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Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na+ loading and stomatal density
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Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na+ loading and stomatal density Sergey Shabala a,∗ , Yuda Hariadi a,c , Sven-Erik Jacobsen b a b c
School of Agricultural Science, University of Tasmania, Private Bag 54, Hobart, Tas 7001, Australia Faculty of Science, University of Copenhagen, Højbakkegård Allé 13, 2630 Tåstrup, Denmark Department of Physics, Faculty of Mathematics and Natural Sciences, University of Jember, Jember 68121, East Java, Indonesia
a r t i c l e
i n f o
Article history: Received 25 September 2012 Received in revised form 30 January 2013 Accepted 30 January 2013 Available online xxx Key words: Osmotic adjustment Potassium Sodium Transpiration Xylem sap
a b s t r a c t Quinoa is regarded as a highly salt tolerant halophyte crop, of great potential for cultivation on saline areas around the world. Fourteen quinoa genotypes of different geographical origin, differing in salinity tolerance, were grown under greenhouse conditions. Salinity treatment started on 10 day old seedlings. Six weeks after the treatment commenced, leaf sap Na and K content and osmolality, stomatal density, chlorophyll fluorescence characteristics, and xylem sap Na and K composition were measured. Responses to salinity differed greatly among the varieties. All cultivars had substantially increased K+ concentrations in the leaf sap, but the most tolerant cultivars had lower xylem Na+ content at the time of sampling. Most tolerant cultivars had lowest leaf sap osmolality. All varieties reduced stomata density when grown under saline conditions. All varieties clustered into two groups (includers and excluders) depending on their strategy of handling Na+ under saline conditions. Under control (non-saline) conditions, a strong positive correlation was observed between salinity tolerance and plants ability to accumulate Na+ in the shoot. Increased leaf sap K+ , controlled Na+ loading to the xylem, and reduced stomata density are important physiological traits contributing to genotypic differences in salinity tolerance in quinoa, a halophyte species from Chenopodium family. © 2013 Elsevier GmbH. All rights reserved.
Introduction Salinity tolerance is a complex multigenic trait, both genetically and physiologically (Flowers, 2004), with less than 25% of salt-regulated genes being salt stress-specific (Ma et al., 2006). While recent success in creating salt tolerant wheat in Australia by introgressing a Na+ transporter TmHKT1;5-A (Munns et al., 2012) suggests that the problem is tangible, pyramiding key physiological traits conferring the most essential characteristics (Flowers and Yeo, 1995; Shabala and Mackay, 2011) remains the preferred option in plant breeding for salinity tolerance. The identity and relative importance of these traits may come from studies on halophytes. Another strategy to cope with increased salinity problems in agriculture is to directly utilise halophytes which are naturally salt tolerant species. Significant genetic variability in salinity stress tolerance exists between halophytes. While dicotyledonous halophytes show optimal growth at concentrations of around 150 mM NaCl, monocotyledonous halophytes generally grow optimally in the absence of salt or, if growth is stimulated, this is achieved at much lower (50 mm or less) concentration of NaCl (Glenn et al.,
∗ Corresponding author. Tel.: +61 3 62267539; fax: +61 3 62262642. E-mail address: [email protected] (S. Shabala).
1999; Flowers and Colmer, 2008). Furthermore, such genetic variability is often observed even within one halophyte species. The latter may be illustrated using quinoa as an example. Being classified as a highly salt tolerant species (Hariadi et al., 2011; Jacobsen, 2011; Razzaghi et al., 2011; Adolf et al., 2013) and capable of growing under salt concentrations of 40 dS m−1 similar to those found in seawater (Jacobsen, 2003), quinoa genotypes display significant variability in agronomical and physiological responses when grown under saline conditions (Adolf et al., 2012). The physiological basis for this genetic variability in salinity tolerance in quinoa, as well as in other halophytic species, is not fully understood. Quinoa seems to use several mechanisms in order to acclimate to a saline environment (Wilson et al., 2002). In the cotyledonous stage, high adaptability to soil salinity may be due to improved metabolic control based on ion absorption, osmolyte accumulation and osmotic adjustment (Ruffino et al., 2010). Quinoa also accumulates salt ions in tissues and thereby adjusts leaf water potential, enabling the plant to maintain cell turgor under saline conditions (Jacobsen, 2003; Hariadi et al., 2011). Organic osmolytes such as soluble sugars and proline are also accumulated in quinoa (Aguilar et al., 2003; Rosa et al., 2009). The breadth of geographical distribution of quinoa cultivars opens prospects of selection, adaptation and breeding of cultivars for the use under different environmental conditions (Jacobsen,
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Please cite this article in press as: Shabala S, et al. Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na+ loading and stomatal density. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.01.014
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1997). Trials of quinoa cultivation have been performed in several countries all over the world, and large differences were observed with respect to growth period and yield, depending in particular on the latitude (Jacobsen, 2003; Bertero et al., 2004; Christiansen et al., 2010;). The objective of this study was to elucidate key mechanisms involved in salt tolerance by comparing cultivars of different geographical origin, differing in the extent of salinity tolerance. This knowledge may be instrumental in further understanding of salt tolerance mechanisms and can facilitate the selection of varieties with improved tolerance to salinity.
7 2
= 0.51 r R= 0. 72*
6 5
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2
4 3 2 1
Materials and methods
0 40
Plant material and growth conditions
60
80
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Biomass, % control Fourteen quinoa (Chenopodium quinoa Willd.) genotypes were grown under greenhouse conditions at the University of Tasmania between December 2007 and April 2008 as described in Hariadi et al. (2011) by irrigating plants with saline water (so-called Method 2 in that work). A short description of the varieties is given in Table 1. Once seedlings had germinated under control conditions, and reached 10 d of age, salinity treatment began. Salt treatment was given in 40 mM/day increments until the final concentration (400 mM) was reached. Samples and measurements were taken 8 weeks after start of the treatment. For detailed description of the genotypes used in this study, see Adolf et al. (2012).
Fig. 1. Correlation between quinoa salinity tolerance (estimated as relative shoot fresh weight under saline conditions) and plants’ ranking according to the natural habitat. “% control” on X-axis refers to plants grown under non saline conditions.
