al., 2015). This range tends to

#### Edited by:

Francesca Sparvoli, Consiglio Nazionale delle Ricerche, Italy

#### Reviewed by:

Albino Maggio, University of Naples Federico II, Italy Antonio M. De Ron, Spanish National Research Council – Misión Biológica de Galicia, Spain

> \*Correspondence: Kevin M. Murphy<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science

Received: 16 December 2015 Accepted: 22 May 2016 Published: 03 June 2016

#### Citation:

Wu G, Peterson AJ, Morris CF and Murphy KM (2016) Quinoa Seed Quality Response to Sodium Chloride and Sodium Sulfate Salinity. Front. Plant Sci. 7:790. doi: 10.3389/fpls.2016.00790

be higher than the protein content of wheat, barley, and rice, which were reported to be 10.5–14%, 8–14%, and 6–7%, respectively (Orth and Shellenberger, 1988; Shih, 2006; Cai et al., 2013). Additionally, quinoa has a well-balanced complement of essential amino acids. Specifically, quinoa is rich in lysine, which is considered the first limiting essential amino acid in cereals (Taylor and Parker, 2002). Protein quality such as Protein Efficiency Ratio is similar to that of casein (Ranhotra et al., 1993). Furthermore, with a lack of gluten protein, quinoa can be safely consumed by gluten sensitive/intolerant population (Zevallos et al., 2014).

Quinoa shows exceptional adaptation to harsh environments such as drought and salinity (González et al., 2015). Soil salinity reduces crop yields and is a worldwide problem. In the United States, approximately 2.2 million hectares of cropland in 48 States were occupied by saline soils, while another 30.8 million hectares are at risk of becoming saline (United States Department of Agriculture [USDA], 2011). The salinity issue leads producers to grow more salt-tolerant crops, such as quinoa.

Many studies have focused on quinoa's tolerance to soil salinity, with a particular emphasis on plant physiology (Ruiz-Carrasco et al., 2011; Adolf et al., 2012; Cocozza et al., 2013; Shabala et al., 2013) and agronomic characteristics such as germination rate, plant height, and yield (Prado et al., 2000; Chilo et al., 2009; Razzaghi et al., 2012; Peterson, 2013; Peterson and Murphy, 2015). For instance, Razzaghi et al. (2012) showed that the seed number per m2 and seed yield did not decrease as salinity increased from 20 to 40 dS m−1 in the variety Titicaca. Ruiz-Carrasco et al. (2011) reported that under 300 mM NaCl, germination and shoot length were significantly reduced, whereas root length was inhibited in variety BO78; variety PRJ biomass was less affected and exhibited the greatest increase in proline concentration. Jacobsen et al. (2000) suggested that stomatal conductance, leaf area, and plant height were the characters in quinoa most sensitive to salinity. Wilson et al. (2002) examined salinity stress of salt mixtures of MgSO4, Na2SO4, NaCl, and CaCl2 (3 – 19 dS m−1 ). No significant reduction in plant height and fresh weight were observed. In a comparison of the effects of NaCl and Na2SO4 on seed yield, quinoa exhibited greater tolerance to Na2SO4 than to NaCl (Peterson and Murphy, 2015).

Few studies have focused on the influence of salinity on seed quality in quinoa. Karyotis et al. (2003) conducted a field experiment in Greece (80 m above sea level, latitude: 39.7◦N). With the exception of Chilean variety 'No. 407,' seven other varieties exhibited significant increases in protein (13–33%) under saline-sodic soil, with electrical conductivity (EC) of 6.5 dS m−1 . Seed minerals contents of phosphorous, iron, copper, and boron did not decrease under saline conditions. Koyro and Eisa (2008) found a significant increase in protein and a decrease in total carbohydrates under high salinity (500 mM). Increasing total soluble sugar, sucrose, and glucose were observed under salinity stress because of starch hydrolysis (Ruffino et al., 2010). The same study found a significant decrease in lipids and relative water content under salinity. Pulvento et al. (2012) indicated that fiber and saponin contents increased under saline conditions with well water/sea water ratio of 1:1 compared to those under non-saline soil.

Protein is one of the most important nutritional components of quinoa seed. The content and quality of protein contribute to the nutritional value of quinoa. Additionally, seed hardness is an important trait in crops such as wheat and soybeans. For instance, kernel hardness highly influences wheat end-use quality (Morris, 2002) and correlates with other seed quality parameters such as ash content, semolina yield, and flour protein content (Hrušková and Švec, 2009). Hardness of soybean influenced water absorption, seed coat permeability, cookability, and overall texture (Zhang et al., 2008). Quinoa seed hardness was correlated with the texture of cooked quinoa, influencing hardness, chewiness, and gumminess, and potentially consumer experience (Wu et al., 2014). Furthermore, seed density is also a quality index and is negatively correlated with the texture of cooked quinoa such as hardness, cohesiveness, chewiness, and gumminess (Wu et al., 2014). Hence, protein content, seed hardness, and seed density were selected as indexes of quinoa seed quality in this study.

Chilean lowland varieties have been shown to be the most well-adapted to temperate latitudes (Bertero, 2003), and therefore they have been extensively utilized in quinoa breeding programs in both Colorado State University and Washington State University (Peterson and Murphy, 2015). The previous study found the varieties of CO407D, UDEC-1, Baer, and QQ065 exhibited extremely high tolerance to Na2SO4 and relatively high tolerance to NaCl in terms of agronomic performance such as yield, plant height, and leaf greenness (Peterson and Murphy, 2015). However, quinoa seed quality under salinity stress remains to be evaluated, since seed quality is critical to nutrition value as well as consumers' liking of the product. Hence, the objectives of this study were to (1) examine the effect of soil salinity on the protein content, seed hardness, and density of quinoa varieties, (2) determine the effect of different levels of two agronomically important soil salts, NaCl and Na2SO4, on seed quality, (3) determine the different influence of and NaCl and Na2SO4; and (4) test the influence of fertilization level on salinity tolerance of quinoa.

### MATERIALS AND METHODS

### Genetic Material

Quinoa germplasm was obtained from Dr. David Brenner at the USDA-ARS North Central Regional Plant Introduction Station in Ames, IA, USA. The four quinoa varieties, CO407D (PI 596293), UDEC-1 (PI 634923), Baer (PI 634918), and QQ065 (PI 614880), were originally sourced from lowland Chile. CO407D was released by Colorado State University in 1987. UDEC-1, Baer, and QQ065 were varieties from northern, central, and southern locations in Chile with latitudes of 34.63◦ S, 38.70◦ S, and 42.50◦ S, respectively.

### Experimental Design

A controlled environment greenhouse study was conducted using a split-split-plot randomized complete block design with three replicates per treatment. Factors included four quinoa varieties, two fertility levels, and seven salinity treatments

(three concentration levels each of NaCl and Na2SO4). Three subsamples, each representing a single plant, were evaluated for each treatment combination. Quinoa variety was treated as the main plot, salinity level as the sub-plot, and fertilization as the sub-sub-plot. Salinity levels included 8, 16, and 32 dS m−1 of NaCl and Na2SO4. The details of controlling salinity levels were described by Peterson and Murphy (2015). In brief, fertilization was provided by a mixture of alfalfa meal, monoammonium phosphate, and feather meal. The low fertilization level included 1 g of N, 0.29 g of P, and 0.29 g of K per pot; and the high fertilization level included 3 g of N, 0.86 g of P, and 0.86 g of K per pot. Each pot contained about 1 L of Sunshine Mix #1 (Sun Gro Horticulture, Bellevue, WA, USA) (dry density of 100 g/L, water holding capacity of ca. 480 g/L potting mix). The entire experiment was conducted twice, with the planting dates of September 10th, 2011 and October 7th, 2011.

### Seed Quality Tests

Quinoa was harvested at maturity. As described in the previous study (Peterson and Murphy, 2015), seeds were first stripped by hand from the inflorescences, and then threshed in a singlehead thresher (Precision Machine Company, Lincolin, NE, USA). The resulting material was cleaned using a Clipper Office Tester (Seedburo, Des Plaines, IL, USA) before the seed quality tests.

Protein content of quinoa was determined using the Dumas combustion nitrogen method (LECO Corp., Joseph, Mich., USA) (AACCI Method 46-30.01) (AACC International, 1995). A factor of 6.25 was used to convert nitrogen to protein. Seed hardness was determined using the Texture Analyzer (TA-XT2i) (Texture Technologies Corp., Scarsdale, NY, USA) and a modified rice kernel hardness method (Krishnamurthy and Giroux, 2001). A single quinoa kernel was compressed until the point of fracture using a 1 cm2 cylinder probe traveling at 5 mm/s. Repeat measurements were taken on nine random kernels. The seed hardness was recorded as the average peak force (kg) of the repeated measures.

Seed density was determined using a pycnometer (Pentapyc 5200e, Quantachrome Instruments, Boynton Beach, FL, USA). Quinoa seed was placed in a closed micro container, and compressed nitrogen was suffused into the container. Pressure in the container was recorded both with and without nitrogen. The volume of the quinoa sample was calculated by comparing the standard pressure obtained with a stainless steel ball. Density was the seed weight divided by the displaced volume. Seed density was collected on only the second greenhouse experiment.

### Statistical Analysis

Data were analyzed using the PROC GLM procedure in SAS (SAS Institute, Cary, NC, USA). Greenhouse experiment repetition was treated as a random factor in protein content and seed hardness analysis. Variety, salinity, and fertilization were treated as fixed factors. Fisher's LSD Test was used to access multiple comparisons. Pearson correlation coefficients between protein, hardness, and density were obtained via PROC CORR procedure in SAS, using the treatment means.

### RESULTS

### Protein

Variety, salinity, and fertilization all exhibited highly significant effects on protein content (P < 0.001) (**Table 1**). The greatest contribution to variation in seed protein was due to fertilization (F = 402.5). In contrast, salinity alone had a relatively minor effect, and the varieties responded similarly to salinity as evidenced by a non-significant interaction. Significant interactions, however, were found in variety × fertilization, as well as in salinity × fertilization, both of which are addressed in later paragraphs. It is worth noting that the two experiments produced different seed protein contents (F = 48.09, P < 0.001) and an experiment × variety interaction was observed (F = 14.94, P < 0.001) (data not shown). Upon closer examination, this interaction was caused by variety QQ065, which produced an overall mean protein content of 12.9% in Experiment 1 and 14.9% in Experiment 2. Protein contents of the other three varieties were essentially consistent across the two experiments.

