Variation in salinity tolerance of four lowland genotypes of quinoa (Chenopodium quinoa Willd.) as assessed by growth, physiological traits, and sodium transporter gene expression

Plant Physiology and Biochemistry 49 (2011) 1333e1341

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Research article

Variation in salinity tolerance of four lowland genotypes of quinoa (Chenopodium quinoa Willd.) as assessed by growth, physiological traits, and sodium transporter gene expression Karina Ruiz-Carrasco a,1, Fabiana Antognoni b,1, Amadou Konotie Coulibaly c, Susana Lizardi d, Adriana Covarrubias d, Enrique A. Martínez e, Marco A. Molina-Montenegro e, Stefania Biondi b, Andrés Zurita-Silva d, * a

Dipartimento di Colture Arboree, Università di Bologna, Bologna, Italy Dipartimento di Biologia evoluzionistica sperimentale, Università di Bologna, Bologna, Italy c Université du Mali IPR, IFRA Katibougou, Katibougou, Mali d Centro de Estudios Avanzados en Zonas Áridas (CEAZA), Universidad de La Serena, Av. Raúl Bitrán s/n, PO Box 554, La Serena, Coquimbo, Chile e Centro de Estudios Avanzados en Zonas Áridas (CEAZA), Facultad de Ciencias del Mar, Universidad Católica del Norte, Larrondo 1281, Coquimbo, Chile b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 June 2011 Accepted 12 August 2011 Available online 23 August 2011

Chenopodium quinoa (Willd.) is an Andean plant showing a remarkable tolerance to abiotic stresses. In Chile, quinoa populations display a high degree of genetic distancing, and variable tolerance to salinity. To investigate which tolerance mechanisms might account for these differences, four genotypes from coastal central and southern regions were compared for their growth, physiological, and molecular responses to NaCl at seedling stage. Seeds were sown on agar plates supplemented with 0, 150 or 300 mM NaCl. Germination was significantly reduced by NaCl only in accession BO78. Shoot length was reduced by 150 mM NaCl in three out of four genotypes, and by over 60% at 300 mM (except BO78 which remained more similar to controls). Root length was hardly affected or even enhanced at 150 mM in all four genotypes, but inhibited, especially in BO78, by 300 mM NaCl. Thus, the root/shoot ratio was differentially affected by salt, with the highest values in PRJ, and the lowest in BO78. Biomass was also less affected in PRJ than in the other accessions, the genotype with the highest increment in proline concentration upon salt treatment. Free putrescine declined dramatically in all genotypes under 300 mM NaCl; however (spermidine þ spermine)/putrescine ratios were higher in PRJ than BO78. Quantitative RT-PCR analyses of two sodium transporter genes, CqSOS1 and CqNHX, revealed that their expression was differentially induced at the shoot and root level, and between genotypes, by 300 mM NaCl. Expression data are discussed in relation to the degree of salt tolerance in the different accessions. Ó 2011 Elsevier Masson SAS. All rights reserved.

Keywords: Chenopodium quinoa Chilean lowland genotypes Halophyte Polyamines Proline Salt tolerance Sodium transporters

1. Introduction Variations of environmental components are widely indicated as a major threat to global food security [1]. It has been estimated that over 930 million hectares of arable lands are currently affected by salinity worldwide [2], with a range of soils defined as saline (e.g., acidicesodic, alkalineesodic) [3]. Considering the current models of climate change, lands under salinization are expected to increase during this century [1], especially in those areas where

* Corresponding author. Tel.: þ56 51204378; fax: þ56 51334741. E-mail address: [email protected] (A. Zurita-Silva). 1 Both authors contributed equally to this work. 0981-9428/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2011.08.005

a drastic decrease in precipitation events has been predicted, or is already occurring as in northern Chile [4]. Salt stress is one of the main abiotic factors limiting crop productivity and plant distribution worldwide, since most species are salt sensitive [5]. While improvement in land and water management can help to solve the problem, gaining knowledge about the salt tolerance of crop species is regarded as being of paramount importance [6]. Salt imposes osmotic stress by reducing the soil water potential thereby limiting water uptake; moreover accumulated ions, particularly Naþ and Cl, can become toxic and interfere with various metabolic processes [7]. Despite this, some plants (tolerant glycophytes and halophytes) can tolerate high levels of salt, in both the soil-environment and in tissues, by efficient morphological, physiological and molecular mechanisms,