5% significance level. All analyses were done in R version 2.15.0 (R Development Core Team, 2012). Results Salinity stress tolerance in quinoa correlates with their natural habitat
Sampling and measurements Young and fully developed leaves were used for all measurements. The maximal photochemical efficiency of PSII was estimated by measuring the chlorophyll fluorescence Fv/Fm ratio using a pulse-amplitude modulation portable fluorometer (Mini-PAM, Heinz Walz GmbH, Effeltrich, Germany) as described in Smethurst et al. (2008). Leaf sap osmolality was measured using a vapour pressure osmometer (Vapro; Wescor Inc, Logan, UT, USA). Xylem sap samples were collected using a Scholander-type pressure chamber (Plant Moisture Systems, Santa Barbara, CA, USA) followed by analysis on flame photometer as described in Shabala et al. (2010). Leaf sap Na+ and K+ content were quantified using frozen-thawed samples as described by Cuin et al. (2009). A minimum of six individual leaves from six different plants was used for each measurement. Leaf imprints were taken from abaxial leaf surface using nail polish. These imprints were later examined under a high magnification (200×) microscope, and stomatal density (number of cells per surface area) was counted. The sample size for each treatment and genotype was 15 (three fields of view per sample × five biological replications). Agronomical characteristics (shoot fresh (FW) and dry (DW) weight) were also measured at the time of harvest. Data analysis All varieties used in this study were ranked according to their salinity tolerance based on their natural habitat (e.g. from areas with different salinity) using a six-point scale (those originating from non-saline regions ranked as 1; those from highly saline areas ranked as 6). These ranks were then used as a tolerance index and correlated with all measured data. The statistical significance of the measured mean differences was determined by using Student’s t-test. Trend lines were added using Excel 2003 package. The statistical significance of correlations between data set was calculated using Pearson’s r-values test. In case of an interaction, the main effects were combined into a new classification and the Tukey’s range test was applied as post-ANOVA test with an evaluation on a
Based on the geographic origin (natural habitat), all genotypes were ranked from 1 (lowest) to 6 (highest) in terms of their expected salinity tolerance (Table 1). The three varieties originating from the salt desert of the Southern altiplano in Bolivia, were ranked as 5 and 6 (genotypes #15, #16, and #10). Four Danish varieties (#3706, #5206, #19 and #20) were adapted to a lowsalinity habitat and, as such, ranked as 1. Other varieties were ranked in between. Strong positive correlation (r = 0.72; significant at P < 0.01) was found between the cultivars’ ranking and maintenance of biomass (shoot fresh weight) production under salinity (Fig. 1) suggesting that genetic variability in salinity stress tolerance in quinoa is determined by their natural habitat. Leaf Na+ but not K+ content is affected by habitat All varieties have clustered into two distinct groups based on their ability to accumulate Na+ in the shoot (Fig. 2). The three most salt tolerant genotypes (#10, #16 and #15) had lowest amounts of Na in leaves under saline conditions (Fig. 2) and were, therefore, defined as “excluders” (Cluster 1 in Fig. 2B). All other (eleven) varieties have accumulated a substantial amounts of Na+ in the shoot (Cluster 2) and were accordingly defined as “includers”. Within this cluster, a positive correlation (r = 0.53; significant at P < 0.05) was observed between amount of accumulated Na+ and plant salinity tolerance (estimated as relative biomass of salt-grown plants expressed as % of control; trend line in Fig. 2B). When plants from both clusters were analysed together, a weak negative correlation (r = −0.41) was reported between leaf sap Na+ content and salinity tolerance in quinoa. Interestingly, however, a highly significant positive correlation (r = 0.72; significant at P < 0.01) between leaf sap Na+ and salinity tolerance was found for plants grown under control conditions (Fig. 2C). Thus, tolerant varieties appear to accumulate more sodium when not exposed to extreme salinities. Plants grown under saline (400 mM NaCl) conditions had nearly twice as much K+ in the leaf sap, compared with control plants (Fig. 3). There was no significant correlation (at P < 0.05) between
Please cite this article in press as: Shabala S, et al. Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na+ loading and stomatal density. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.01.014
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Table 1 Origin, photoperiod sensitivity, and ranking for salinity tolerance of quinoa varieties used in this experiment (modified from Adolf et al., 2012). Genotype
Name
Origin
Photoperiod sensitivity
Rankinga
3706 5206 19 20 36 3 5 28 4 23 25 15 16 10
3706 5206 KVL 37 KVL 52 Amarilla de Marangani Kamiri ˜ Sayana Misa quinua Huariponcho Pasankalla rosada Chullpi roja Pandela rosada Huallata Utusaya
Denmark Denmark Denmark Denmark Peru (Cusco) Bolivia Bolivia Peru (Puno) Peru (Puno) Peru (Puno) Peru (Puno) Bolivia (Southern altiplano) Bolivia (Southern altiplano) Bolivia (Southern altiplano)
Daylength neutral Daylength neutral Daylength neutral Daylength neutral Short day Short day Short day Short day Short day Short day Short day Short day Short day Short day
1 1 1 1 2 3 3 3 4 4 4 5 5 6
a
Salinity ranking scores are: 1 – lowest, 6 – highest.
leaf K+ and salinity tolerance, neither under control or saline conditions.
xylem Na+ content. A strong negative correlation (r = −0.73; significant at P < 0.01) was observed between xylem Na+ content and salinity tolerance, so xylem Na+ was lowest in tolerant cultivars. Due to methodological issues, it was not possible to collect samples from all genotypes (some varieties were too fragile to withstand the pressure required to get xylem sap collected by the Scholander method). Salinity treatment also increased significantly the amount of K+ in the xylem sap (Fig. 6). It may be interpreted as an important feature to adapt to osmotic stress imposed by salinity and enable efficient water transport into the shoot. The above increase in xylem K+ was consistent with increased “free” K+ in leaf sap under saline conditions (Fig. 3). Xylem K+ did not correlate with plant salinity tolerance, but showed strong positive correlation (r = 0.70; significant at P < 0.01) with salinity tolerance under control conditions,
Leaf sap osmolality correlates negatively with salinity tolerance Leaf sap osmolality increased 3-fold between control and NaClgrown plants (Fig. 4). A strong negative correlation (r = −0.67; significant at P < 0.01) was seen between the overall osmolality in plants grown at 400 mM NaCl and plant salinity tolerance. At the same time, leaf sap osmolality strongly correlated with amount of Na+ in quinoa xylem (r = 0.62, significant at P < 0.05; Table 2). Negligible small amounts of Na (<0.1 mM) were detected in control samples (Fig. 5). 400 mM NaCl treatment significantly increased
1000
Control 800
a
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a
ab abc abc
600
abc abc
400
abc
A
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abc
Leaf Na+, mM
bc
bc
200
c 0 #3706#5206 #19
#20
#36
#3
#5
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#4
#23
#25
#16
#15
#10
Genotype 1000
Cluster 2
C
7
B
800
6 5
600
Rr2 == 0. 0.16 53*
400
4
r = 0.72*
3 2
200
1
Cluster 1 0
0 60
70
80
90
100
60
70
80
90
100
Biomass, % control Fig. 2. Leaf sap Na+ concentration in quinoa plants grown under control and saline conditions. Open symbols – controls; closed symbols – saline grown plants. Mean ± SE (n = 6). Panels B and C show correlations (Pearson’s r-values) between leaf sap Na+ and salinity tolerance (estimated as relative biomass of salt-grown plants expressed as % of control) for saline and control conditions, respectively. Values labelled with asterisk are significant at P < 0.05.