Across all salinity and fertilization treatments, the variety protein means ranged from 13.0 to 16.7% (data not shown). As expected, high fertilization resulted in an increase in protein content across all varieties. The mean protein contents under high and low fertilization were 15.8 and 13.6%, respectively (**Table 2**). The means of Baer and CO407D were the highest, 15.1 and 14.9%, respectively. QQ065 contained 14.1% protein, significantly lower than the other varieties.

Even though salinity effects were relatively smaller than fertilization and variety effects, salinity still had a significant effect on protein content (**Table 1**). The two types of salt exhibited different impacts on protein (**Table 2**). Protein content did not differ according to different concentrations of NaCl with means (across varieties and fertilization levels) from 14.7 to 14.9%. Seed from 32 dS m−1 Na2SO4, however, contained higher protein (15.2%) than that from 8 dS m−1 and 16 dS m−1 Na2SO4 (14.4 and 14.2%, respectively).

A significant interaction of salinity × fertilization was detected, indicating that salinity differentially impacted seed

†Significant at the 0.10 probability level. ∗Significant at the <0.05 probability level. ∗∗Significant at the 0.01 probability level. ∗∗∗Significant at the <0.001 probability level.

1Different letters in a given column indicate significant differences (P < 0.05).

protein content under high and low fertilization level (**Figure 1**). Within the high fertilizer treatment, seed protein content from 32dS m−1 Na2SO4 was significantly higher (16.7%) than all other samples, which did not differ from each other (∼13%). Within the low fertilizer treatment, seed protein content from 8 dS m−1 and 16 dS m−1 Na2SO4 were significantly lower than those from the NaCl treatments and 32dS m−1 Na2SO4.

The significant interaction between variety and fertilization (**Table 1**) was due to the different response of QQ065. Protein mean of QQ065 from high fertilization was 14.4%, lower than the other varieties. CO407D, UDEC-1, and Baer exhibited a decline of 16–18% in protein under low fertilization, while QQ065 dropped only 5%.

### Hardness

Variety exhibited the greatest influence on seed hardness (F = 210.6, P < 0.001), whereas fertilization did not show any significant effect (**Table 1**). Salinity exhibited a moderate effect (F = 2.0, P = 0.09). Varieties responded consistently to salinity under various fertilization levels, since neither variety × salinity nor salinity × fertilization interaction was significant. However, a variety × fertilization interaction was observed, which will be discussed in a later paragraph. Similar to the situation in protein content, experiment repetition exhibited a significant influence on seed hardness. Whereas the hardness of CO407D was consistent across the two greenhouse experiments, the hardness of other three varieties all decreased by 8–9%.

Mean hardness was significantly different among varieties. CO407D had the hardest seeds with hardness mean of 10.0 kg (**Table 3**). UDEC-1 was softer at 9.4 kg, whereas Baer and QQ065 were the softest and with similar hardness means of 7.7 and 7.4 kg, respectively.

Salinity exhibited a moderate impact on seed hardness (P = 0.09). The highest hardness mean was observed under 16 dS m−1 Na2SO4, whereas the lowest was under 8 dS m−1 NaCl, with means of 8.9 and 8.3 kg, respectively.

A significant fertilization × variety interaction was found for seed hardness. The hardness of UDEC-1 and Baer did not

TABLE 3 | Salinity level and composition, variety, and fertilization effects on quinoa seed hardness (kg).

1Hardness was significant at the 0.09 probability level. 2Different letters in a given column indicate significant differences (P < 0.05).

differ across fertilization level, whereas CO407D was harder under low fertilization, and QQ065 was harder under high fertilization.

### Seed Density

Variety and salinity both significantly affected seed density, whereas fertilization did not show a significant influence (**Table 1**). The greatest contribution to variation in seed density was due to variety (F = 22.82). Salinity exhibited a relatively smaller effect, yet still significant (F = 2.82, P < 0.05). Neither variety × salinity interaction nor salinity × fertilization interaction was observed, which indicated that varieties similarly responded to salinity under high and low fertilization levels. An interaction of variety × fertilization was found and the details are presented below.

Across all salinity and fertilization treatments, CO407D had the highest mean density, 0.80 g/cm3 , followed by Baer with 0.69 g/cm3 (**Table 4**). UDEC-1 and QQ065 had the lowest and similar densities (∼0.65 g/cm3 ).

With regard to salinity effect, the Na2SO4 treatments exhibited differential influence on seed density. Density means did not significantly change due to the increased concentration of NaCl, which ranged from 0.68 to 0.71 g/cm3 (**Table 4**). The samples from 8 dS m−1 Na2SO4 soil had lower density (0.66 g/cm3 ) than those from 16 dS m−1 and 32 dS m−1 Na2SO4, 0.74 and 0.72g/cm3 , respectively.

A significant variety × fertilization interaction was found. With closer examination, UDEC-1 and Baer yielded higher density seeds under high fertilization, whereas CO407D and QQ065 did not differ in density between fertilization treatments.

TABLE 4 | Salinity level and composition, variety, and fertilization effects on quinoa seed density (g/cm3 ).

1Different letters in a given column indicate significant differences (P < 0.05).

## Density Correlation coefficients among seed protein content, hardness,

and density are shown in **Table 5**. No significant correlation was detected between protein content and seed hardness. However, both protein content and hardness were correlated with seed density. The overall correlation coefficient was low (r = 0.19, P = 0.03) between density and protein. A marginally significant correlation was found between density and protein content of the seeds from NaCl salinized soil under low fertilization. No correlation was found between density and protein content of the seeds from NaCl salinized soil under high fertilization, or Na2SO4 salinized soil.

Correlations of Protein, Hardness, and

The overall correlation coefficient was r = 0.38 (P < 0.0001) between density and hardness. The low fertilization samples from both NaCl and Na2SO4 soil showed significant correlations between density and hardness, with coefficients of r = 0.51 and 0.47 (both P < 0.005). The high fertility samples did not exhibit any correlation between density and hardness.

### Correlation with Yield, Leaf Greenness Index, Plant Height, and Seed Minerals Contents

Correlation between seed quality and yield, leaf greenness index, plant height, and seed mineral concentration were obtained using data from Peterson and Murphy (2015) (**Table 6**). Seed hardness was significantly correlated with yield and plant height (r = 0.35 and 0.31, respectively). Protein content and density, however, did not correlate with yield, leaf greenness, or plant height. Correlations were found between quality indices and the concentration of different minerals. Protein was negatively correlated with Cu and Mg (r = −0.52 and −0.50, respectively). Hardness was negatively correlated with Cu, P, and Zn (r = −0.37, −0.56, −0.29, respectively), but was positively correlated with Mn (r = 0.57). Density was negatively correlated with Cu (r = −0.35).

### DISCUSSION

### Protein

Although salinity exhibited a significant effect on seed protein content, the impact was relatively minor compared to fertilization and variety effects. In another words, over a wide range of saline soil, quinoa can grow and yield seeds with relatively stable protein content.

Protein content of quinoa growing under salinized soil ranged from 12.7 to 16.7% (data not shown), within the general range of protein content under non-saline conditions, which was 12– 17% (Rojas et al., 2015). Saline soil did not cause a significant decrease in seed protein. It is interesting to notice that the samples from 32 dS m−1 Na2SO4 tended to contain the highest protein, especially in variety QQ065. The studies of Karyotis et al. (2003) and Koyro and Eisa (2008) also indicated that protein content significantly increased under high salinity (NaCl) whereas total carbohydrate decreased. In contrast, Ruffino et al.

#### TABLE 5 | Correlation coefficients of protein, hardness, and density of quinoa seed.

ns, Not significant. †Significant at the 0.10 probability level. ∗Significant at the <0.05 probability level. ∗∗∗Significant at the <0.001 probability level.

#### TABLE 6 | Correlation coefficients of quinoa seed quality, and agronomic performance and seed mineral contenta .

aAgronomic and mineral contents taken from Peterson and Murphy (2015). †Significant at the 0.10 probability level. ∗Significant at the <0.05 probability level. ∗∗Significant at the 0.01 probability level. ∗∗∗Significant at the <0.001 probability level.

(2010) found that quinoa protein decreased under 250 mM NaCl salinity in a growth chamber experiment. It is reasonable to conclude that salinity exhibits contrasting effects on different quinoa genotypes.

Na2SO4 level exhibited a significant influence on protein content, whereas NaCl level did not. In the study of Koyro and Eisa (2008), however, seed protein content of the quinoa variety Hualhuas (origin Peru) increased under the highest salinity level of 500 mM NaCl compared to lower NaCl levels (0 – 400 mM). This disagreement of NaCl influence may be due to diversity of genotypes. It is worth noting that quinoa protein contents in this paper were primarily above 13% based on a wet weight basis (asis-moisture of approximately ∼8–10%) even under saline soil and low fertilization level. This protein content is generally equal to or higher than that of other crops such as barley and rice (Wu, 2015). In conclusion, quinoa maintained high and stable protein content under salinity stress.

Besides seed quality, grain yield is another significant factor when quinoa is grown in marginal environments. The yield data were reported in the previous study (Peterson and Murphy, 2015). Under 32 dS m−1 Na2SO4, the yield of CO407D, UDEC-1, and Baer decreased by 24.5, 10.8, and 11.6%, respectively, and the yield decline was much lower than a barley variety Albacete (yield decline of 82.4%). The yield decline of QQ065 was 54.5% under 32 dS m−1 Na2SO4. In the same soil condition, both CO407D and QQ065 exhibited increased protein content. Hence, if only protein content is considered, QQ065 contained the highest protein under high Na2SO4; however, if both yield and protein content are considered, then CO407D is the variety more suitable for severe Na2SO4 affected areas since it exhibited a moderate yield decline and a significant increase in protein content. It also implies the importance to evaluate both agronomy performance and seed quality when studying crops' adaption to extreme environments.

### Hardness

Quinoa seed hardness was only moderately affected by salinity (P = 0.09), indicating that quinoa primarily maintained seed texture when growing under a wide range of saline soil. CO407D exhibited the hardest seed (10.0 kg), whereas Baer and QQ065 were relatively soft (7.4 – 7.7 kg). A previous study indicated a hardness range of 5.8 – 10.9 kg among 11 quinoa varieties and 2 commercial samples (Wu et al., 2014). The commercial samples had hardness values of 6.2 and 7.1 kg. Since commercial samples generally maintain stable quality and indicate an acceptable level for consumers, seed hardness around 7 kg, as in Baer and QQ065, should be considered as acceptable quality. The hardness of CO407D was close to that of the colored variety 'Black' (10.0 kg), which had a thicker seed coat than that of the yellow seeded varieties. It was reported that a thicker seed coat is related to harder texture (Fraczek et al., 2005).