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which attenuate (or avoid) salt-induced stress [6,8,9]). Such responses depend mainly on the inherent salt tolerance of each species (or variety), as well as on ontogenetic stage [7]. In fact, several studies have shown that even halophytes are salt-sensitive during the stages of seed germination and seedling emergence [10]. Despite this, reports on salt tolerance mechanisms in halophyte seedlings are scarce, even though seedling survival and performance are crucial for successful crop establishment. Under salinity stress, the accumulation of sugars and other compatible solutes (e.g., proline) allows plants to maintain the cellular turgor pressure necessary for cell expansion under stress conditions; they also act as osmo-protectants. Proline is also considered the only osmolyte able to scavenge free radicals thereby ensuring membrane stabilization, and preventing protein denaturation during severe osmotic stress [11]. The protective role of proline in plants under water deficit or salinity conditions has been reported in several species [12e14]. Recently, it has been demonstrated that proline supplements enhanced salt tolerance in olive plants by improving photosynthetic activity, and increasing the activity of enzymes involved in the antioxidant defense system [12]. The accumulation of nitrogen-containing compounds is likewise correlated with plant salt tolerance, and polyamines (PAs) are amongst these [15]. At physiological pH, PAs are cationic and can, therefore, establish strong ionic interactions with negatively charged nucleic acids, membrane phospholipids, and cell wall components. Through these interactions, PAs regulate the structure and function of biological macromolecules. Putrescine (Put), spermidine (Spd) and spermine (Spm) are the most commonly found PAs in plants; they are regarded as essential for normal growth and development [16]. Moreover, enhanced accumulation of PAs appears to be a general response to stress [17]. Indeed, due to their purported protective nature (mainly via membrane stabilization, and free radical scavenging), PAs may represent bioindicators of stress tolerant lines [18]. The protective function of PAs was corroborated by several studies indicating that engineered plants over-expressing PA biosynthetic enzymes have increased resistance toward environmental challenges [19]. PAs also seem to play an important role as regulators of ion channels [16]. In plants, intracellular Kþ and Naþ homeostasis is important for the activities of many cytosolic enzymes, and for maintaining membrane potential and an appropriate osmoticum for cell turgor/ expansion. Under high external NaCl concentrations, the maintenance of Kþ and Naþ homeostasis becomes even more crucial if salt stress is to be attenuated [20]. Salinity tolerance can be related to three main mechanisms of Naþ homeostasis: Naþ exclusion from the shoot, Naþ tissue tolerance, and osmotic tolerance [21,8,7]. Pivotal genes related to Naþ transport have been cloned in several species, and their role in salt tolerance assessed by over-expression of the gene, or its homologue from other species [22,23]. AtNHX1 is the gene encoding the tonoplast-localized vacuolar Naþ/Hþ antiporter [21]. Over-expression of NHX1, regarded as being responsible for Naþ and or Kþ compartmentation in the vacuole [24], or of the plasma membrane Salt Overly Sensitive 1 (SOS1) gene [25,23] increased NaCl tolerance of plants by controlling ion homeostasis under saline stress conditions. With regard to halophytes, five Naþ/Hþ antiporters were recently cloned, and analyzed in response to NaCl stress in the facultative halophyte Mesembryanthemum crystallinum [26], while two homologous SOS1 loci were cloned and characterized in Chenopodium quinoa Willd. (quinoa) [27]. The latter represents the first molecular characterization of salttolerance genes in a halophytic species of the Amaranthaceae, as well as the first comparative analysis of coding and non-coding DNA sequences of the two homologous genomes of quinoa. Quinoa is an Andean native crop grown primarily for its edible seeds. The plant is regarded as one of the crops that might sustain

food security in this century for its excellent nutritional features, such as a high protein content and unique amino acid composition [28]. Moreover, it exhibits remarkable tolerance against several abiotic stresses such as salt [29] and drought [4], and very recently to a combination of salinity and drought in a Danish bred cultivar [30], which has led it to be considered an emerging stress-tolerant model plant. Thus, quinoa is one of the few species that are naturally adapted to extreme environmental conditions, including drought, salinity and frequent frosts, typical of the Andean highlands [31]. It can also grow under arid and saline conditions at sea level near the coast, an important feature for increasing production of this crop in Chile [4]. Chilean quinoa has been characterized as morphologically and genetically highly diverse as a result of artificial/natural selection [32], but the potential offered by its high biodiversity still needs to be fully exploited. Considering the broad range of growth responses observed under salt stress among Peruvian accessions [33], the same diversity could be expected among Chilean genotypes. Based on previous studies regarding some physiological parameters related to drought and salinity adaptation in a quinoa cultivar from the southern Altiplano of Bolivia, the species has been described as a facultative halophyte, and several mechanisms associated with tolerance to abiotic/salt stress have been investigated [34,35]. However, the physiological and molecular responses to high salinity in Chilean genotypes, the least studied within the Andean region with one exception [29], have yet to be analyzed. For instance, the expression of CqSOS1 upon application of NaCl was recently investigated in a cultivar also originating from the salt flats (salares) of the Bolivian Altiplano using 30-day old plants grown hydroponically [27], but the expression of the CqNHX1 gene implicated in the regulation of ion homeostasis under salt stress is studied here for the first time in quinoa. This work examines whether the genetic variability in quinoa is associated with different degrees of salinity tolerance, on the basis of morphological (germination, growth) and physiological (proline, polyamines) responses under salt stress. The aim was to link these modifications to the expression levels of sodium transporter genes, and ultimately, to understand whether the parameters analyzed contribute to salt tolerance in seedlings of four Chilean lowland and coastal genotypes occurring along a latitudinal gradient, an issue that has not been previously addressed with genotypes of central latitudes of the country. 2. Results 2.1. Germination and growth Seed germination was evaluated in the accessions PRJ, PRP, UDEC9 and BO78 (Table 1), four days after sowing on half-strength MS medium supplemented or not with 150 or 300 mM NaCl. Under control conditions (0 mM NaCl), germination percentages were similar in all four accessions, and treatment with 150 mM NaCl did not significantly affect germination in any of them (Fig. 1). By Table 1 Genotypes and original location of Chenopodium quinoa used in the present study. Three different drought indexes were applied in order to range the sites hierarchically. All indexes consider precipitation, temperature and soil texture. The closer the index is to zero, the higher the potential osmotic/salt stress. Accession

Location

PRJ PRP UDEC9 BO78

34 ,500 S; 34 ,600 S; 35 ,730 S; 39 ,100 S;