Please cite this article in press as: Shabala S, et al. Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na+ loading and stomatal density. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.01.014
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#3
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C
r = 0.09
r = 0.38
300
150
40
60
80
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40
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80
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Biomass, % control Fig. 3. Leaf sap K+ concentration in quinoa plants grown under control and saline conditions. Open symbols – controls; closed symbols – saline grown plants. Mean ± SE (n = 6). Panels B and C show correlations (Pearson’s r-values) between leaf sap Na+ and salinity tolerance (estimated as relative biomass of salt-grown plants expressed as % of control) for saline and control conditions, respectively. Data labelled with different low case letters are significantly different at P < 0.05).
with higher xylem K+ content observed in the most tolerant cultivars. Again, due to above methodological issues, it was not possible to collect samples from all genotypes.
Effect of habitat on chlorophyll fluorescence and stomatal characteristics All 14 cultivars had Fv/Fm values above 0.8 (Fig. 7), suggesting that PSII is not a main target of salinity stress, and PSII is well protected in all varieties. Salinity treatment, however, had a major effect on stomata density (Fig. 8), with 20–30% decrease observed in most varieties. A strong positive correlation between stomata density and plant salinity tolerance was found, both in control and under saline conditions (r = 0.56 and 0.59, accordingly; both significant at P < 0.05). Stomatal density under saline conditions also showed strong positive correlation with leaf K+ content and negative correlation with both xylem and leaf Na+ , as well as with leaf sap osmolality (all significant at P < 0.05; Table 2).
Discussion Quinoa varieties display different strategies of dealing with salt stress Salinity is a complex physiological trait, composed of many subcomponents. Plants can deal with potential cytosolic Na+ toxicity by one of the following means: (1) restricting Na+ from uptake by roots; (2) reducing Na+ loading into xylem; (3) retranslocating Na+ from the shoot; and (4) its sequestering in vacuoles (Munns and Tester, 2008). While these strategies differ dramatically between species (Flowers and Colmer, 2008; Munns and Tester, 2008), reports on intra-species variability are not as numerous. Nonetheless, in our early work on lucerne we have shown that two salt tolerant varieties, Ameristand and L33, employed two different strategies of dealing with salinity (Smethurst et al., 2008). Variety Ameristand was a classical “excluder”, while variety L33 (of a similar tolerance) has accumulated substantial amounts of Na+ in the shoot without negative consequence to cell metabolism as a result of efficient Na+ sequestration in vacuoles. Here we report
Table 2 Correlation matrix (Pearson’s r-values) calculated for a range of physiological characteristics measured in 14 quinoa varieties grown under saline (400 mM NaCl) conditions. Osm – leaf sap osmolality; SD – stomatal density. Values labelled with asterisk are significant at P < 0.05.
Leaf Na Leaf K Osm Xylem Na Xylem K SD
Leaf Na
Leaf K
Osm
Xylem Na
Xylem K
SD
n/a – – – – –
−0.43 n/a – – – –
0.35 0.2 n/a – – –
0.41 0.26 0.62* n/a – –
0.31 0.2 −0.18 0.3 n/a –
−0.55* 0.47* −0.46* −0.61* 0.21 n/a
Please cite this article in press as: Shabala S, et al. Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na+ loading and stomatal density. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.01.014
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NaCl
5
Control
1400
A
1200
Leaf osmolality, mOsm kg-1
1000 800 600 400 200 0 #3706#5206 #19
#20
#36
#3
#5
#28
#23
#4
#25
#16
#15
#10
Genotype 550
1500
B 1400
500
1300
450
1200
400
1100
C
350
r = -0.67*
r = -0.37
300
1000 40
60
80
100
40
60
80
100
Biomass, % control Fig. 4. Leaf sap osmolality in quinoa plants grown under control and saline conditions. Open symbols – controls; closed symbols – saline grown plants. Mean ± SE (n = 6). Panels B and C show correlations (Pearson’s r-values) between leaf sap Na+ and salinity tolerance (estimated as relative biomass of salt-grown plants expressed as % of control) for saline and control conditions, respectively.
a similar phenomenon for halophytic species of quinoa. As evident from Fig. 2B, all genotypes split into two distinct clusters. Three most tolerant varieties belonging to cluster 1 clearly follows an exclusion strategy, accumulating very small amounts of Na+ in the shoot. The major bulk of plants (eleven varieties in cluster 2), however, accumulate substantial quantities of Na+ in the shoot. A positive correlation (r = 0.53) observed between amount of accumulated Na+ and plant salinity tolerance suggests that the dominant mechanism contributing to salinity tolerance in this cluster may be vacuolar Na+ sequestration.
Plants in Cluster 1 had lowest amounts of sodium in leaves under saline conditions (Fig. 2). These genotypes originated from Bolivian Altiplano and were, therefore, similar to ones used by Maughan et al. (2009). The fact that these varieties do not increase SOS1 Na+ excluding activity in roots upon salinity treatment (Maughan et al., 2009) but nonetheless accumulate very small amounts of Na+ in the shoot suggests that either control of xylem loading or Na+ retrieval from the shoot may be involved. Unfortunately experiments by Maughan et al. (2009) were conducted on bulk roots and did not allow differentiate SOS1 expression between root epidermis and stellar tissue.