Even though the greenhouse is a highly controlled environment, and the two experiments were conducted in similar seasons (planted in September and October, respectively), seed protein and hardness were nevertheless different across the two experiments. However, ANOVA indicated modest-to-no significant interactions with salinity and fertilization such that responses to salinity and fertilization were consistent with little or no change in rank order. Even though the experiment × variety interaction was significant, the F-values were relatively low compared to the major effects such as variety and fertilization, and neither of them exhibited cross-over interaction. This is a particularly noteworthy result for breeders, farmers, and processors.

### Density

The range of seed density under salinity, 0.55 – 0.89 g/cm3 , was comparable to the density range of 13 quinoa samples (0.58 – 0.76 g/cm3 ) (Wu et al., 2014). Generally, CO407D had higher seed density (0.71 – 0.89 g/cm3 ), which indicated that seed density of this variety was affected by salinity stress. In contrast, the density of QQ065 did not change according to salinity type or concentration, which indicated a stable quality under saline soil.

### Correlations

The correlation between seed hardness and density was only significant under low fertilization, but not under high fertilization. The high fertilization level in the greenhouse experiment exceeded the amount of fertilizer that would normally be applied in field environments, whereas the low fertilization level was closer to the field situation. Therefore, correlation between hardness and density may still exist in field trials.

### CONCLUSION

fpls-07-00790 June 1, 2016 Time: 12:11 # 7

This study confirmed quinoa's tolerance to salinity stress in terms of seed quality. Under saline soil conditions, quinoa did not show any marked decrease in seed quality such as protein content, hardness, and density. Protein content even increased under high Na2SO4 concentration (32 dS m−1 ). Varieties exhibited great differential reactions to fertilization and salinity levels. QQ065 maintained a similar level of hardness and density, whereas seed of CO407D was both harder and higher density under salinity stress. If only seed quality is considered, then QQ065 is the most well-adapted variety in this study. Additionally, the influences of NaCl and Na2SO4 were different. The higher concentration of Na2SO4 tended to increase protein content and seed density, whereas NaCl concentration did not exhibit any significant difference on those quality indexes. In other words, quinoa can be grown in areas severely affected by Na2SO4 and still produce high protein seeds, which can be a good protein resource for vegetarians or malnourished populations.

In the present study, protein content, seed hardness, and density were selected as quinoa seed quality indexes due to their significance in nutritional value and end-use quality. Besides those factors, other seed components such as essential amino acids, starch, fiber, and minerals are also important to nutrition value. Their variation under different salt types should be investigated in the future. Hence, when quinoa is applied to

REFERENCES

a specific salinity area, the right varieties can be utilized not only with the consideration of yield but also the health benefits to local people. When addressing malnutrition in developed countries, the profile of essential amino acids, protein quality (such as protein efficiency ratio), and deficient minerals should be specifically evaluated. Furthermore, this study was based on greenhouse experiments. The soil conditions and environment in the field are much more complex than the soil medium used in the greenhouse. Based on the conclusion from this research, further field study should be conducted using the varieties best adapted to specific salt type and salinity level.

### AUTHOR CONTRIBUTIONS

AP set up the experiment design in the greenhouse and grew, harvested, and processed quinoa samples. GW collected seed quality data such as protein content, seed hardness, and density. AP and GW together processed the data. GW also drafted the manuscript. KM and CM edited the manuscript.

### FUNDING

The research was funded by USDA Organic Research and Extension Initiative, project number NIFA:GRANT11083982.

### ACKNOWLEDGMENT

The authors acknowledge Alecia Kiszonas for assisting in the data analysis.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Wu, Peterson, Morris and Murphy. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# New Insight into Quinoa Seed Quality under Salinity: Changes in Proteomic and Amino Acid Profiles, Phenolic Content, and Antioxidant Activity of Protein Extracts

Iris Aloisi 1 †, Luigi Parrotta1 †, Karina B. Ruiz 1, 2, Claudia Landi 3 , Luca Bini 3 , Giampiero Cai 3 , Stefania Biondi 1 \* and Stefano Del Duca1 \*

1 Department of Biological, Geological and Environmental Sciences, University of Bologna, Bologna, Italy, 2 Departamento de Producción Agrícola, Universidad de Chile, Santiago, Chile, 3 Department of Life Sciences, University of Siena, Siena, Italy

#### Edited by:

José J. Pueyo, Consejo Superior de Investigaciones Científicas, Spain

#### Reviewed by:

Sara Maldonado, University of Buenos Aires, Argentina Sergey Shabala, University of Tasmania, Australia

#### \*Correspondence:

Stefania Biondi<EMAIL_ADDRESS>Stefano Del Duca<EMAIL_ADDRESS>
† These authors have contributed equally to this work.

#### Specialty section:

This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science

Received: 30 December 2015 Accepted: 28 April 2016 Published: 18 May 2016

#### Citation:

Aloisi I, Parrotta L, Ruiz KB, Landi C, Bini L, Cai G, Biondi S and Del Duca S (2016) New Insight into Quinoa Seed Quality under Salinity: Changes in Proteomic and Amino Acid Profiles, Phenolic Content, and Antioxidant Activity of Protein Extracts. Front. Plant Sci. 7:656. doi: 10.3389/fpls.2016.00656 Quinoa (Chenopodium quinoa Willd) is an ancient Andean seed-producing crop well known for its exceptional nutritional properties and resistance to adverse environmental conditions, such as salinity and drought. Seed storage proteins, amino acid composition, and bioactive compounds play a crucial role in determining the nutritional value of quinoa. Seeds harvested from three Chilean landraces of quinoa, one belonging to the salares ecotype (R49) and two to the coastal-lowlands ecotype, VI-1 and Villarrica (VR), exposed to two levels of salinity (100 and 300 mM NaCl) were used to conduct a sequential extraction of storage proteins in order to obtain fractions enriched in albumins/globulins, 11S globulin and in prolamin-like proteins. The composition of the resulting protein fractions was analyzed by one- and two-dimensional polyacrylamide gel electrophoresis. Results confirmed a high polymorphism in seed storage proteins; the two most representative genotype-specific bands of the albumin/globulin fraction were the 30- and 32-kDa bands, while the 11S globulin showed genotype-specific polymorphism for the 40- and 42-kDa bands. Spot analysis by mass spectrometry followed by in silico analyses were conducted to identify the proteins whose expression changed most significantly in response to salinity in VR. Proteins belonging to several functional categories (i.e., stress protein, metabolism, and storage) were affected by salinity. Other nutritional and functional properties, namely amino acid profiles, total polyphenol (TPC) and flavonoid (TFC) contents, and antioxidant activity (AA) of protein extracts were also analyzed. With the exception of Ala and Met in R49, all amino acids derived from protein hydrolysis were diminished in seeds from salt-treated plants, especially in landrace VI-1. By contrast, several free amino acids were unchanged or increased by salinity in R49 as compared with VR and VI-1, suggesting a greater tolerance in the salares landrace. VR had the highest TPC and AA under non-saline conditions. Salinity increased TPC in all three landraces, with the strongest increase occurring in R49, and enhanced radical scavenging capacity in R49 and VR. Overall, results show that salinity deeply altered the seed proteome and amino acid profiles and, in general, increased the concentration of bioactive molecules and AA of protein extracts in a genotype-dependent manner.

Keywords: antioxidant activity, Chenopodium quinoa, polyphenols, salt stress, seed storage proteins

### INTRODUCTION

Quinoa (Chenopodium quinoa Willd., Amaranthaceae) is an Andean seed-producing crop cultivated since ca. 7000 years around Lake Titicaca in the Andean highlands (altiplano) from where it spread as far north as Ecuador and down to southern Chile, and from 3800 m above sea level to coastal and lowlands areas. This diversification in terms of native habitats and the wide genetic diversity has led to the identification of five ecotypes: salares (salt flats), highlands, inter-Andean valleys, yungas, and coastal-lowlands. The salares of the Andes are found principally in southern Bolivia, northern Chile, and Argentina. These highland deserts are extremely arid; temperatures often fall well below freezing and quinoa is the only crop that can grow under these edapho-climatic conditions (Fuentes et al., 2009). Other landraces (local varieties) are adapted to totally different environments. For example, in central and southern Chile, quinoa can grow at sea level; here annual rainfall, distributed throughout the year, ranges from 400 to 1500–2000 mm and soils have a high water retention capacity. Thus, quinoa has attracted the attention of scientists since over a decade mainly for its extreme tolerance and adaptability to unfavorable environmental conditions, such as salinity, drought, and frost (Jacobsen et al., 2003). However, different accessions, landraces, and cultivars of quinoa have been shown to exhibit different degrees of tolerance to abiotic, in particular salt stress (Gómez-Pando et al., 2010; Adolf et al., 2012; Peterson and Murphy, 2015).

Consumption of seeds is the most common use of quinoa, which is, therefore, considered a "cereal-like" crop. European and North American consumers are increasingly aware of the exceptional nutritional qualities of quinoa seeds that, together with sprouts, are now considered "functional foods" (Vega-Gálvez et al., 2010). This is because seeds have high protein content and contain all the essential amino acids (Abugoch et al., 2008). In quinoa seeds, protein and lipid reserves are accumulated in embryonic protein and lipid bodies, respectively, which occupy most of the cell (Prego et al., 1998). The classification of seed storage proteins dates from the turn of the century, when Osborne (1924) classified them into groups on the basis of their extraction and solubility, convention still used nowadays for storage proteins from plant seeds in general. These proteins are soluble in water (albumins), dilute saline solution (globulins), alcohol mixtures (prolamins), and dilute acid or alkali (glutelins). The major seed storage proteins include albumins, globulins, and prolamins (Shewry et al., 1995). The main protein fractions in quinoa seeds are globulins and albumins. Brinegar and Goundan (1993) characterized the 11S storage protein, chenopodin, and showed that it is composed of two subunit groups bridged by a disulfide bond, the acidic A subunit group (32–39 kDa) and the basic B subunit group (22– 23 kDa). Moreover, they reported that the 11S and 2S (8–9 kDa) polypeptides represented ca. 35 and 37%, respectively of total proteins (Brinegar and Goundan, 1993; Brinegar et al., 1996). The genomic and amino acid sequences of the 11S globulin of quinoa have been reported (Balzotti et al., 2008).

Quinoa seeds are also rich in bioactive compounds, such as vitamins (vitamin B2, vitamin E), carotene, tocopherols, and other molecules exerting antioxidant properties (e.g., phenolics) that scavenge harmful radicals (Pa´sko et al., 2009; Hirose et al., 2010; Repo-Carrasco-Valencia et al., 2010; Miranda et al., 2014). Diversity in these nutritional traits across genotypes has been reported (González et al., 2011; Vidueiros et al., 2015). In seeds of six quinoa genotypes from three geographical areas of Chile, significant differences in all the parameters analyzed were reported, with VR showing the highest content of protein, and vitamins E and C (Miranda et al., 2013, 2014). Seeds were also described as good sources of antioxidant compounds, although phenolic content, antioxidant and antimicrobial activities also varied among genotypes.