72 ,010 W 71 ,360 W 72 ,530 W 72 ,680 W

Lang index

de Martonne index

Thornthwaite index

452 493 611 1973

284 298 366 1072

431 452 551 1598

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Fig. 1. Germination percentage of four C. quinoa accessions (four days after sowing) grown on half-strength MS medium supplemented with either 0, 150 or 300 mM NaCl. Values are means of three experiments  2S.E. Different letters indicate significant differences (P < 0.05).

contrast, treatment with the higher salt concentration had a significant and dramatic inhibitory effect only on the southern genotype BO78 (F2,72 ¼ 132.52; p < 0.001), and decreased germination by about 15e30% in the central genotypes PRJ, PRP, and UDEC9 (Fig. 1). Thus, seed germination was significantly affected by NaCl treatment depending upon genotype (F6,72 ¼ 35.23; p < 0.001, Table 2). Root and shoot lengths were measured after 15 days of growth on Petri dishes. At 150 mM NaCl, a slight (approximately 20%) reduction of shoot length relative to untreated controls was observed in all accessions except UDEC9 (Fig. 2A). In the presence of 300 mM NaCl, the inhibitory effect was notably higher, reaching about 70% reduction relative to controls in PRJ, PRP and UDEC9; no further inhibition in shoot length was observed in BO78 (Fig. 2A). Thus, shoot inhibition by NaCl was significantly different among genotypes (F3,72 ¼ 16.09; p < 0.001, Table 2). Variation in root length (Fig. 2B) was significantly affected by salt treatment (F2,72 ¼ 46.65; p < 0.001), and exhibited a different response in the various genotypes (F3,72 ¼ 13.11; p < 0.0041, Table 2). At 150 mM NaCl, only a very small reduction (<10% less than controls) was observed in PRJ and PRP, and an increment in UDEC9 and BO78 (Fig. 2B). However, at 300 mM NaCl, the strongest inhibition (about 70%) of root length, which was significantly different from that of the other accessions, occurred in BO78 (Fig. 2B), while PRJ, PRP and UDEC9 were inhibited by less than 40% at this concentration. Thus, the genotype  treatment interaction factor was significant (F6,72 ¼ 35.33; p < 0.005, Table 2). The root-to-shoot length ratio (Fig. 3) of each genotype was not affected by treatment with 150 mM NaCl as compared with controls (F1,345 ¼ 132.65; p ¼ 0.13). However, under 300 mM NaCl,

Fig. 2. Seedling growth parameters of four C. quinoa genotypes (15 days after sowing) grown on half-strength MS medium supplemented with either 0, 150 or 300 mM NaCl. Shoot growth inhibition (panel A) and root growth inhibition (panel B) in NaCl-treated seedlings relative to controls. Values are means of three experiments  2S.E. Different letters indicate significant differences (P < 0.05).

the root/shoot ratio was differentially affected in the different genotypes (F3,345 ¼ 1.23; p < 0.001, Table 2). In PRJ, the ratio was significantly higher than in controls and 150 mM NaCl-treated seedlings, and higher than in the other three accessions; the lowest ratio among genotypes was in BO78, which was also the only accession in which the ratio decreased relative to controls and 150 mM treatment (Fig. 3). Total dry biomass production (Fig. 4) was different between genotypes under control conditions, with PRP showing the lowest

Table 2 Significance of the variation source (salt treatment, genotype, genotype  treatment interaction) in germination and seedling growth of four different quinoa genotypes under control or saline (150 or 300 mM NaCl) conditions. Source

Germination

Root length

Shoot length

R/S ratio

Biomass

Salt treatment (T) Genotype (G) G  T interaction

** * **

** * **

** ** **

NS * *

** ** NS

Asterisks indicate a significant difference between NaCl treatments and the control at P < 0.05 (*) and P < 0.01 (**) according to Tukey’s test. NS, not significant.

Fig. 3. Root-to-shoot length ratio of four C. quinoa genotypes (15 days after sowing) grown on half-strength MS medium supplemented with either 0, 150 or 300 mM NaCl. Values are means of three experiments  2SE. Different letters indicate significant differences (P < 0.05).

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Fig. 4. Seedling dry biomass (expressed as g dry weight/seedling) of four C. quinoa genotypes (15 days after sowing) grown on half-strength MS medium supplemented with either 0, 150 or 300 mM NaCl. Values are means of three experiments  2S.E. Different letters indicate significant differences (P < 0.05).

value. At 150 mM NaCl, PRJ exhibited a higher biomass than the other three accessions, but none of the genotypes were significantly affected by this salt treatment. Under 300 mM NaCl, a significant decrease compared with controls was observed in all genotypes, except PRP (F2,68 ¼ 18.10; p < 0.001). Although inhibited relative to the control, PRJ maintained the highest biomass as compared with the other genotypes also under this treatment (F2,68 ¼ 17.3; p < 0.001), while no differences were found among PRP, UDEC9 and BO78 under either of the two salt concentrations (Fig. 4). Salt treatment and genotype effects were significant, but not the genotype  treatment interaction (Table 2). 2.2. Proline and polyamine concentrations Proline concentrations were similar in all accessions under control conditions (0 mM NaCl) with the largest difference occurring between BO78 and PRP (Fig. 5). When seedlings were exposed to 150 mM NaCl (data not shown), none of the genotypes exhibited an increase in their proline concentrations compared with controls. By contrast, under the 300 mM salt treatment, a significant increase