Molecular identity of Na+ transporters in quinoa Active Na+ exclusion from uptake by roots is known to be mediated by a Na+ /H+ exchanger located at the plasma membrane of epidermal root cells (Blumwald et al., 2000), encoded by SOS1 (salt overly sensitive) gene in Arabidopsis (Shi et al., 2000). Functional SOS1 analogues have been reported in different species (MartínezAtíenza et al., 2007; Mullan et al., 2007; Cuin et al., 2011) including quinoa (Maughan et al., 2009; Ruiz-Carrasco et al., 2011). The latter authors have also looked at expression patterns of tonoplast NHX Na+ /H+ exchangers mediating Na+ sequestration in vacuoles. Significant genotypic difference in expression levels for both genes was found (Ruiz-Carrasco et al., 2011). Interestingly, while expression of cqSOS1 was 3–4 times stronger in root compared to leaf tissue under control conditions, saline treatment (450 mM NaCl) caused SOS1 up-regulation in leaf, but not in the root tissue (Maughan et al., 2009), suggesting that Na+ exclusion from uptake by quinoa root plays only a minor role in overall salinity tolerance in this species. This is consistent with the major bulk of our data (plants in Cluster 2; Fig. 2B).
Increased leaf ion content is essential for shoot osmotic adjustment Plants grown under saline (400 mM NaCl) conditions had twice as much K+ in the leaf sap, compared with control plants (Fig. 3). This observation is counterintuitive, in the light of widely reported phenomena of potassium deficiency imposed by salinity (Marschner, 1995), however the results are consistent with other publications on quinoa (Orsini et al., 2011) and some salttolerant glycophytes, e.g. barley (Shabala et al., 2010). In the latter work, this phenomenon was interpreted as a higher demand for “free” potassium (as opposed to “structural” potassium) for osmotic adjustment purposes. Plants need to maintain positive shoot turgor to enable growth of new tissues; the process that requires osmotic adjustment. Being able to efficiently sequester Na+ in vacuoles, halophytes rely heavily on Na+ for these purposes (Glenn et al., 1999; Flowers and Colmer, 2008). This is further corroborated by observation of a strong (r = 0.72; significant at P < 0.01) positive correlation between
Please cite this article in press as: Shabala S, et al. Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na+ loading and stomatal density. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.01.014
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0.18
B
12
C
0.15
10
r = -0.73*
0.12
r = 0.10
8 0.09
6
0.06
4
0.03
2 0
0 60
70
80
90
100
60
70
80
90
100
Biomass, % control Fig. 5. Xylem Na+ concentrations in quinoa plants grown under control and saline conditions. Open symbols – controls; closed symbols – saline grown plants. Xylem sap was collected by using the Scholander-type pressure bomb and analysed on flame photometer. Mean ± SE (n = 6). Panels B and C show correlations (Pearson’s r-values) between leaf sap Na+ and salinity tolerance (estimated as relative biomass of salt-grown plants expressed as % of control) for saline and control conditions, respectively.
salinity tolerance and shoot Na+ content in plants grown under control conditions (Fig. 2C), and a similar, but weaker, correlation in plants in Cluster 2 under saline condition (r = 0.53; Fig. 2B). However, high concentrations of Na+ in vacuoles need to be balanced by equally high amounts of osmolytes in the cytosol, to prevent water movement between two compartments. This can be done by accumulating either organic or inorganic osmolytes. As the former is energetically an expensive option that will cause yield penalty (Raven, 1985; Chen et al., 2007), accumulation of K+ is much more preferred. In addition, hyperosmotic stress exerted by saline conditions results in a decrease in the xylem pressure (i.e. a build-up of tension; see Fig. 10 in Shabala et al., 2010). At pressures below vacuum, vessels may become dysfunctional by cavitation; the cavitation frequency strongly increases with xylem tension (Wegner and Zimmermann, 1998). The pressure drop can be counterbalanced, hence, cavitation can be prevented, by the accumulation of K+ or Na+ in the xylem sap. Control of xylem loading as component of salinity tolerance mechanism Molecular mechanisms of Na+ loading into xylem remain elusive, mainly due to uncertainty of thermodynamic requirements for this process (Tester and Davenport, 2003; Shabala and Mackay, 2011). SOS1 transporters are known to be expressed in xylem parenchyma tissue (Shi et al., 2002, 2003), and it was suggested that the xylem Na+ loading in halophytic plants is an active process that requires an enhanced operation of SOS1 Na+ /H+ antiporters at the xylem parenchyma interface (Shabala and Mackay, 2011). It was argued that rapid Na+ loading into the xylem during the initial stages of salinity stress may be the preferred mechanism,
beneficial for shoot osmotic adjustment (Shabala et al., 2010), assuming this process is tightly controlled. Recent studies on barley (relatively salt tolerant crop) showed that transgenic plants overexpressing HvHKT2;1 transporters had higher Na+ concentrations in the xylem, enhanced translocation of Na+ to the leaves, and superior salt tolerance compared with the wild type (Mian et al., 2011). In this work, a strong negative correlation (r = −0.73; significant at P < 0.005) between xylem Na+ content and plant salinity tolerance was found (Fig. 5). This explains clustering between two groups of genotypes reported in Fig. 2 and suggests that control of xylem Na+ is more important for salinity tolerance in quinoa compared with root Na+ exclusion from uptake. It should be commented, however, that reported data represents one particular “snapshot” taken 8 weeks after stress onset. By that time, most varieties have finished their growth and were in reproductive stage of development. It has been reported before that plant sensitivity to salinity is increased after transition from vegetative to reproductive stage (Rao et al., 2008; Samineni et al., 2011). Thus, prevention of Na+ delivery to the shoot at that stage may be crucial for plant metabolism. This explains a strong positive correlation between quinoa ability to control xylem Na+ content and salinity stress tolerance in plants (Fig. 5B). The situation may be rather different, however, for early stages of plant growth. In barley, tolerant varieties load more Na+ into the xylem during the first weeks, presumably for quickly reaching an osmotic adjustment, but then they restrict Na+ translocation to the shoot (Shabala et al., 2010). Sensitive barley varieties have lower rate of Na+ loading but do it “non-stop”. Hence, after some time (e.g. weeks) they accumulate more Na+ in the shoot. This may be also the case for quinoa. Hence, importance of timing of measurements is emphasised.