While many studies have been performed to investigate the tolerance of different quinoa genotypes to abiotic stress (mainly salinity) in terms of agronomic performance (growth, yield) and morpho/physiological mechanisms underlying salt tolerance (Adolf et al., 2013; Ruiz et al., 2016b), fewer have been devoted to the effects of high salinity on the nutritional quality of quinoa seeds. Increases or no effects in total protein content have been reported in most cases (Karyotis et al., 2003; Hariadi et al., 2011; Pulvento et al., 2012; Miranda et al., 2013). The effects of drought and salinity on seed phenolic content were investigated and only limited changes in these compounds under reduced irrigation (with or without salinity) were found (Gómez-Caravaca et al., 2012). Recently, increased total polyphenolics content (TPC) and antioxidant activity (AA) in methanol extracts of quinoa seeds harvested from plants grown under salinity has been observed, suggesting that stressful conditions may positively affect the seed's content of these important bioactive compounds (Ruiz et al., 2016a). Flour and the protein concentrate (PC) of seeds of amaranth, a close relative of quinoa, have been shown to contain polyphenols and to possess AA (Escudero et al., 2011). To date, these parameters have not been investigated in quinoa protein extracts.

The purpose of the present work was to investigate changes in the amino acid and protein profiles of seeds obtained from quinoa plants grown under saline conditions. In general, the capacity to accumulate more storage proteins in seeds plays an important role in the initial stages of the next generation, especially germination (Koyro and Eisa, 2008). Our purpose, however, was to conduct a more detailed investigation on the changes occurring under salinity in the relative amounts of different protein fractions (albumin/globulin fraction) and, more specifically, in spot patterns of individual proteins. The proteomic analysis reveals which proteins are involved in the Aloisi et al. Salinity Effects on Quinoa Proteins

salt-stress response in quinoa, thus contributing to a better understanding of the complex metabolic network involved in stress responses of halophytes (Koyro et al., 2013). In addition, the TPC and AA of protein extracts were evaluated in control and salinized seeds. These features strongly contribute to the nutraceutical properties of quinoa (Repo-Carrasco-Valencia et al., 2010; Abderrahim et al., 2015; Tang et al., 2015), may influence seed longevity (Sano et al., 2015), and can ultimately lead to the production of PCs to be used in the food industry and as dietary complements for their high protein level, functional properties, and low content of antinutritional factors (Cordero-De-Los-Santos et al., 2005; Escudero et al., 2011; Castel et al., 2014). To date, the phenolics content and AA of quinoa seed protein extracts have not yet been examined. Given the strong genotypic differences reported for all aspects of quinoa's responses to saline conditions (Adolf et al., 2012; Ruiz et al., 2016b), including nutritional aspects (Miranda et al., 2014; Abderrahim et al., 2015; Tang et al., 2015; Vidueiros et al., 2015), these parameters were comparatively analyzed in different genotypes. Thus, three Chilean landraces originating from contrasting habitats, namely R49, belonging to the salares ecotype, and two landraces belonging to the coastallowlands ecotype but from different latitudes and altitudes (VI-1, Villarrica), were examined. Seeds were harvested from plants grown in a pot experiment under saline (100 or 300 mM NaCl) and non-saline (0 mM NaCl) conditions.

### MATERIALS AND METHODS

### Plant Material

Seeds of three Chilean landraces of C. quinoa (Willd.), one belonging to the salares ecotype (R49) and two, VI-1 and Villarrica (VR), to the coastal-lowlands ecotype were collected along an altitudinal gradient from the arid northern highland with saline soils (R49, 3800 m a.s.l.) to sea level, and along a latitudinal gradient of ca. 2500 km, from ca. 34◦ S (VI-1) down to the rainier southern region (ca. 39◦ S, VR) with higher precipitation and non-saline soils (Peterson and Murphy, 2015). All seeds were obtained from the National Seed Bank of Chile managed by INIA-Intihuasi (Vicuña, Chile).

Vernalized seeds were sown in 20-L plastic pots containing a garden soil:sand (1:1) mixture. When plants had four to six wellexpanded leaves (ca. 34 days after sowing) salt treatment was started by irrigating pots weekly with 0, 100, or 300 mM NaCl solutions; all the pots (control and salt-treated) were also watered weekly with 100–200 mL water supplemented with Phostrogen (N:P:K 10:10:27; 0.4 g L−1 ; Bayer Garden, Cambridge, UK). Plants were grown (October to April) in a greenhouse under natural daylight conditions; the temperature was maintained at 23 ± 3 ◦C. Seeds were collected at maturity starting from 91 days and up to 140 days after the first salt treatment, depending on the landrace, weighed, and stored in an air-tight container at 4◦C until use.

### Chemicals

All chemicals were obtained from Sigma-Aldrich (Milan, Italy) unless otherwise indicated.

### Seed Flour Preparation

In order to remove saponins, quinoa seeds were washed repeatedly with cold water until there was no more foam in the wash water, and then dried at 50◦C up to 15 ± 3% moisture. The dried seeds were ground to a fine powder using a mortar and pestle. The resulting flour was defatted with hexane under continuous stirring overnight and then air-dried at room temperature. Flours were stored at 4◦C until use.

### Preparation of Protein Fractions

Protein fractions were prepared by a solvent-based sequential extraction following the methods of Ju et al. (2001), Bergamo et al. (2011), Džunková et al. (2011), and Zevallos et al. (2012), with slight modifications. Briefly, the albumin/globulin fraction was obtained by suspending 50 mg of flour in 300 µl of 5% NaCl; the suspension was then homogenized for 5 min and centrifuged at 5500 g for 10 min. The procedure was repeated twice and the supernatants were collected. The flour was further extracted for prolamin-like proteins following the same procedure but using 250 µl of 60% (v/v) aqueous ethanol. The crude acid-soluble globulins and the 11S-enriched fraction were simultaneously isolated following Thanh et al. (1975). Briefly, defatted quinoa flour was extracted with 63 mM Tris-HCl buffer containing 10 mM β-mercaptoethanol, pH 7.8, for 1 h and then centrifuged for 15 min at 9500 × g. The extraction buffer was adjusted to pH 6.6, dialyzed against 63 mM Tris-HCl containing 10 mM βmercaptoethanol, pH 6.6, at 4◦C for at least 4 h and centrifuged for 20 min at 9500 × g. The precipitate corresponded to the crude 11S fraction while the supernatant, which corresponded to the crude acid-soluble globulin fraction, was further purified by adjusting the pH to 4.8. After centrifugation, the precipitate was dispersed in water and the pH was raised to 7.0. For assaying radical scavenging activity, extraction of the 11Senriched fraction was performed by omitting β-mercaptoethanol both in the extraction and dialyzing buffers (Thanh et al., 1975). Protein concentration was determined spectrophotometrically at 562 nm using the bicinchoninic acid kit and bovine serum albumin (BSA) as standard.

### SDS-PAGE and Gel Staining

All one-dimensional electrophoretic runs were performed with the Mini Protean III apparatus (Bio-Rad Laboratories, Segrate, Italy). Proteins (40 µg lane−1 ) were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (1970). For the glutenin-like enriched fraction, 2 M urea was added to both stacking and resolving gels. The molecular mass standard was the Biomol (Hamburg, Germany) BLUEplus prestained Protein Ladder (10–180 kDa). Gels were fixed at room temperature in 50% methanol: 5% glacial acetic acid for 20 min, then in 50% methanol for 10 min. After fixing, gels were washed twice in deionized water (10 min each) and stained with silver as previously described (Shevchenko et al., 1996) with minor modifications. Protein profiles were densitometrically analyzed using the AIDA software 4.14 (raytest Isotopenmessgeräte GmbH, Straubenhardt, Germany).

### Protein Precipitation

For protein precipitation, 4 vol of 20% trichloroacetic acid (TCA) and 0.007% β-mercaptoethanol in cold acetone were added to samples, mixed and kept at −20◦C for at least 45 min. Proteins were pelleted by centrifugation at 15,000 × g for 15 min at 4◦C and then washed with cold acetone containing βmercaptoethanol. This step was repeated at least three times and residual acetone was finally removed by air-drying. The last pellet was resuspended with buffer for 2DE analysis (see below) and the protein concentration of samples was determined using a commercial kit (2-D Quant Kit, GE HealthCare, Milan, Italy), performed as described in the instruction manual and using BSA as reference.

### Two-Dimensional Electrophoresis (2DE)

For 2DE analyses, 11-cm IPG Strips with a 3–10 pH gradient (Bio-Rad) were used in combination with 10% Criterion XT gels (Bio-Rad). Strips were rehydrated in the solubilization buffer (40 mM Tris, 8 M urea, 2 M thiourea, 2% CHAPS, traces of bromophenol blue) to which 18 mM DTT and 20 µl ml−1 IPG buffer were added. Samples were dissolved to 1 mg ml−1 concentration in the solubilization buffer. Strips were rehydrated overnight in an Immobiline Dry Strip Reswelling Tray covered with a Dry Strip Cover PlusOne (GE HealthCare). Strips were run using a Protean IEF cell (Bio-Rad) through eight different steps:

Strips were stored at −80◦C or used immediately. In both cases, they were equilibrated for 15 min in equilibration buffer (50 mM Tris-HCl, pH 8.8 containing 6 M urea, 30% glycerol, 2% SDS, bromophenol blue, 10 mg ml−1 dithiothreitol). Proteins were then separated in the second dimension based on a Bis-Tris buffer system (pH 6.4) that uses discontinuous chloride and MES or MOPS ion fronts to form moving boundaries to stack and then separate denatured proteins by size. Molecular weight standards of the Precision series (Bio-Rad) were run in parallel. Gels were stained with Bio-Safe Coomassie blue (Bio-Rad) as described in the instruction protocol.

### Spot Analysis

Images of gels were captured using the Fluor-S Multi-Imager (Bio-Rad). The exposure time was 5–7 s for gels stained with Coomassie blue. Analysis of spots in 2DE gels was performed using the Spot Detection Wizard of PDQuest (Bio-Rad) by selecting the weakest protein spot and the larger protein clusters. Subsequently, spot analysis was improved manually by adding unidentified spots and by removing incorrect signals. After creating a Master (virtual) gel, spots were matched to determine qualitative and quantitative differences. Further analysis of spots was done using the Spot and Match set tools. The intensity of protein spots was normalized in relation to the total abundance of effective spots. After normalization and background subtraction, gels from control and treated samples were used to create a match set, which allowed the differential expression analysis between treated and control samples. Spots were considered as up- or down-regulated if their amount changed at least by a factor of 2. All samples were analyzed in duplicate.