occurred in all genotypes (F1,16 ¼ 144.13; p < 0.001), with PRJ showing the highest proline accumulation compared to controls as well as to other accessions, followed by PRP and, finally, UDEC9 and BO78. The genotype  treatment interaction was not significant (Table 3). Free PA content was evaluated in seedlings treated or not with NaCl for 7 days. In untreated seedlings, free Put was the most abundant PA, followed by Spd and Spm; no significant differences among accessions were observed in the concentration of any of the three PAs (Fig. 6A). The most evident effect of 300 mM NaCl on the PA profile was a drastic reduction (from ca. 5-fold up to 8.5 fold in BO78) of Put levels occurring across all genotypes (F1,16 ¼ 139.7; p < 0.001; Fig. 6A). The response was similar, or slightly less pronounced, with 150 mM NaCl (data not shown). By contrast, in the presence of 300 mM salt, Spd levels were not affected, and Spm levels always increased, with PRP showing the highest (3-fold) increase (Fig. 6A). The (Spd þ Spm)/Put ratio increased dramatically (up to 10-fold) in response to salinity (F1,16 ¼ 144.13; p < 0.001) with BO78 showing a significantly lower ratio than the other accessions (Fig. 6B). The genotype  treatment interaction was not significant (F3,16 ¼ 0.72; p ¼ 0.55; Table 3), because all accessions increased the ratio to a similar degree. 2.3. Salt transporter gene expression The transcript levels of CqSOS1A, CqSOS1B and CqNHX1 were determined in both shoot and root tissues of all four quinoa genotypes exposed to 300 mM NaCl compared to their corresponding untreated controls (0 mM NaCl), and normalized to the expression level of CqEF-1a. On the whole, shoots displayed more pronounced changes in the regulation of the three genes analyzed under salt-stress conditions in comparison to roots for all genotypes (F1,48 ¼ 538.37; p < 0.001). Thus, in root tissues, low or no induction in UDEC9 and BO78, and a significant down-regulation in PRP was observed for CqSOS1A (F3,16 ¼ 36.16; p < 0.001; Fig. 7A) and CqSOS1B (F3,16 ¼ 49.67; p < 0.001; Fig. 7C). A significant upregulation of these two genes occurred only in PRJ (Fig. 7A, C). PRP was also the only genotype that exhibited no CqNHX1 induction in roots (F3,16 ¼ 55.79; p < 0.001), whereas the gene was strongly up-regulated in PRJ, and to a lesser, though significant (p < 0.05) extent, in BO78 and UDEC9 (Fig. 7E). By contrast, CqSOS1A was strongly and significantly upregulated by salt treatment in shoots of PRP (ca. four times more than UDEC9 and BO78, Fig. 7B). In shoots, the expression level of CqSOS1B was again most strongly induced in PRP (about 4-fold relative to the other genotypes) by 300 mM NaCl, while in salttreated PRJ and BO78 a lower, but significant, induction also occurred (Fig. 7D). Under salt treatment, all genotypes, except BO78, exhibited a strong induction of CqNHX1 in shoots tissues (Fig. 7F); indeed, PRJ, PRP and UDEC9 displayed an 8-fold higher relative expression compared to the southern genotype BO78. A comparative analysis of the three genes indicates that saline conditions induced stronger increments in CqNHX1 than in CqSOS1B and even less in CqSOS1A (F2,48 ¼ 353,40; p < 0.001). The Table 3 Significance of the variation source (salt treatment, genotype, genotype  treatment interaction) in proline and polyamine concentrations of four different quinoa genotypes under control or saline (300 mM NaCl) conditions.

Fig. 5. Proline concentration (mmol/g FW) in seedlings of four C. quinoa genotypes (7 days after sowing) grown on half-strength MS medium supplemented with either 0 or 300 mM NaCl. Values are means of three experiments  2S.E. Different letters indicate significant differences (P < 0.05).

Source

Proline

Put

Spd

Spm

Spd þ Spm/Put

Salt treatment (T) Genotype (G) G  T interaction

** NS NS

** NS NS

NS NS NS

* ** NS

** NS NS

Asterisks indicate a significant difference between NaCl treatments and the control at P < 0.05 (*) and P < 0.01 (**) according to Tukey’s test. NS, not significant.

K. Ruiz-Carrasco et al. / Plant Physiology and Biochemistry 49 (2011) 1333e1341

Fig. 6. Free putrescine spermidine and spermine concentrations in 7-day old seedlings of four C. quinoa ecotypes grown on half-strength MS medium supplemented with either 0 or 300 mM NaCl (panel A). Values are means of three experiments  2S.E. Different letters indicate significant differences (P < 0.05). Polyamine [(Spd þ Spm)/ Put] ratios in control and salt-stressed ecotypes of C. quinoa (panel B). Values are means of three experiments  2S.E. Different letters indicate significant differences (P < 0.05).

genotypic response was also significantly different, with the highest levels of induction occurring in PRJ, followed by UDEC9 and BO78 (F3,48 ¼ 87,42; p < 0.001), while a significant down-regulation of genes (CqSOS1A and B) was observed only in roots of PRP. Overall, the genotypes responded differentially to 300 mM NaCl in terms of the transcript accumulation of CqSOS1A, CqSOS1B and CqNHX1 in shoot and root tissues (F6,48 ¼ 60.17; p < 0.001, Table 4). 3. Discussion Four lowland Chilean genotypes of C. quinoa coming from sites with distinct pedo-climatic characteristics located along a NeS latitudinal gradient in coastal-central and southern Chile were assessed for their response to imposed salt stress at early stages of plant development. Seed germination was strongly inhibited by 300 mM NaCl only in the accession collected from the southernmost provenance (BO78), whereas germination was unaffected in the genotypes from more northern locations. Accordingly, a lesser reduction in seedling root growth was observed in the latter genotypes as compared with BO78, with PRJ showing the highest tolerance. The sensitivity of BO78 was further confirmed by transferring young (2-day old) seedlings from control to NaClcontaining media and monitoring root growth over one week in order to avoid the interference with salt effects on germination (data not shown). In fact, rapid responses to salt (occurring within