Please cite this article in press as: Shabala S, et al. Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na+ loading and stomatal density. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.01.014
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Genotype 70
4
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3.5
50
r = -0.19
40 30
r = 0.70*
3 2.5
20 2
10 0
1.5 40
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100
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Biomass, % control Fig. 6. Xylem K+ concentrations in quinoa plants grown under control and saline conditions. Open symbols – controls; closed symbols – saline grown plants. Xylem sap was collected by using the Scholander-type pressure bomb and analysed on flame photometer. Mean ± SE (n = 6). Panels B and C show correlations (Pearson’s r-values) between leaf sap K+ and salinity tolerance (estimated as relative biomass of salt-grown plants expressed as % of control) for saline and control conditions, respectively.
Changes in stomatal density as a mechanism contributing to improved water use efficiency in quinoa Another interesting observation in this study was the phenomenon of salinity-induced reduction in stomatal density observed in quinoa leaves in all 14 varieties (Fig. 8). It is normally expected that salinity reduces cell growth rate (Munns and Tester, 2008), and this results in the larger number of cells per surface area (i.e. increased cell density). This is obviously not the case for quinoa (Fig. 8). These findings confirm our previous observations on a few selected quinoa genotypes (Adolf et al., 2012; Shabala et al., 2012) and are consistent with reports from other cultivars of quinoa (Orsini et al., 2011; Adolf et al., 2013). Also consistent with these findings, Omami et al. (2006) have reported that in
0 mM
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0.85
Fv/Fm
0.8 0.75 0.7 0.65 0.6 #3706 #5206 #19
#20
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#16
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Genotype Fig. 7. Chlorophyll fluorescence characteristics (Fv/Fm ratio) of quinoa plants grown under control (open symbols) and saline (closed symbols) conditions. Mean ± SE (n = 10).
a related species amaranth (Amaranthus sp.), an accession grown under salinity and not surviving 200 mM NaCl, had thinner leaves, more stomata per unit leaf area, and larger stomatal apertures compared with other, more tolerant amaranth species. A freshwater species of Spartina had high stomatal densities, while salt marsh species had significantly lower stomatal densities (Maricle et al., 2009). These observations emphasise the importance of reducing stomatal density as an important adaptive tool under saline conditions. It should be added that, while all varieties have reduced their stomata density as a part of their adaptation to saline environment, tolerant varieties still had higher stomata density compared with sensitive ones (Fig. 8). This may be explained by the fact that positive correlation between stomata density and salinity tolerance is also observed in genotypes under control (non-saline) conditions (Fig. 8C). When salinity stress is imposed, all varieties reduce their stomata density by 20–30%, to deal with the reduced water availability. However, as tolerant varieties had much higher stomata density at beginning (prior to stress onset), this difference is preserved under saline conditions as well. It may be suggested that these varieties are better in their ability to control guard cell functioning and thus can afford having more stomata, both under control and saline conditions. We believe that changes in stomata density may represent a fundamental mechanism by which plant may optimise water productivity under saline conditions. A substantial amount of water evaporated from the leaf surface may bypass stomata and occur through the cuticle. Depending on conditions, this non-stomatal component can be as high as 28% of the water transpired through stomata (Boyer et al., 1997). Cuticular transpiration is usually concentrated in the area surrounding stomata, where there are
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Biomass, % control Fig. 8. Stomata density (number of cells per filed of view) in quinoa plants grown under control and saline conditions. Open symbols – controls; closed symbols – saline grown plants. Mean ± SE (n = 15). Panels B and C show correlations (Pearson’s r-values) between stomatal density and salinity tolerance (estimated as relative biomass of salt-grown plants expressed as % of control) for saline and control conditions, respectively.
more and larger cuticular pores (Marschner, 1995). While stomatal conductance is a dynamic process that can be rapidly regulated by ion fluxes into and out of guard cells, cuticular transpiration relies almost exclusively on existing (passive) hydraulic permeability of the leaf surface and, hence, cannot rapidly adjust to changing conditions. Thus, even at much more modest rates of cuticular transpiration, reducing the density of stomata, and the more numerous cuticular pores associated with them, would be of benefit to osmotically stressed plants (Shabala et al., 2012). However, in quinoa when tolerant varieties had higher stomata density, it may be a compensatory mechanism, e.g. more capacity to fix CO2 , with less yield penalties.
Conclusion Responses to salinity differed greatly among the varieties. Least affected were varieties from the Bolivian altiplano whose natural habitat is highly saline. All varieties reduced stomatal density and increased leaf sap K+ concentrations when exposed to high salinity. Tolerant varieties were more efficient in controlling the amounts of Na+ loading into xylem.
Acknowledgements This project was supported by the ARC Discovery and ARC Linkage grants to Prof. Sergey Shabala. We are grateful for the financial support from the EU 7th Framework Programme through the project “Sustainable water use securing food production in dry areas of the Mediterranean region”. Dr. Yuda Hariadi was a recipient of the Australian Endeavour Fellowship.
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Contents lists available at SciVerse ScienceDirect
Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na+ loading and stomatal density Sergey Shabala a,∗ , Yuda Hariadi a,c , Sven-Erik Jacobsen b a b c
School of Agricultural Science, University of Tasmania, Private Bag 54, Hobart, Tas 7001, Australia Faculty of Science, University of Copenhagen, Højbakkegård Allé 13, 2630 Tåstrup, Denmark Department of Physics, Faculty of Mathematics and Natural Sciences, University of Jember, Jember 68121, East Java, Indonesia
a r t i c l e
i n f o
Article history: Received 25 September 2012 Received in revised form 30 January 2013 Accepted 30 January 2013 Available online xxx Key words: Osmotic adjustment Potassium Sodium Transpiration Xylem sap
a b s t r a c t Quinoa is regarded as a highly salt tolerant halophyte crop, of great potential for cultivation on saline areas around the world. Fourteen quinoa genotypes of different geographical origin, differing in salinity tolerance, were grown under greenhouse conditions. Salinity treatment started on 10 day old seedlings. Six weeks after the treatment commenced, leaf sap Na and K content and osmolality, stomatal density, chlorophyll fluorescence characteristics, and xylem sap Na and K composition were measured. Responses to salinity differed greatly among the varieties. All cultivars had substantially increased K+ concentrations in the leaf sap, but the most tolerant cultivars had lower xylem Na+ content at the time of sampling. Most tolerant cultivars had lowest leaf sap osmolality. All varieties reduced stomata density when grown under saline conditions. All varieties clustered into two groups (includers and excluders) depending on their strategy of handling Na+ under saline conditions. Under control (non-saline) conditions, a strong positive correlation was observed between salinity tolerance and plants ability to accumulate Na+ in the shoot. Increased leaf sap K+ , controlled Na+ loading to the xylem, and reduced stomata density are important physiological traits contributing to genotypic differences in salinity tolerance in quinoa, a halophyte species from Chenopodium family. © 2013 Elsevier GmbH. All rights reserved.