### Protein Identification by Mass Spectrometry

Protein identification was performed as previously described (Hellman et al., 1995; Soskic´ et al., 1999). Spots of interest were manually excised, destained in 2.5 mM ammonium bicarbonate and 50% (v/v) acetonitrile and then dehydrated in acetonitrile. Gel pieces were rehydrated in trypsin solution and in-gel protein digestion was performed by an overnight incubation at 37◦C. For MALDI-TOF MS, 1.25 µl of each protein digest was directly spotted onto the MALDI target and air-dried. After drying, 0.75 µl of matrix solution [5 mg ml−1 α-cyano-4-hydroxycynnamic acid in 50% (v/v) acetonitrile and 0.5% (v/v) trifluoroacetic acid] was added to samples, which were allowed to dry again. Acquisition of mass spectra was performed using an Ultraflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Billerica, MA, United States) in reflector positive mode. Spectra were analyzed by Flex Analysis software v. 3.0. Peptide mass fingerprinting (PMF) database searching was carried out in NCBInr and/or Swiss-Prot/TrEMBL databases set for Viridiplantae (Green Plants) using Mascot (Matrix Science Ltd., London, UK, http://www.matrixscience.com) online available software. The search settings were as follows: mass tolerance was set at 100 ppm, trypsin as the digestion enzyme with one allowed missed cleavage and oxidation of methionine as a variable modification. In order to accept identifications, the number of matched peptides, the extent of sequence coverage, and the probabilistic score were considered. Peptide digests that did not give unambiguous identifications were subjected to peptide sequencing by tandem mass spectrometry. MS/MS analysis was performed on the Ultraflex III MALDI-TOF/TOF instrument. Two to three PMF peaks showing a high intensity were CID (Collision Induced Dissociation) fragmented using Argon as collision gas, and MALDI-TOF/TOF tandem MS was performed in LIFT mode by software controlled data acquisition. Fragmented ions were analyzed using the Flex Analysis software v. 3.0. The MS/MS database search was carried out in NCBInr and/or Swiss-Prot/TrEMBL databases using the on-line MASCOT MS/MS ion search software. The following parameters were applied for the database search: taxonomy: Viridiplantae (Green Plants), trypsin specificity, one missed cleavage allowed, peptide precursor mass tolerance: ±100 ppm, fragment mass tolerance ±0.6 Da, peptide precursor charge state +1, carbamidomethylation of cysteine as a fixed modification, oxidation of methionine as a possible modification. Protein identification was considered significant based on Mascot ion score, peptide coverage by "b" and "y" ions, and expected value.

### Amino Acid Analysis

Analysis of free amino acids and amino acids derived from complete hydrolysis of proteins in quinoa flour was performed in triplicate essentially as described by Silvanini et al. (2014) with some modifications. To analyze the content of free amino acids, 0.5 ml dH2O and 75 µl trifluoroacetic acid were added to 20 mg of quinoa flour. After mixing, samples were centrifuged at 12,000 g for 5 min at room temperature. Supernatants were dried by Speed-Vac and residues were dissolved in 20 µl of 20 mM HCl. The content of protein-derived amino acids (PAAs) was determined by mixing each flour sample with 6 N HCl and phenol crystal (around 1 mg to avoid oxidation) followed by incubation for 24 h at 110◦C. After heating, samples were centrifuged at 4000 rpm for 15 min at room temperature. The supernatant was dried under vacuum and the residue was dissolved in 20 µl of 20 mM HCl. Both free amino acids and those derived from hydrolysed proteins were derivatized according to the AccQ-Tag protocol (Waters, Milford, MA, USA). For HPLC analysis, a C18 AccQ-Tag column (3.9 × 150 mm; Waters, Milford, MA, USA) was used. A gradient elution was performed using a phosphate buffer solution as eluent A and acetonitrile:water 60:40 (v/v) as eluent B. The temperature was set at 37◦C; the flow rate was 1 ml min−1 . The fluorescent detector parameters were set as follows: λex = 250 nm, λem = 395 nm, gain 1, eufs 100. All data are expressed as mg 100 g−1 flour.

### Extraction of Total Proteins

Extraction of total proteins was performed essentially as described by Escudero et al. (2004). After adding 15 ml of double distilled water to an aliquot (50 mg) of the flour and continuous stirring for 30 min at room temperature (RT), the pH was taken to 9.0 with 0.1 N NaOH. After further stirring for 30 min, the homogenate was centrifuged at 4500 × g for 20 min at RT. The supernatant was collected and taken to pH 5.0 with 0.1 N HCl. After stirring for another 20 min and centrifugation at 4500 × g at 4◦C, the supernatant was discarded and the pellet re-suspended in 500 µl of 63 mM Tris-HCl, pH 8.0. Protein concentration was determined spectrophotometrically at 562 nm using the bicinchoninic acid kit and BSA as standard.

### Total Phenolics and Flavonoid Contents

The Folin-Ciocalteu (FC) assay was performed according to Singleton and Rossi (1965) with some modifications; 50 µl of protein extract (1 mg protein ml−1 ) were mixed with 250 µl of FC reagent (previously diluted 10-fold with distilled water) and 500 µl distilled water. The reaction mixture was incubated for 1 min at room temperature and then 800 µl of 20% (w/v) Na2CO3 was added. After incubation at 40◦C for 30 min, the absorbance was measured spectrophotometrically at 760 nm (Jasco V-530, Jasco Corporation, Tokyo, Japan). The total polyphenolic content (TPC) was evaluated from a gallic acid standard curve and was expressed as mg gallic acid equivalents (GAE) g−1 seed DW. The Prussian blue method (Hagerman and Butler, 1994) was also applied to determine total polyphenols. After adding 12 µl of 0.1 M FeNH4(SO4)2 to 100 µl protein extract (1 mg protein ml−1 ), the mixture was incubated for 20 min at RT. Subsequently, 12 µl of 8 mM K3Fe(CN)6 were added, and after 5 min at room temperature, the optical density of the mixture was determined at 720 nm. Gallic acid was used as standard to make a calibration curve and data were expressed as mg GAE g−1 DW.

The total flavonoid content (TFC) was determined with AlCl3 according to Liu et al. (2002) with slight modifications using rutin as standard. The seed protein extract (50 µl) was added to 450 µl of 100% methanol followed by 500 µl of 2% (w/v) AlCl3 in methanol. This reaction mixture was incubated for 15 min at room temperature. Finally, the absorbance of the reaction mixture was measured spectrophotometrically at 430 nm. Data were expressed as mg rutin equivalents (RE) g−1 DW.

### Radical Scavenging Activity

Free radical scavenging capacity of the protein extracts was determined by using the ABTS assay performed according to Arnao et al. (2001) with slight modifications. The ABTS›+ radical cation was generated by oxidizing a 2.0 mM aqueous solution of ABTS with 70 mM K2S2O8 and incubating in the dark for 24 h at room temperature. The reaction mixture contained 1.0 ml of ABTS›+ (diluted with methanol in order to obtain an absorbance of 0.7 at 734 nm) and different amounts (12.5, 25, 50, 100 µl) of sample solutions (1 mg ml−1 total protein or 11S fraction) or Tris buffer for the blank. The absorbance at 734 nm was measured after a reaction time of 20 min. Trolox equivalents per g DW of seeds were calculated using a standard curve prepared with a range of Trolox concentrations (0–30 µM) in order to calculate the Total Antioxidant Activity (TAC) value for the samples.

### Statistical Analysis

Two independent experiments were performed. Each experiment consisted of three pots per treatment (0, 100, and 300 mM NaCl), each containing one plant per landrace, and set up according to a randomized block design. Flour preparation, protein extractions, SDS-PAGE, and AIDA analyses were performed at least twice. TPC, TFC, and AA data were performed in triplicate from two separate protein extractions. To determine the overall significances, a two-way factorial analysis of variance (two-way ANOVA) was used with salt treatment and landrace as factors. Mean comparisons were made by applying Tukey's post-hoc test using InfoStat software (www.infostat.com.ar). Differences were considered significant at P < 0.05.

## RESULTS

### Seed Storage Protein Profiles and Spot Analysis

Sequential extraction of the different protein fractions and isolation of the major storage proteins were performed on seeds of the three quinoa landraces (R49, VI-I, and VR) grown with/without 100 or 300 mM NaCl. A combination of SDS-PAGE and 2DE was used to, first of all, identify the fractions that were most affected by salt, and second the proteins that were most involved in the salt-induced response. The protein fraction analyzed was the one enriched in albumins/globulins. A general feature was the reduction of these storage proteins, expressed as mg protein mg−1 flour, after the 300 mM NaCl treatment. In particular, the albumin/globulin fraction declined by ca. 12, 7, and 15% in R49, VI-I, and VR, respectively (data not shown).

As shown in **Figure 1**, the proteins were resolved into distinct bands that spanned a broad range of apparent molecular weights from 15 to >55 kDa. The image analysis software identified 10 major bands of 49 (1), 45 (2), 42 (3), 40 (4), 32 (5), 30 (6), 25 (7), 22 (8), 19 (9), and 16 (10) kDa. Some difference in the band patterns among genotypes was evident: in R49 the 32-kDa (5) band was absent while VI-I seemed to lack the 30-kDa band (6); VR had both the 32- (5); and the 30-kDa (6) bands. Comparing the protein profiles in control and salt-treated seeds by SDS-PAGE revealed that the NaCl treatment induced significant changes in the protein patterns and that the three landraces were differentially affected by salinity. In R49, the profile was very similar in seeds from control and 100 mM NaCl-treated plants; in the 300 mM NaCl treatment, the high molecular weight bands (1–4) appeared to be slightly increased, while the low molecular weight bands (6–10) decreased significantly relative to controls (**Figure 1B**). Under saline conditions (300 mM NaCl), VI-I showed a decrease in different proteins; the decline was most evident in the low molecular-weight bands except for 8 and 9 (5–10; **Figure 1C**). Finally, in VR, both salt treatments decreased the intensity of bands 5, 6, and 7 in a concentration-dependent manner (**Figure 1D**).

Subsequently, a proteomic profile of the albumin/globulin fraction was performed by 2DE in control and salt-treated samples of landrace VR. This genotype was chosen because, in addition to changes in the SDS-PAGE band pattern, under salinity it also exhibited the highest TPC, TFC, and AA (see below) and the greatest percentage decrease in the albumin/globulin fraction. Two-DE gels revealed significant differences in protein composition between the two samples. Following the analysis of master gels obtained from albumin/globulin fractions, the nine spots whose abundance changed at least two-fold during treatments were selected and processed for identification by MALDI-TOF MS (**Figure 2**). Spots were numbered arbitrarily and correspond to numbers indicated in the identification list. As shown in **Table 1**, the spots analyzed by MS revealed specific correspondence to peptides in the protein database. They were clustered into seven functional categories: stress (chaperone/folding), transcription factors, respiration, photosynthesis, storage proteins, metabolism, and cell division. The spot intensities show that significant changes, either increases or decreases, occurred in response to salinity (**Figure 3**).