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a few hours of exposure) often resemble responses to low water potential imposed using non-ionic solutes, whereas longer-term responses that occur over a period of days or weeks are more salt-specific [7]. Several studies have shown that even halophytes are salt sensitive during the stages of seed germination and seedling emergence [10]. Moreover, halophyte seed germination displays a high degree of inter- and intra-specific variability [29], thus germination and root length are useful traits to screen germplasm collections for salt tolerance in many species [7]. Germination rates as high as 75% have been reported for quinoa at a salt concentration of 57 mS cm1 [31]. In a study of a Peruvian variety of quinoa [36], plants showed a significant reduction in growth but were able to complete their life cycle and produce seeds even at the salinity level of seawater (approximately 500 mM NaCl). However, in a comparative study between quinoa selections from the Altiplano region of northern Chile and from the southern humid region with non-saline soils, Delatorre-Herrera and Pinto [29] observed a contrasting response to salinity. Application of 400 mM NaCl reduced seed germination percentages by 53% in the northern, most tolerant selection (Amarilla), and by as much as 90% in the less tolerant southern selection (Hueque). Plant responses to salt stress mainly involve changes in ion uptake/translocation/compartmentation, and in metabolic and/or gene expression patterns aimed at maintaining cellular homeostasis. These lead to varying types and degrees of adaptation, ultimately resulting in modified growth rate or differential root-to-shoot biomass allocation [37]. In BO78, shoot growth was inhibited by 300 mM NaCl much less than root growth, and less than in the other genotypes, suggesting that there was a lower accumulation of Naþ in the shoot than in the root, possibly due to reduced translocation. Conversely, root length was most strongly reduced in BO78. This resulted in a strongly diminished root-to-shoot length ratio in this accession as compared with the others under the higher salt treatment; again, PRJ could be distinguished because its root/shoot ratio was highest, a feature normally associated with enhanced tolerance, and because its dry biomass also remained higher than in the other genotypes. Plants often allocate a greater proportion of their biomass to the organs that are involved in acquiring the resources that are scarcest [38]. In general, this supports the notion that salt tolerance demands a root system capable of sustaining or inducing growth under challenging conditions. This is indeed the case for PRJ and PRP that responded by allocating more resources to the root than UDEC9 and BO78. General inhibition of shoot growth with continued root growth has been regarded as a morphological adaptation to salt or water stress, and an increase in the root/shoot ratio was observed in the halophyte Suaeda maritima under salt stress [39]. Taken together, all the growth parameters analyzed (germination, root length, root/shoot ratio, dry biomass) indicate that BO78 is a salt-sensitive genotype, and PRJ the most tolerant amongst those presently examined. Overall, the response of the four Chilean accessions is in line with their geographical origin and, hence, with the gradient of temperature, precipitation and soil composition (Table 1). It is noteworthy that germination, seedling root and shoot length, and dry biomass were scarcely affected or unaffected by application of 150 mM NaCl, or even enhanced (root length), thus confirming the halophytic nature of quinoa. This is in accord with results obtained by Hariadi et al. [34], who found optimal longterm plant growth and biomass in pot-grown plants at NaCl levels between 100 and 200 mM. In the four lowland genotypes investigated, proline did not accumulate when seedlings were exposed to moderate NaCl concentration (150 mM, data not shown), suggesting that this salinity treatment did not impose a stressful condition, as expected in a halophytic (or facultative halophytic) species whose germination and growth were likewise not inhibited by the same treatment.

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Fig. 7. Expression levels of CqSOS1A (panels A and B), CqSOS1B (panels C and D), and CqNHX1 (panels E and F) in roots (panels A, C and E) and shoots (panels B, D and F) of 7-day old seedlings of four C. quinoa genotypes grown on half-strength MS medium supplemented with either 0 or 300 mM NaCl. Values are the means (S.D.) of three biological replicates of relative expression levels (log2 fold change) of 300 mM NaCl-treated samples over controls, normalized to the expression level of CqEF1-a. Asterisks indicate significant differences (P < 0.05) relative to the control within each genotype; different letters indicate differences among NaCl-treated genotypes.

In fact, proline concentrations after exposure to 150 mM NaCl were below control levels (data not shown), perhaps indicating that low salt treatment plants was more beneficial than no added salt, in agreement with Maughan et al. [27] and Hariadi et al. [34]. Conversely, 300 mM NaCl induced an accumulation of proline in all accessions; these could be distinguished into those that exhibited a moderate increase (UDEC9, BO78), and those that accumulated this osmolyte 3- to 5-fold more than control levels (PRP, PRJ). Considering that this compatible solute acts as an osmoprotectant with a positive function in mitigating abiotic stress Table 4 Significance of the variation source (tissue, genotype, tissue x genotype interaction) in CqSOS1A, CqSOS1B and CqNHX1 gene expression of four different quinoa genotypes under control or saline (300 mM NaCl) conditions. Source

CqSOS1A

CqSOS1B

CqNHX1

Tissue (T) Genotype (G) T  G interaction

** ** **

** ** **

** ** **

Asterisks indicate a significant difference between NaCl treatments and the control at P < 0.05 (*) and P < 0.01 (**) according to Tukey’s test.