Introduction Salinity tolerance is a complex multigenic trait, both genetically and physiologically (Flowers, 2004), with less than 25% of salt-regulated genes being salt stress-specific (Ma et al., 2006). While recent success in creating salt tolerant wheat in Australia by introgressing a Na+ transporter TmHKT1;5-A (Munns et al., 2012) suggests that the problem is tangible, pyramiding key physiological traits conferring the most essential characteristics (Flowers and Yeo, 1995; Shabala and Mackay, 2011) remains the preferred option in plant breeding for salinity tolerance. The identity and relative importance of these traits may come from studies on halophytes. Another strategy to cope with increased salinity problems in agriculture is to directly utilise halophytes which are naturally salt tolerant species. Significant genetic variability in salinity stress tolerance exists between halophytes. While dicotyledonous halophytes show optimal growth at concentrations of around 150 mM NaCl, monocotyledonous halophytes generally grow optimally in the absence of salt or, if growth is stimulated, this is achieved at much lower (50 mm or less) concentration of NaCl (Glenn et al.,
∗ Corresponding author. Tel.: +61 3 62267539; fax: +61 3 62262642. E-mail address: [email protected] (S. Shabala).
1999; Flowers and Colmer, 2008). Furthermore, such genetic variability is often observed even within one halophyte species. The latter may be illustrated using quinoa as an example. Being classified as a highly salt tolerant species (Hariadi et al., 2011; Jacobsen, 2011; Razzaghi et al., 2011; Adolf et al., 2013) and capable of growing under salt concentrations of 40 dS m−1 similar to those found in seawater (Jacobsen, 2003), quinoa genotypes display significant variability in agronomical and physiological responses when grown under saline conditions (Adolf et al., 2012). The physiological basis for this genetic variability in salinity tolerance in quinoa, as well as in other halophytic species, is not fully understood. Quinoa seems to use several mechanisms in order to acclimate to a saline environment (Wilson et al., 2002). In the cotyledonous stage, high adaptability to soil salinity may be due to improved metabolic control based on ion absorption, osmolyte accumulation and osmotic adjustment (Ruffino et al., 2010). Quinoa also accumulates salt ions in tissues and thereby adjusts leaf water potential, enabling the plant to maintain cell turgor under saline conditions (Jacobsen, 2003; Hariadi et al., 2011). Organic osmolytes such as soluble sugars and proline are also accumulated in quinoa (Aguilar et al., 2003; Rosa et al., 2009). The breadth of geographical distribution of quinoa cultivars opens prospects of selection, adaptation and breeding of cultivars for the use under different environmental conditions (Jacobsen,
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1997). Trials of quinoa cultivation have been performed in several countries all over the world, and large differences were observed with respect to growth period and yield, depending in particular on the latitude (Jacobsen, 2003; Bertero et al., 2004; Christiansen et al., 2010;). The objective of this study was to elucidate key mechanisms involved in salt tolerance by comparing cultivars of different geographical origin, differing in the extent of salinity tolerance. This knowledge may be instrumental in further understanding of salt tolerance mechanisms and can facilitate the selection of varieties with improved tolerance to salinity.
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Biomass, % control Fourteen quinoa (Chenopodium quinoa Willd.) genotypes were grown under greenhouse conditions at the University of Tasmania between December 2007 and April 2008 as described in Hariadi et al. (2011) by irrigating plants with saline water (so-called Method 2 in that work). A short description of the varieties is given in Table 1. Once seedlings had germinated under control conditions, and reached 10 d of age, salinity treatment began. Salt treatment was given in 40 mM/day increments until the final concentration (400 mM) was reached. Samples and measurements were taken 8 weeks after start of the treatment. For detailed description of the genotypes used in this study, see Adolf et al. (2012).
Fig. 1. Correlation between quinoa salinity tolerance (estimated as relative shoot fresh weight under saline conditions) and plants’ ranking according to the natural habitat. “% control” on X-axis refers to plants grown under non saline conditions.