Three spots (1, 2, and 9) correspond to proteins involved in stress responses or as support in protein folding. In this category, spot 1 corresponds to a stromal chloroplast 70-kDa heat shockrelated protein of Glycine soja, and to a putative heat shock protein of Ricinus communis. Analysis of the spot 2 sequence revealed a correspondence to several proteins with similar function: a GRP-78/luminal-binding protein of Spinacia oleracea, a luminal-binding protein of Beta vulgaris subsp. vulgaris, a luminal-binding protein 5 of Jatropha curcas, an endoplasmic reticulum chaperone binding protein of Lycium chinense, and a putative heat shock protein of R. communis. The intensity of

spots 1 and 2 increased after salt treatment. Spot 9 corresponded to peptidyl-prolyl cis-trans isomerase CYP20-1 of Amborella trichopoda, a protein involved in enhancing the correct folding of proteins; it decreased in seeds of salt-stressed plants as compared with controls. The second functional category is represented by spot 3, which corresponds to an Ethylene Insensitive3-like 1 protein of Phoenix dactylifera, a positive regulator in the ethylene response pathway. Its expression level decreased after treatment with NaCl. A protein involved in respiration was also identified (although only in the NaCl-treated samples). Spot 4 was matched to a mitochondrial beta-subunit of ATP synthase in different organisms (Fragaria vesca subsp. vesca, Oryza sativa and Pyrus × bretschneideri). Spot 5 was identified as a plastid movement impaired protein of Medicago truncatula; the protein showed an increased level in the NaCl-treated sample. Mass spectrometry analysis revealed that spot 6 corresponded to the 11S seed storage globulin A of C. quinoa. The expression level of this protein showed a drastic decrease in samples treated with NaCl. Spot 7 found homology with a pantothenate kinase of Micromonas sp. This protein converts pantothenate to phosphopantothenate, using ATP as phosphate donor and was significantly up-regulated under salinity. Finally, spot 8 matched to a helicase protein of Tetrahymena thermophile, a protein involved in the unwinding of DNA or RNA helix. The expression level of this protein was drastically increased in samples treated with NaCl.

### Profiles and Spot Analysis of Globulin Fractions

As the albumin/globulin fraction from seed proteins of quinoa was strongly affected by salinity, a further attempt was made to isolate and analyze the crude acid-soluble globulins and the 11S- (chenopodin) enriched fractions by SDS-PAGE. As shown in **Figure 4**, proteins of the crude acid-soluble globulin fraction were resolved into distinct bands that spanned a broad range of apparent molecular weights from 15 to >90 kDa. Results showed that the protein profiles were represented by eight major and distinct bands, common to all three landraces, with molecular weights of 90, 68, 45, 43, 31, 20, 18, and 15 kDa. Comparing the protein profiles in seeds from control and salt-treated plants revealed that the NaCl treatment did not induce significant changes in the protein composition; however, seeds of plants grown under 300 mM NaCl showed a general reduction in these storage proteins. In particular, the crude acid-soluble globulin fraction declined by 22.5, 13.9, and 25.3% in R49, VI-I, and VR, respectively.

As shown by 2DE followed by mass spectrometry of the albumin/globulin fraction, a general reduction of chenopodin, the major 11S storage protein, was also evident (**Figure 5**). Proteins of the 11S-enriched fractions were resolved into distinct bands giving the typical electrophoretic pattern of chenopodin, in which subunits A (32–42 kDa) and B (22–23 kDa) were clearly evident (**Figure 5A**). By image analysis, five major bands of 42 (1), 40 (2), 32 (3), 24 (4), and 22 (5) kDa were identified. As already reported for the albumin/globulin fraction, protein band patterns showed some differences among genotypes: the 42-kDa (1) band was absent in R49, VI-I did not present the 40-kDa (2) band and VR had both. Comparing the protein profiles in control and salt-treated seeds by SDS-PAGE revealed that the NaCl treatment induced a significant reduction in the 11S-enriched fraction and that the three landraces were differentially affected by salinity (**Figures 5B–D**).

As previously done for the albumin/globulin fraction, a proteomic profile of the 11S fraction was obtained by 2DE. Only landrace VR was analyzed because, as outlined above, all of the five main bands were affected by salt treatment. The comparison between control and salt-treated samples revealed

For each spot, number, protein name, organism, accession number, principal functions, theoretical pI, molecular mass (MW), and Mascot search data are shown.

cis-trans isomerization

oligopeptides.

some of its effects via an inhibitory action on PPIase

 Binds cyclosporin

 A (CsA). CsA mediates

 of proline imidic peptide bonds in

remarkable differences in protein composition (**Figure 6**). Fifteen spots whose expression levels changed significantly were further analyzed by mass spectrometry. They revealed a specific correspondence to proteins found in databases (**Table 2**). As in the case of the albumin/globulin fraction, the proteins were clustered into five main functional categories: stress (chaperone/folding), storage proteins, photosynthesis, respiration, and ion transport. The spot intensities in seeds of plants grown under non-saline or saline (300 mM NaCl) conditions are represented in **Figure 7**. Three different proteins (spots 1, 2, and 4) are involved in stress (chaperone/folding). Two were identified as hypothetical and predicted proteins; however, subsequent BLAST analysis revealed that they corresponded to heat shock 70-kDa protein 6, chloroplastic-like of Gossypium raimondii and heat shock cognate 70-kDa protein 1 of Aegilops tauschii, respectively. The third protein was identified as a heat shock protein 90 of Nicotiana tabacum. In all cases, salt stress increased the accumulation of these proteins compared to controls.

For the storage category, nine spots corresponding to the 11S seed storage globulin of C. quinoa and a predicted-legumin Blike of B. vulgaris subsp. vulgaris were identified. The expression levels of these proteins changed significantly under salinity. In the case of 11S globulin, the salt-treated sample showed a significant down-regulation or a complete disappearance of spots 6, 9, 10, 11, 12, and 13. On the other hand, the expression level of spots 7 and 8 increased in salt-treated samples compared to controls. Spot 3 corresponds to a chloroplastic RuBisCO large subunit-binding protein, subunit alpha of Brassica napus. This photosynthetic protein increased after salt treatment. For the respiration category, spot 5 had a significant correspondence to mitochondrial ATP synthase subunit beta of Prunus mume and its intensity increased during stress. The ion transport category was represented by spot 14, which corresponds to a predicted cation/H(+) antiporter 6B of Camelina sativa whose intensity increased during stress.

### Prolamin-Like Proteins

Detecting and separating quinoa prolamin-like proteins was quite problematic because these polypeptides are scarcely soluble. The ethanol-soluble prolamin-like proteins were scarce and consisted of low-molecular weight polypeptides (**Figure 8**). Comparing the protein profiles in control and salt-treated seeds by SDS-PAGE revealed that the NaCl treatment did not induce changes in the pattern of proteins possibly related to gluten.

### Amino Acid Profiles

The HPLC analysis of free amino acids and of amino acids derived from the complete hydrolysis of proteins (PAAs) from control and salt-treated seeds of quinoa revealed that some (Asp, Ser, Glu, Gly) were below the limit of detection in most samples

and were present only as free amino acids in VI-1 controls (**Figure 9D**, inset). Overall, the PAA profile was quite different from that of the free amino acids, both quantitatively (PAAs were much more abundant) and qualitatively (**Figure 9**).

In seeds of control plants, the profile derived from protein hydrolysis showed that Pro was the most abundant PAA in R49 and VR, followed by Cys and Tyr in R49, and by Ala, Cys, Tyr and Met in VR; in VI-1, Pro content was comparable to that of the other landraces, but the most abundant amino acid was Thr (several fold higher than the other amino acids, **Figures 9A,C,E**). In control plants of VI-1, by far the most abundant essential free amino acids were His and Thr, followed by essential and

in the Y-axes of each graph because they were optimized to highlight the different intensity of spots between samples. In all cases, the most intense spot was used as reference for calibrating the scale of Y-axes.

non-essential amino acids Cys, Tyr, Leu, Glu; in R49 and VR these amino acids were much less abundant or not detectable (**Figures 9B,D,F**). Pro was the most represented free amino acid in these two landraces followed by Cys. Free Lys content was also ca. three-fold higher in VI-1 than in the other landraces.

Salt differentially affected the amino acid composition of the three landraces. Again, the PAA profile differed substantially from that of free amino acids also in salt-treated samples (**Figures 9A,C,E**). The non-essential PAAs Ala, Pro, and Cys decreased strongly in all landraces relative to controls, except for Ala in R49, which increased. In R49 there were no changes in some essential amino acids (Lys, Ile, Phe) while other essential amino acids (Thr, Val) declined. Met was the only essential PAA that increased in response to salinity, but only in R49. In VR, all essential PAAs either decreased (Met, Lys, Ile) or remained unchanged; only Leu increased slightly (30% above control value). The strongest overall salt-induced reduction in amino acids was observed in VI-1.

It is noteworthy that in R49 free Thr was induced 60-fold by salinity; other free essential amino acids, namely Val, Lys, and Ile were also significantly enhanced (ca. 2.6−, 2.1−, 3.6-fold) while Leu increased by 25% (**Figures 9B,D,E**). By contrast, in VR and VI-1, all free essential amino acids were down-regulated, with the exception of Phe in VR, which increased by ca. 7.5-fold. Amongst the non-essential amino acids Ala, Pro, and Cys, there were no or very limited reductions (20–40%) in all three landraces and Pro even increased by 30 and 60% in R49 and VI-1, respectively. In VI-1, most free amino acids were undetectable in salt-treated samples showing a similar trend to the PAAs; in this landrace, Thr disappeared under salinity, whereas in R49 and VR the opposite occurred.