[11], the southern genotype BO78 appears again to be the less salt-tolerant, and the coastal-central genotype PRJ the most salttolerant accession. Different from other assessed traits, PA concentrations followed the same pattern in all salt-treated quinoa genotypes, namely a sharp decline in Put levels, accompanied by an increase in the (Spd þ Spm)-to-Put ratio. The fact that Put was the PA which declined most is in favor of the idea that Spd/Spm may be more closely linked with vital physiological processes; hence, their levels are more tightly regulated than those of Put [40]. A reduction in Put levels under long-term salt stress has been reported in other species exposed to salinity stress [41]; the inverse relationship with tissue levels of Naþ or Kþ are in accord with its purported role in maintaining the cation/anion balance. On the other hand, the protective role of PAs in plants exposed to abiotic stresses is well established [42], and reports point specifically to the importance of Spd and/or Spm in salt tolerance [18,19,43]. Since barley seedlings treated with exogenous Spd maintained higher Kþ/Naþ ratios under salt stress by limiting Naþ transport into shoots [44], ion channel regulation by PAs is now regarded as an important mechanism in salt stress tolerance. Interestingly, the (Spd þ Spm)/Put ratio was significantly

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lower in BO78 than in the other analyzed genotypes. In accord with other parameters, and its provenance from the least stress-prone environment, and since Spd/Spm are regarded as protective molecules, this observation confirms the highest sensitivity status of this southern genotype. Thus, whereas highest proline accumulation distinguishes the most tolerant accession (PRJ) from the others, the PA response, instead, distinguishes the most sensitive (BO78) from the others. In general, a salt-tolerant genotype might be characterized by elevated levels of both proline and PAs (Spd/Spm), with both exerting protective functions. In the present study, CqSOS1A, and especially CqSOS1B, were, on the whole, more strongly up-regulated by salt stress in shoots than in roots. This behavior is in accord with the results of Maughan et al. [27] who reported that CqSOS1 expression was significantly upregulated by 450 mM NaCl treatment in leaf tissue only, but not in roots. Likewise, in the facultative halophyte M. crystallinum, realtime qPCR analyses revealed an induction by NaCl treatment of McSOS1; a physiological role of McSOS1, together with McNhaD and McNHX1, in Naþ compartmentation during plant adaptation to high salinity was proposed [26]. Indeed, in shoots of PRP, an accession displaying a fairly high level of salt tolerance as assessed by germination, growth and physiological parameters, CqSOS1A and B were strongly induced. By contrast, the more salt-sensitive genotype BO78 showed no alteration in the transcript levels of these two genes (except for a slight up-regulation of CqSOS1B in shoots). Interestingly, in the roots of salt-treated plants of PRP a strong down-regulation of both genes was observed, whereas CqSOS1 expression was enhanced only in the roots of PRJ, the most salttolerant genotype. Indeed, a recent study of four ecotypes of Arabidopsis thaliana revealed an inverse correlation between AtSOS1 expression and plant Naþ content, supporting its role in efflux from the plant [45]. Thus, up-regulation of this gene in PRJ roots suggests that tolerance might be associated with reduced Naþ translocation also in quinoa. NHX1 gene expression levels have not been hitherto investigated in C. quinoa. Present results indicated that significant induction of CqNHX1 expression by NaCl occurred in shoots of all genotypes analyzed, except in the most salt sensitive one (i.e., BO78). At the root level, CqNHX1 transcript levels followed a different pattern in response to treatment with 300 mM NaCl: an up-regulation of this gene was observed in the most tolerant accession (PRJ), but not in the other tolerant one (PRP), suggesting that their tolerance may depend upon different mechanisms of ion (Naþ and/or Kþ) uptake/exclusion, translocation and compartmentation. Compartmentation of Naþ into vacuoles is regarded as a critical mechanism to avoid the toxic effects of Naþ in the cytosol while providing additional osmoticum for water uptake and turgor maintenance. This function has been attributed to tonoplastlocalized NHX-like antiporters. Moreover, it seems plausible that cation/proton transporters of the NHX family are also instrumental in the Hþ-linked Kþ transport that mediates active Kþ uptake at the tonoplast for the unequal partitioning of Kþ between vacuole and cytosol [46]. Thus, under salt or osmotic stress, NHX proteins may exert a protective function through the vacuolar compartmentation of Kþ as well as Naþ thereby preventing toxic Naþ/Kþ ratios in the cytosol. This has recently been demonstrated by over-expressing AtNHX1 in tomato plants [24]. Likewise, in M. cristallinum, the correlation between the kinetics of changes in the transcript levels of McNHX1 followed that of Naþ accumulation over a period of 14 days [26], suggesting the pivotal role of NHX1-mediated Naþ accumulation in leaves under salt stress. Similarly, Hariadi et al. [34] emphasized the role of inorganic ions for osmotic adjustment in quinoa under saline conditions. Thus, the stronger salt-induced up-regulation of CqNHX1 in the shoots of the more tolerant accessions than in the salt-sensitive BO78, and its maximum