5% significance level. All analyses were done in R version 2.15.0 (R Development Core Team, 2012). Results Salinity stress tolerance in quinoa correlates with their natural habitat
Sampling and measurements Young and fully developed leaves were used for all measurements. The maximal photochemical efficiency of PSII was estimated by measuring the chlorophyll fluorescence Fv/Fm ratio using a pulse-amplitude modulation portable fluorometer (Mini-PAM, Heinz Walz GmbH, Effeltrich, Germany) as described in Smethurst et al. (2008). Leaf sap osmolality was measured using a vapour pressure osmometer (Vapro; Wescor Inc, Logan, UT, USA). Xylem sap samples were collected using a Scholander-type pressure chamber (Plant Moisture Systems, Santa Barbara, CA, USA) followed by analysis on flame photometer as described in Shabala et al. (2010). Leaf sap Na+ and K+ content were quantified using frozen-thawed samples as described by Cuin et al. (2009). A minimum of six individual leaves from six different plants was used for each measurement. Leaf imprints were taken from abaxial leaf surface using nail polish. These imprints were later examined under a high magnification (200×) microscope, and stomatal density (number of cells per surface area) was counted. The sample size for each treatment and genotype was 15 (three fields of view per sample × five biological replications). Agronomical characteristics (shoot fresh (FW) and dry (DW) weight) were also measured at the time of harvest. Data analysis All varieties used in this study were ranked according to their salinity tolerance based on their natural habitat (e.g. from areas with different salinity) using a six-point scale (those originating from non-saline regions ranked as 1; those from highly saline areas ranked as 6). These ranks were then used as a tolerance index and correlated with all measured data. The statistical significance of the measured mean differences was determined by using Student’s t-test. Trend lines were added using Excel 2003 package. The statistical significance of correlations between data set was calculated using Pearson’s r-values test. In case of an interaction, the main effects were combined into a new classification and the Tukey’s range test was applied as post-ANOVA test with an evaluation on a
Based on the geographic origin (natural habitat), all genotypes were ranked from 1 (lowest) to 6 (highest) in terms of their expected salinity tolerance (Table 1). The three varieties originating from the salt desert of the Southern altiplano in Bolivia, were ranked as 5 and 6 (genotypes #15, #16, and #10). Four Danish varieties (#3706, #5206, #19 and #20) were adapted to a lowsalinity habitat and, as such, ranked as 1. Other varieties were ranked in between. Strong positive correlation (r = 0.72; significant at P < 0.01) was found between the cultivars’ ranking and maintenance of biomass (shoot fresh weight) production under salinity (Fig. 1) suggesting that genetic variability in salinity stress tolerance in quinoa is determined by their natural habitat. Leaf Na+ but not K+ content is affected by habitat All varieties have clustered into two distinct groups based on their ability to accumulate Na+ in the shoot (Fig. 2). The three most salt tolerant genotypes (#10, #16 and #15) had lowest amounts of Na in leaves under saline conditions (Fig. 2) and were, therefore, defined as “excluders” (Cluster 1 in Fig. 2B). All other (eleven) varieties have accumulated a substantial amounts of Na+ in the shoot (Cluster 2) and were accordingly defined as “includers”. Within this cluster, a positive correlation (r = 0.53; significant at P < 0.05) was observed between amount of accumulated Na+ and plant salinity tolerance (estimated as relative biomass of salt-grown plants expressed as % of control; trend line in Fig. 2B). When plants from both clusters were analysed together, a weak negative correlation (r = −0.41) was reported between leaf sap Na+ content and salinity tolerance in quinoa. Interestingly, however, a highly significant positive correlation (r = 0.72; significant at P < 0.01) between leaf sap Na+ and salinity tolerance was found for plants grown under control conditions (Fig. 2C). Thus, tolerant varieties appear to accumulate more sodium when not exposed to extreme salinities. Plants grown under saline (400 mM NaCl) conditions had nearly twice as much K+ in the leaf sap, compared with control plants (Fig. 3). There was no significant correlation (at P < 0.05) between
Please cite this article in press as: Shabala S, et al. Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na+ loading and stomatal density. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.01.014
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Table 1 Origin, photoperiod sensitivity, and ranking for salinity tolerance of quinoa varieties used in this experiment (modified from Adolf et al., 2012). Genotype
Name
Origin
Photoperiod sensitivity
Rankinga
3706 5206 19 20 36 3 5 28 4 23 25 15 16 10
3706 5206 KVL 37 KVL 52 Amarilla de Marangani Kamiri ˜ Sayana Misa quinua Huariponcho Pasankalla rosada Chullpi roja Pandela rosada Huallata Utusaya
Denmark Denmark Denmark Denmark Peru (Cusco) Bolivia Bolivia Peru (Puno) Peru (Puno) Peru (Puno) Peru (Puno) Bolivia (Southern altiplano) Bolivia (Southern altiplano) Bolivia (Southern altiplano)
Daylength neutral Daylength neutral Daylength neutral Daylength neutral Short day Short day Short day Short day Short day Short day Short day Short day Short day Short day
1 1 1 1 2 3 3 3 4 4 4 5 5 6
a
Salinity ranking scores are: 1 – lowest, 6 – highest.
leaf K+ and salinity tolerance, neither under control or saline conditions.
xylem Na+ content. A strong negative correlation (r = −0.73; significant at P < 0.01) was observed between xylem Na+ content and salinity tolerance, so xylem Na+ was lowest in tolerant cultivars. Due to methodological issues, it was not possible to collect samples from all genotypes (some varieties were too fragile to withstand the pressure required to get xylem sap collected by the Scholander method). Salinity treatment also increased significantly the amount of K+ in the xylem sap (Fig. 6). It may be interpreted as an important feature to adapt to osmotic stress imposed by salinity and enable efficient water transport into the shoot. The above increase in xylem K+ was consistent with increased “free” K+ in leaf sap under saline conditions (Fig. 3). Xylem K+ did not correlate with plant salinity tolerance, but showed strong positive correlation (r = 0.70; significant at P < 0.01) with salinity tolerance under control conditions,
Leaf sap osmolality correlates negatively with salinity tolerance Leaf sap osmolality increased 3-fold between control and NaClgrown plants (Fig. 4). A strong negative correlation (r = −0.67; significant at P < 0.01) was seen between the overall osmolality in plants grown at 400 mM NaCl and plant salinity tolerance. At the same time, leaf sap osmolality strongly correlated with amount of Na+ in quinoa xylem (r = 0.62, significant at P < 0.05; Table 2). Negligible small amounts of Na (<0.1 mM) were detected in control samples (Fig. 5). 400 mM NaCl treatment significantly increased
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Biomass, % control Fig. 2. Leaf sap Na+ concentration in quinoa plants grown under control and saline conditions. Open symbols – controls; closed symbols – saline grown plants. Mean ± SE (n = 6). Panels B and C show correlations (Pearson’s r-values) between leaf sap Na+ and salinity tolerance (estimated as relative biomass of salt-grown plants expressed as % of control) for saline and control conditions, respectively. Values labelled with asterisk are significant at P < 0.05.
Please cite this article in press as: Shabala S, et al. Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na+ loading and stomatal density. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.01.014
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r = 0.38
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Biomass, % control Fig. 3. Leaf sap K+ concentration in quinoa plants grown under control and saline conditions. Open symbols – controls; closed symbols – saline grown plants. Mean ± SE (n = 6). Panels B and C show correlations (Pearson’s r-values) between leaf sap Na+ and salinity tolerance (estimated as relative biomass of salt-grown plants expressed as % of control) for saline and control conditions, respectively. Data labelled with different low case letters are significantly different at P < 0.05).
with higher xylem K+ content observed in the most tolerant cultivars. Again, due to above methodological issues, it was not possible to collect samples from all genotypes.