### Total Phenolic and Flavonoid Content, and Antioxidant Activity

In the total proteins extracted from quinoa seeds, there was a genotype-dependent and salt-dependent variation in TPC as determined by the FC assay (**Figure 10A**). Under non-saline conditions, VR had the highest values followed by VI-1and R49. In all three landraces, seeds from plants grown with 300 mM NaCl had higher TPC than those grown without NaCl. The strongest increase (3.5-fold) was observed in R49 while in VR

and VI-1 the increase was lower (ca. 60–70% above control levels). Using the Prussian blue method, polyphenols were below detection limit in R49 and VI-1 control and treated seeds; in VR controls, polyphenols were confirmed to be substantially more abundant than in the other two landraces (0.17 ± 0.007 GAE mg−1 DW) and dramatically enhanced (five-fold) under salinity (data not shown). Flavonoid concentrations were similar in R49 and VI-1 but lower in VR; however, salinity enhanced TFC only in VR (**Figure 10B**). The radical scavenging capacity of the seed protein extracts assayed with the ABTS method indicated that TAC was significantly different between landraces, with VR exhibiting higher values as compared with R49 and VI-1. It was slightly but significantly higher in seeds from salt-treated plants of R49 and VR as compared with controls (**Figure 10C**). The 11S globulin-enriched fraction, extracted without using β-mercaptoethanol, also revealed some ABTS radical scavenging capacity. Values were much lower (0.005–0.006 mM Trolox equivalent g−1 DW) than those of the total protein extract and did not change significantly in salt-treated vs. control samples (data not shown).

### DISCUSSION

Quinoa seeds are regarded as one of the most nutritionally well-balanced plant foods under cultivation, especially for their protein content and excellent balance between carbohydrates, lipids, and proteins. The increasing interest in quinoa as a crop is also centered on its remarkable adaptability to harsh environmental conditions such as low water content and high

alkalinity of saline soils. Soil salinity is, in fact, among the major factors limiting crop yield and productivity worldwide and is expected to increase in the future. Several comparative studies have shown that the extent to which these and other parameters are affected by salinity in quinoa is strongly genotype-dependent (Gómez-Pando et al., 2010; Adolf et al., 2012).

In the framework of our current investigations on salinity tolerance in quinoa, we have measured growth, yield, germination capacity, leaf TPC, and seed quality in the three Chilean landraces R49, VI-I, and VR exposed to two levels of salinity (Ruiz et al., 2016a). In the present work, we aimed to delve further into the effects of salt treatment on the seed proteome and on the polyphenol content, and AA of seed extracts. The PC of Amaranthus cruentus seeds were reported to have improved nutritional quality as compared with flour through increased content of factors that directly or indirectly influence lipid metabolism and enhance antioxidant protection (Escudero et al., 2011). The AA of PCs obtained through different pilot-scale industrial processes from A. mantegazzianus seeds was recently evaluated in order to optimize production for the food industry (Castel et al., 2014).

### Proteomic Changes

Preliminary analyses were conducted on the three different Chilean landraces in order to unveil genotype-specific responses. In fact, seed protein electrophoresis has been utilized in taxonomy for the explanation of the origin and evolution of a number of cultivated plants (Ahmad and Slinkard, 1992; Jha and Ohri, 1996; Nath et al., 1997; Ghafoor et al., 2002). Seed protein profiles of 40 wild and cultivated taxa of Chenopodium were congruent with taxonomic position, crossability relationships, and other biochemical characters, confirming SDS-PAGE as a powerful tool in solving taxonomic problems (Bhargava et al., 2005). More recently, Džunková and co-workers showed that both classical SDS-PAGE and chip electrophoresis analysis of seed storage proteins were useful methods for studying amaranth taxonomy in order to assess differences among different species and even among accessions (Džunková et al., 2011). They suggested that specific fractions, including albuminand glutelin-enriched fractions, instead of whole extracts, could better reveal differences among landraces. In the present study, the best candidates for SDS-PAGE-mediated polymorphism analysis were the most abundant seed storage proteins, i.e., the albumin/globulin fraction. In fact, results confirm a high polymorphism in seed storage proteins both in the position and in the intensity of some bands. The two most representative genotype-specific bands of this fraction were the 30- and 32-kDa bands.

Because of their low concentrations, prolamins were not suitable for this type of analysis, both in quinoa, herein analyzed, and amaranth and Džunková et al. (2011). While the globulins of amaranth did not differ in band intensity and position (Džunková et al., 2011), we showed that chenopodin, the 11S globulin of quinoa, displayed a high polymorphism in the three landraces analyzed, with genotype-specific protein bands of 40 and 42-kDa. Balzotti et al. (2008) reported the phylogenetic relationships between quinoa and 49 other species by using the coding DNA sequence for the well-conserved 11S basic subunit. On the basis of amino acid alignments, more than 74% sequence identity between amaranth and quinoa was revealed.

To better understand the effects of salt stress on quinoa seeds, we further investigated the composition of storage proteins after irrigation with 300 mM NaCl as compared with untreated controls. Both quantitative and qualitative differences in protein expression as a result of salt treatment were found. The albumin/globulin fraction, and in particular the 11S globulin, was the most affected fraction in terms of reduced percentage content as well as altered profile, both in SDS-PAGE and 2DE. Proteomic analysis revealed increases in the expression of proteins known to be involved directly in ER lumenal protein folding and in the assembly of proteins (Shewry et al., 1995), thus indirectly in the response to cellular stresses. Thus, despite a general decrease in protein content, up-regulation of some specific proteins, such as proteins involved in specific and fundamental cellular processes (photosynthesis and metabolism) as well as in stress adaptation, was observed by analyzing in detail the selected landrace (VR).

An interesting finding was that the expression level of heat shock-related protein (HSP70 and HSP90) and of a GRP-78/Luminal-binding protein (an endoplasmic reticulum chaperone involved in response to different types of stress) increased after salt treatment. Abiotic stresses, such as salinity, usually cause protein dysfunction. Maintaining proteins in their functional conformations thereby preventing the aggregation of non-native proteins is particularly important for cell survival under stress. Heat-shock proteins (HSPs)/chaperones are responsible for protein folding, assembly, translocation, and degradation in many normal cellular processes; they also stabilize proteins and membranes, and can assist in protein refolding under stress conditions (Wang et al., 2004). Data presented in this paper are supported by previous findings showing that at late developmental stages, when seeds undergo dehydration, LEA proteins, also regarded as chaperonins, are synthesized as part of the developmental program. Moreover, salinity triggers an abscisic acid (ABA)-mediated response that induces dehydrin accumulation in embryo cells (especially in the nucleus). This could imply a role for dehydrins as protective molecules for DNA when the cells are undergoing the normal dehydration process. Under salinity, however, dehydration could be drastically exacerbated, thereby favoring chaperonin accumulation (Burrieza et al., 2012). In Arabidopsis thaliana cell suspension cultures, the expression of HSP70 also increased under salt stress (Ndimba et al., 2005). Worthy of notice was the reduced content of a member of the peptidylprolyl cis-trans isomerase family, also called cyclophilins, known to assist protein folding by accelerating the isomerization of Xaa-Pro bonds, which is a rate-limiting step in the folding process of some proteins. This could be explained by the general decrease of proteins, including the substrates of cyclophilins.

Other proteins of quinoa seeds that responded to salinity are involved in the photosynthetic machinery, i.e., RuBisCO and plastid movement impaired protein. The RuBisCO enzyme is sensitive to salt stress as revealed by enhanced expression in leaves of salt-treated Triticum durum (Caruso et al., 2008). In developing embryos of B. napus L. (oilseed rape), RuBisCo acts without the Calvin cycle. This improves the carbon efficiency of developing green seeds in a metabolic context able to increase the efficiency of carbon use during the formation of oil (Schwender et al., 2004). Although photosynthesis is usually negatively affected by salinity, the enhanced expression of RuBisCO without the Calvin cycle could be involved in plant adaptation to salt stress at the seed level.

The Plastid Movement Impaired protein is required for regulation of chloroplast position in cells in order to reduce their avoidance. Unfortunately, it is difficult to assign a specific function in seed tissues to this protein directly involved in movement of plastids in response to light and normally expressed in leaves. We can only speculate that salt stress and, more generally, abiotic stress cause changes in the expression level of this protein. Salt stress also seemed to perturb proteins involved in respiration. In fact, the expression of mitochondrial ATP synthase was up-regulated in seeds of salt-treated quinoa plants. Mitochondrial ATP synthase is a key enzyme in plant metabolism and upregulation of its activity might be required in response to stressful conditions such salt stress. Increased production of ATP might fuel all the metabolic pathways generally involved in plant homeostasis. In addition, it has been proposed that increased activity of ATP synthase creates the driving force for Na+ transport by the Salt Overly Sensitive1 (SOS1) membrane-localized proteins, whose activity is currently regarded as a strong basis for salt tolerance in plants (Zhu, 2003). Over-expression of an ATP synthase gene in Arabidopsis suspension-cultured cellsincreased resistance to salt, drought, oxidative, and cold stresses (Zhang et al., 2008). Upregulation of SOS1 gene expression was also reported for Chilean landraces of quinoa grown on saline media (Ruiz-Carrasco et al., 2011).

Salinity also affected three other main protein categories. Increased expression levels were observed for ion transport proteins (e.g., a cation/H+ antiporter), and for pantothenate kinase, and helicase carboxy-terminal domain protein involved in metabolism and cell division, respectively. The overexpression of these proteins during stress, and more specifically salt stress, has been reported in several studies. In particular, expression of the cation antiporter was found to increase in salt-treated tomato plants; the protein plays an important role in adaptation to salinity by improving K+ accumulation (Rodriguez-Rosales et al., 2008). Pantothenate kinase activity plays a critical role in regulating intracellular coenzyme A (CoA) levels in bacteria and animals. In plants, pantothenate kinase activity was first reported in stroma of spinach chloroplasts, but little is known about the role of this enzyme (Tilton et al., 2006). Pantothenate, also known as vitamin B5 or "anti-stress vitamin," is part of the water-soluble B vitamin group. It is the key precursor of CoA and acyl carrier protein, which are essential co-factors for many metabolic enzymes (Raman and Rathinasabapathi, 2004). It was suggested that an increased level of CoA, mediated by an increased activity of pantothenate kinase, may be responsible for improved plant growth and stress resistance (Rubio et al., 2008). The main function of helicase is to bind ATP and to catalyze the reaction that drives the unwinding of DNA or RNA helix. Over-expression of this protein is one of the first cellular responses to stress conditions. Thus, pea DNA helicase was stimulated by salinity and cold stress in both shoots and roots (Vashisht et al., 2005). Moreover, DNA helicase 45 mRNA was induced in pea seedlings in response to high concentrations of salt and overexpression of this protein in transgenic tobacco plants conferred salinity tolerance, suggesting an important target pathway for manipulating stress tolerance in crop plants (Sanan-Mishra et al., 2005).