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induction in the roots of the most tolerant genotype (PRJ), may be in accord with the notion that vacuolar Naþ/Kþ accumulation is an important tolerance mechanism in this species. In conclusion, this work points to the existence of genotypespecific responses to high salinity in quinoa, thus confirming, at physiological and molecular levels, the high genetic diversity existing among Chilean lowland genotypes, and its potential for crop improvement. Some coastal-central genotypes (like PRJ) are highly tolerant to salt stress probably due to the agricultural practices of small-scale farmers who cultivate quinoa close to the estuaries facing the Pacific Ocean where soil salt content increases periodically, thereby contributing to selection toward higher salt tolerance. Results indicate, for the first time in this species, that PAs may be useful markers of salt tolerance. Finally, gene expression analyses of sodium antiporters CqSOS1 and CqNHX1 confirm the different response to NaCl in shoots vs. roots, but also introduce the notion that they are differentially regulated in more salt tolerant vs less salt tolerant genotypes. This calls for further studies aimed at investigating the mechanisms of Naþ and Kþ xylem loading, transport to the shoot, and vacuolar compartmentation in conferring different degrees of salinity tolerance. 4. Materials and methods 4.1. Plant material and growth conditions C. quinoa Willd. seeds were collected from four geographical locations in coastal-central and southern Chile along a latitudinal gradient with contrasting soils and precipitation regimes (decreasing salt content and increasing precipitation from northern to southern locations). This latitudinal gradient goes from central (ca. 34.5 S) to southern (ca. 39.1 S) Chile, and corresponds to a decreasing drought and salinity gradient (Table 1). Initially, a small number of seeds (four to five) per individual plant, collected from a relatively large number of sampled plants (over 100) per site, provided the initial pool of seeds. Seeds from each population analyzed (accessions PRJ, PRP, UDEC9 from the centre, and BO78 from the south [32]) were pooled, and randomized before performing the experimental treatments. All seeds were obtained from the National Seed Bank of Chile managed by INIA-Intihuasi (Vicuña, Chile) where plants were generated from the initial seed pool, and grown under controlled conditions [4]. Since maternal effects have been shown to affect some ecophysiological traits in many crop species, the F2 of all populations was used. Seeds were surface-sterilized with 70% (v/v) ethanol for 5 min followed by 10% (v/v) commercial bleach for 5 min, and rinsed five times in sterile water. After stratification at 4  C for three days to synchronize germination, seeds were sown on 12  12 cm Petri dishes containing autoclaved half-strength Murashige and Skoog medium (MS) [47] supplemented with 0.5% sucrose and 1% (w/v) Phytagel (Sigma-Aldrich), with or without NaCl. Plates (five per treatment) containing 12e14 seeds each were then arranged vertically in growth chambers at 21  C (2) under a 16/8-h light/ dark photoperiod (50 mmol m2 s1 irradiance) for up to 15 days. Preliminary experiments were performed at five different NaCl concentrations, i.e., 0, 75, 150, 300 and 500 mM, in order to determine the sub-lethal concentration. Based on these results, the concentrations used for further experiments were 150 and 300 mM NaCl, since the highest concentration caused almost total inhibition of germination. 4.2. Germination and seedling growth parameters In vitro germination percentage was recorded at four days after sowing (DAS) on MS media supplemented with either 0, 150 or

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300 mM NaCl. Additionally, shoot and root lengths were measured at 15 DAS. To obtain final dry biomass, both control and treated 15-day old seedlings were oven-dried in paper sacks at 60  C until no differences in weight were registered. 4.3. Proline quantification Tissue proline concentration was determined in seedlings grown on 0, 150 and 300 mM NaCl following Bate’s [48] method with slight modifications. About 100 mg of frozen shoots were ground in 1.2 ml of 3% sulphosalicylic acid and the homogenate centrifuged at 16,000 g at room temperature for 20 min. An aliquot of the supernatant (1 mL) was added to 2 mL ninhydrin reagent [2.5% ninhydrin in glacial acetic acid-distilled water-85% orthophosphoric acid (6:3:1)]. The reaction mixtures were kept in a water bath at 90  C for 1 h to develop the colour. Test tubes were then cooled in an ice-bath, and 2 mL toluene was added to separate the chromophore. Absorbance of the toluene phase was read in a spectrophotometer at 525 nm, and proline concentration was calculated by comparing sample absorbancies with the standard proline curve. 4.4. HPLC analysis of polyamines PA analyses were performed on 7 day-old seedlings grown in the absence or in the presence of 300 mM NaCl. The samples (about 0.02 g DW) were homogenized in 10 volumes of 4% (v/v) cold perchloric acid and centrifuged at 14,000  g for 30 min at 4  C. Aliquots (0.2 mL) of the supernatant and standard solutions of putrescine, spermidine and spermine were derivatized with dansylchloride (5 mg mL1 acetone), extracted with toluene, and analyzed by High-Performance Liquid Chromatography (HPLC, Jasco, Tokyo, Japan) with a reverse phase C18 column (Spherisorb ODS2, 5 mM particle diameter, 4.6  250 mm, Phenomenex, Torrance, CA, USA). 4.5. CqNHX1 cloning For cloning a CqNHX1 gene, RNA was extracted, as described by Meisel et al. [49], from whole 7-day old quinoa seedlings (PRP genotype) germinated on MS/2 medium during four days, and then transferred to 300 mM NaCl for induction. RNA was treated with DNaseI, according to the manufacturer’s instructions (New England Biolabs); SuperScriptÒ One-Step RT-PCR System with PlatinumÒ Taq DNA Polymerase kit (Invitrogen, California, USA) was used to synthesize and amplify directly an orthologous NHX1 gene. The primers were designed from the NHX1 sequence of Chenopodium glaucum (NCBI No. AY371319); the CACC sequence was added at the 50 end of the forward primer for cloning into the pENTR-D-TOPO vector (Invitrogen) by topoisomerization. Based upon sequence conservation across species of the Amaranthaceae family (Atriplex gmelini, C. glaucum, Salicornia europaea and Suaeda maritima), the primers were finally designed to span the full NHX1 Open Reading Frame (ORF) of C. glaucum (from ATG to the stop codon). The oligonucleotide sequences used were: 50 CACCATGTGGTCACAGTTAAGCTCT-30 (sense), and 50 - CTATGTTCTGT CTGCCAAA-30 (antisense). The protocol used for cDNA synthesis and PCR consisted of a first initial cycle of 30 min at 45  C and 2 min at 94  C, and then 40 cycles as follows: 94  C for 30 s, 55  C for 30 s and 72  C for 2 min; finally one cycle at 72  C for 10 min. Five hundred ng of total RNA were used. Once an amplicon was obtained, it was cloned into pGEM-T (the PCR kit used extreme left “A” free); this vector was sent for sequencing, and retrieved sequences were further confirmed through Blastn at GenBank.