Ethylene Insensitive 3-like 1 protein is probably a transcription factor acting as positive regulator in the ethylene signaling pathway. The expression of this protein decreased in salt-treated quinoa seeds. Ethylene plays important roles in multiple aspects of plant growth and development and its biosynthesis is induced by many stresses. However, its role in salt stress is uncertain. Cao et al. (2007) reported that alteration of ethylene signaling in Arabidopsis mutants affected salt-stress responses. El-Iklil and coworkers showed that lower ethylene production was associated with salt tolerance in tomato treated with high salt concentrations (El-Iklil et al., 2000), whereas higher ethylene production has been suggested as an indicator for salt tolerance in rice seeds (Khan et al., 1987). No information is available on the hormonal responses to abiotic stress in quinoa, except for one report on increased ABA levels under drought (Jacobsen et al., 2009).

The category exhibiting the greatest variation in expression levels under salinity was the functional category of seed storage proteins. Storage proteins accumulate in developing seeds as a source of nitrogen, carbon, sulfur, and amino acids for use in germination and growth of developing seedlings. Globulins generally fall into two major groups based on these coefficients: the 7-8S vicilin type and the 11-12S legumin type. Because legumin-type seed storage proteins vary in size, the 11-12S globulins are collectively referred to as legumins in other species (Balzotti et al., 2008). In our study, proteins belonging to this category were the 11S globulin A or B, and the legumin βlike. The 11S globulins are the major storage proteins in most legumes and in many other dicots (e.g., brassicas, composites, and cucurbits) and in some cereals (oats and rice; Shewry et al., 1995). Also known as chenopodin, the 11S globulin is the major seed storage protein of quinoa. We found two different subunits of chenopodin; A and B subunit. The 11S globulin is a hexamer consisting of six pairs of acidic (34–36 kDa) and basic subunits (22–24 kDa), with each subunit pair connected by a disulfide bond (Brinegar and Goundan, 1993). Recently, the quinoa 11S globulin gene has been suggested to belong to a multigene family (Stevens et al., 2006), as in other species (Shewry et al., 1995). These proteins are related to each other based on their primary structures, with homologies reaching 63% between soybean proA1aB1b and pea prolegumin (Tandang-Silvas et al., 2010).

In the present paper, the 11S seed storage globulin displayed the strongest decrease in salt-treated samples as compared with controls; in fact, in all analyses we found a substantial downregulation of these proteins and even a complete absence of the corresponding spots in 2DE gels. The decrease in total protein content (Ruiz et al., 2016a) and in percentage content of the albumin/globulin fraction is thus ascribable to the severe reduction of the major seed storage protein, chenopodin. Decreased protein content with increasing salinity can also be attributed to disturbance in nitrogen metabolism or inhibition of nitrate absorption through reduced water uptake and decreased root permeability (Karyotis et al., 2003; Pulvento et al., 2012). The legumin β-like protein displayed an opposite trend insofar as the spot intensity increased in salt-treated samples. Legumin holoproteins are composed of six nearly identical subunits with molecular weights of 50–60 kDa. Each subunit is composed of two differently sized polypeptide chains. The larger more hydrophilic one (30–40 kDa) is named α-chain, whereas the smaller more hydrophobic one (20 kDa) is named β-chain (Müntz, 1996). A relatively lower decrease in energy-rich storage compounds, such as storage proteins (legumin-like and 11S seed storage protein) was found in tolerant wheat genotypes than in less tolerant and sensitive genotypes upon cold treatment (Kosová et al., 2015). In soybean seeds, β-conglycinin and glycinin are the two major storage proteins; sulfur deficiency caused a substantial decrease in the level of glycinins and a concomitant increase in β-conglycinins; the subunit composition of these proteins was also affected (Gayler and Sykes, 1985). The crude acid-soluble globulin seemed not to be affected by growing quinoa under salinity, but since a crude extract and not a purified or highly enriched fraction (as was done for the 11S globulin) was analyzed, we cannot exclude possible changes in protein expression and content. Prolamin-related proteins likewise did not show evident changes in the presence of salt. This is probably due to the low amount of these peptides in gluten-free seeds such as those of quinoa. However, also in this case, we cannot exclude slight changes in this fraction, which represents a very interesting research topic with respect to gluten allergy also in so-called gluten-free crops.

### Amino Acid Profiles

The amino acid profile of quinoa is one of its major nutritional attributes as it contains all the essential amino acids (Escuredo et al., 2014). The amino acid composition of quinoa seeds of cultivars from the Andean highlands (Bolivia/Argentina) and Argentinian Northwest were shown to vary depending on genotype and area of cultivation (González et al., 2011). Changes in the amino acid profile under drought and salinity, alone or combined, have been described in wild and cultivated barleys (Ahmed et al., 2013), but not in quinoa. In barley, the contents of all amino acids (except Met) were significantly increased relative to controls under drought alone or under combined stress in all genotypes, whereas they decreased or remained unchanged under salinity alone. Present results confirm the strongly genotype-dependent amino acid profile, both under control and salinized conditions, in the three Chilean landraces analyzed. Unexpectedly, the profiles of R49 (salares ecotype) and VR (coastal lowlands ecotype) were more similar than those of VR and VI-1, which belong to the same ecotype.

The non-essential amino acids Ala, Pro, and Cys were generally the most abundant PAAs in all three landraces. With the exception of VI-1, Pro was also the most abundant free amino acid; it was one of the few whose content was unaltered or enhanced by salinity in all three landraces, including VI-1. Induction of free amino acids in response to abiotic stress is thought to play a role in plant stress tolerance because of their role as osmolytes. The accumulation of Gly, Pro, Ala, and Val is regarded as a general response to stress. In particular, the accumulation of branched-chain amino acids (BCAAs)— Val, Leu, Ile—is induced by osmotic stress (Joshi et al., 2010). Ile is synthesized from Thr and Met; these three amino acids are synthesized in a highly regulated network depending on physiological and growth requirements. In the present study, free BCAAs (Val, Leu, Ile) were most strongly induced (twoto almost four-fold relative to controls) by salinity in R49, but either not affected or diminished in the other two landraces. It is noteworthy that Thr was also strongly upregulated by salinity in both R49 and VR, but not in VI-1. Unlike Pro, little is known about the function of the stress-induced response involving BCAAs, even though Pro may represent only 10% of the total free amino acids induced by drought (Shen et al., 1989). The increases in BCAAs of drought-stressed tomato leaves were also higher than that of Pro (Joshi et al., 2010). Moreover, it has been proposed that accumulation of free BCAAs may serve as a substrate for the synthesis of stress-induced proteins and that they may act as signaling molecules to regulate gene expression. Further research is needed in this area particularly for stresstolerant species such as quinoa.

As a general response to abiotic stress, all plants, even halophytes such as quinoa, accumulate amino acids, betaines, sugars, organic acids, and other osmoprotectants (Parida and Das, 2005; Ruiz-Carrasco et al., 2011). Thus, amino acid metabolism, including protease activation, may play an important role in plant tolerance, e.g., under drought stress (Joshi et al., 2010). As observed in the present study, increased contents of at least some amino acids could be the result of protein degradation, although de novo synthesis cannot be excluded. For example, Pro decreased in the protein fraction, but increased in the free form in all three landraces of quinoa under salinity. A better understanding of this process will also provide new insight into the negative vs. beneficial effects of abiotic stress on the nutritional qualities of quinoa.

In many cereals, two essential amino acids, Thr and Met, are less abundant than required for the human diet. In R49, free Thr was strongly induced by salinity; similarly, Met was induced by salt in both fractions (free and PAA) only in R49, suggesting that salt stress could improve the nutritional properties of this landrace. By contrast, Thr was very high in VI-1 under nonsaline conditions and fell below detection limit in seeds from salt-grown plants. In quinoa, the amount of the essential amino acid Lys is higher than that of cereals (Escuredo et al., 2014). In our study, Lys, representing about 10% of total free amino acids in control seeds, was also differentially affected by salinity in a genotype-dependent manner. Overall, this important amino acid was maintained or induced only in R49 in both fractions.

Based on these results, VI-1 seems to be the most sensitive to salinity in terms of amino acid profiles, and R49 the most tolerant, in accord with its origin from a highly stress-prone area. The differential response between the two coastal-lowlands landraces is in agreement with results for other parameters, namely AA, TPC, and TFC.

## Bioactive Molecules and Antioxidant Activity of Quinoa Seed Protein Extracts

Plant-derived antioxidants (phenolic compounds, tocopherols, tocotrienols, ascorbic acid and carotenoids) are essential for counteracting oxidative stress and hence contrasting the insurgence of various diseases. In recent years, the quest for natural antioxidants for dietary as well as cosmetic and pharmaceutical uses is a major objective in plant research. AA is also a matter of great practical interest in food science since oxidation, mainly of lipids and proteins, can lead to the deterioration of quality attributes, such as flavor, aroma, and color. The health benefits of quinoa seeds derive from their nutritional properties (e.g., amino acid composition, minerals, vitamins) but also from their content in bioactive molecules, such as phenolic compounds, and their antioxidant activity. Not surprisingly, therefore, these aspects are increasingly attracting the attention of researchers (Abderrahim et al., 2015; Tang et al., 2015).

We have previously observed that TPC (measured with the FC method) in methanolic extracts of quinoa seed was genotypedependent and enhanced by salinity in landraces R49 and VR (Ruiz et al., 2016a). In R49, increased seed TPC could be related to better germination capacity on saline media. Improved antioxidant defense may also provide protection from salt stress during seedling establishment. Present results confirm this trend also for PCs obtained from the same seeds. Thus, in all three landraces, seed PCs from plants irrigated with 300 mM NaCl had higher phenolic contents than those grown without NaCl. Spectrophotometric redox assays are among the most common methods for determining total phenolics in plant extracts as they detect the easily oxidized phenolic groups. While being the standard method for the determination of TPC, the FC assay is essentially a means of evaluating the redox potential of a plant extract and is, therefore, subject to interference by many compounds (e.g., sugars, proteins, organic acids; Rodriguez-Amaya, 2010). The Prussian blue method is less subject to interference from non-phenolic compounds and was, therefore, used in parallel in the present work to check for the presence of polyphenols in seed protein extracts. This method provided further proof that polyphenols were present. Although in most cases below detection limit or very low, they were dramatically enhanced in VR under salinity, thus confirming results of the FC assay.

Although protein extraction methods can also extract phenolic compounds, it is important to determine whether they maintain their bioactivity (Salgado et al., 2012). Based on the ABTS radical scavenging test, quinoa seed PC did exhibit AA. Values were also significantly different between landraces (highest in VR) and were slightly increased by salinity in R49 and VR, the two landraces in which TPC and flavonoids (the latter only in VR) also increased the most. These data point to some degree of correlation between phenolic content and AA. If, however, we consider the FC assay also as an indicator of redox potential, then it can be inferred that the antioxidant capacity of salt-treated samples was higher than that of controls in all three landraces.