4.6. Quantitative real-time PCR (qRT-PCR) analysis Total RNA was extracted from roots and shoots of 7 day-old seedlings according to Chang et al. [50]. RNA yield and purity were checked by means of UV absorption spectra, whereas RNA integrity was determined by electrophoresis on agarose gel. DNA was removed using the TURBO DNA-freeÔ (Applied Biosystems, California, USA) from 6 mg aliquots of total RNA. The first-strand cDNA was synthesized from 3 mg of the DNaseI-treated RNA by means of the High-Capacity cDNA Kit (Applied Biosystems) using random primers. Real-time PCR (qRT-PCR) was performed in a final volume of 25 mL containing 3 ng of cDNA, 5 pmol of each primer, and 12.5 mL of the Fast SYBR Green PCR master mix (Applied Biosystems) according to the manufacturer’s instructions. The Elongation Factor1a (EF1a) housekeeping gene was used as reference gene to normalize, and estimate up- or down-regulation of the target genes for all qRT-PCR analyses: 50 - GTACGCATGGGTGCTTGACAAACTC-30 (sense); 50 - ATCAGCCTGGGAGGTACCAGTAAT-30 (antisense). The Salt Overly Sensitive 1 (SOS1) related primer sequences were those reported by Maughan et al. [27]. NHX1 sequences were used to amplify CqNHX1 amplicons that were 200-bp long: 50 -GCACTTCTGTTGCTGTGAGTTCCA-30 (sense); 50 -TGTGCCCTGA CCTCGTAAACT- GAT- 30 (antisense). PCRs were carried out with StepOnePlusÔ 7500 Fast (Applied Biosystems) for 30 sec at 95  C and then for 40 cycles as follows: 95  C for 3 s, 60  C for 30 s. Cycle threshold (Ct) values were obtained and analyzed with the 2DDCT method [51]. The relative expression ratio (log2) between each target gene and the reference gene, and fold changes (FC) between salt-treated samples vs corresponding controls were calculated from the qRT-PCR efficiencies and the crossing point deviation using the mathematical model of Pfaffl [52]. Data are the means (S.D.) of three biological replicates. 4.7. Statistical analysis A two-way analysis of variance (ANOVA), followed by Tukey’s post-test, was performed to evaluate the effects of NaCl on germination, growth traits, proline and polyamine concentrations, and gene expression, and to compare genotypes among each other, after testing for normality and homogeneity of variances using the ShapiroeWilkes and Bartlett tests, respectively. Acknowledgements This work was supported by ICGEB-TWAS [CRP.PB/CHI06-01 to A.Z.S.], InnovaChile [06FC01IBC-71 to A.Z.S.], IRSES [PIRSES-GA2008-230862], IMAS [ANR 07 BDIV 016-01], and University of Bologna funds [RFO to F.A. and S.B]. K.R. is the recipient of a PhD fellowship from DCA, Faculty of Agriculture, University of Bologna. We thank Dr Pedro León Lobos of the INIA-Chile National Seed Bank Repository for the quinoa seeds. References [1] N.V. Fedoroff, D.S. Battisti, R.N. Beachy, P.J. M Cooper, D.A. Fischhoff, C.N. Hodges, V.C. Knauf, D. Lobell, B.J. Mazur, D. Molden, M.P. Reynolds, P.C. Ronald, M.W. Rosegrant, P.A. Sanchez, A. Vonshak, J.K. Zhu, Radically rethinking agriculture for the 21st century, Science 327 (2010) 833e834. [2] FAO. FAO land and plant nutrition management service. http://www.fao.org/ ag/agl/agll/spush (accessed 15.11.10). [3] P. Rengasamy, Soil processes affecting crop production in salt-affected soils, Func. Plant Biol. 37 (2010) 613e620. [4] E.A. Martínez, E. Veas, C. Jorquera, R. San Martín, P. Jara, Re-introduction of Chenopodium quinoa Willd. into arid Chile: cultivation of two lowland races under extremely low irrigation, J. Agron. Crop Sci. 195 (2009) 1e10. [5] R. Munns, Physiological processes limiting plant growth in saline soil: some dogmas and hypotheses, Plant Cell Environ. 16 (1993) 15e24.

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