Effect of habitat on chlorophyll fluorescence and stomatal characteristics All 14 cultivars had Fv/Fm values above 0.8 (Fig. 7), suggesting that PSII is not a main target of salinity stress, and PSII is well protected in all varieties. Salinity treatment, however, had a major effect on stomata density (Fig. 8), with 20–30% decrease observed in most varieties. A strong positive correlation between stomata density and plant salinity tolerance was found, both in control and under saline conditions (r = 0.56 and 0.59, accordingly; both significant at P < 0.05). Stomatal density under saline conditions also showed strong positive correlation with leaf K+ content and negative correlation with both xylem and leaf Na+ , as well as with leaf sap osmolality (all significant at P < 0.05; Table 2).
Discussion Quinoa varieties display different strategies of dealing with salt stress Salinity is a complex physiological trait, composed of many subcomponents. Plants can deal with potential cytosolic Na+ toxicity by one of the following means: (1) restricting Na+ from uptake by roots; (2) reducing Na+ loading into xylem; (3) retranslocating Na+ from the shoot; and (4) its sequestering in vacuoles (Munns and Tester, 2008). While these strategies differ dramatically between species (Flowers and Colmer, 2008; Munns and Tester, 2008), reports on intra-species variability are not as numerous. Nonetheless, in our early work on lucerne we have shown that two salt tolerant varieties, Ameristand and L33, employed two different strategies of dealing with salinity (Smethurst et al., 2008). Variety Ameristand was a classical “excluder”, while variety L33 (of a similar tolerance) has accumulated substantial amounts of Na+ in the shoot without negative consequence to cell metabolism as a result of efficient Na+ sequestration in vacuoles. Here we report
Table 2 Correlation matrix (Pearson’s r-values) calculated for a range of physiological characteristics measured in 14 quinoa varieties grown under saline (400 mM NaCl) conditions. Osm – leaf sap osmolality; SD – stomatal density. Values labelled with asterisk are significant at P < 0.05.
Leaf Na Leaf K Osm Xylem Na Xylem K SD
Leaf Na
Leaf K
Osm
Xylem Na
Xylem K
SD
n/a – – – – –
−0.43 n/a – – – –
0.35 0.2 n/a – – –
0.41 0.26 0.62* n/a – –
0.31 0.2 −0.18 0.3 n/a –
−0.55* 0.47* −0.46* −0.61* 0.21 n/a
Please cite this article in press as: Shabala S, et al. Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na+ loading and stomatal density. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.01.014
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1000 40
60
80
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40
60
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Biomass, % control Fig. 4. Leaf sap osmolality in quinoa plants grown under control and saline conditions. Open symbols – controls; closed symbols – saline grown plants. Mean ± SE (n = 6). Panels B and C show correlations (Pearson’s r-values) between leaf sap Na+ and salinity tolerance (estimated as relative biomass of salt-grown plants expressed as % of control) for saline and control conditions, respectively.
a similar phenomenon for halophytic species of quinoa. As evident from Fig. 2B, all genotypes split into two distinct clusters. Three most tolerant varieties belonging to cluster 1 clearly follows an exclusion strategy, accumulating very small amounts of Na+ in the shoot. The major bulk of plants (eleven varieties in cluster 2), however, accumulate substantial quantities of Na+ in the shoot. A positive correlation (r = 0.53) observed between amount of accumulated Na+ and plant salinity tolerance suggests that the dominant mechanism contributing to salinity tolerance in this cluster may be vacuolar Na+ sequestration.
Plants in Cluster 1 had lowest amounts of sodium in leaves under saline conditions (Fig. 2). These genotypes originated from Bolivian Altiplano and were, therefore, similar to ones used by Maughan et al. (2009). The fact that these varieties do not increase SOS1 Na+ excluding activity in roots upon salinity treatment (Maughan et al., 2009) but nonetheless accumulate very small amounts of Na+ in the shoot suggests that either control of xylem loading or Na+ retrieval from the shoot may be involved. Unfortunately experiments by Maughan et al. (2009) were conducted on bulk roots and did not allow differentiate SOS1 expression between root epidermis and stellar tissue.
Molecular identity of Na+ transporters in quinoa Active Na+ exclusion from uptake by roots is known to be mediated by a Na+ /H+ exchanger located at the plasma membrane of epidermal root cells (Blumwald et al., 2000), encoded by SOS1 (salt overly sensitive) gene in Arabidopsis (Shi et al., 2000). Functional SOS1 analogues have been reported in different species (MartínezAtíenza et al., 2007; Mullan et al., 2007; Cuin et al., 2011) including quinoa (Maughan et al., 2009; Ruiz-Carrasco et al., 2011). The latter authors have also looked at expression patterns of tonoplast NHX Na+ /H+ exchangers mediating Na+ sequestration in vacuoles. Significant genotypic difference in expression levels for both genes was found (Ruiz-Carrasco et al., 2011). Interestingly, while expression of cqSOS1 was 3–4 times stronger in root compared to leaf tissue under control conditions, saline treatment (450 mM NaCl) caused SOS1 up-regulation in leaf, but not in the root tissue (Maughan et al., 2009), suggesting that Na+ exclusion from uptake by quinoa root plays only a minor role in overall salinity tolerance in this species. This is consistent with the major bulk of our data (plants in Cluster 2; Fig. 2B).
Increased leaf ion content is essential for shoot osmotic adjustment Plants grown under saline (400 mM NaCl) conditions had twice as much K+ in the leaf sap, compared with control plants (Fig. 3). This observation is counterintuitive, in the light of widely reported phenomena of potassium deficiency imposed by salinity (Marschner, 1995), however the results are consistent with other publications on quinoa (Orsini et al., 2011) and some salttolerant glycophytes, e.g. barley (Shabala et al., 2010). In the latter work, this phenomenon was interpreted as a higher demand for “free” potassium (as opposed to “structural” potassium) for osmotic adjustment purposes. Plants need to maintain positive shoot turgor to enable growth of new tissues; the process that requires osmotic adjustment. Being able to efficiently sequester Na+ in vacuoles, halophytes rely heavily on Na+ for these purposes (Glenn et al., 1999; Flowers and Colmer, 2008). This is further corroborated by observation of a strong (r = 0.72; significant at P < 0.01) positive correlation between
Please cite this article in press as: Shabala S, et al. Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na+ loading and stomatal density. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.01.014
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Biomass, % control Fig. 5. Xylem Na+ concentrations in quinoa plants grown under control and saline conditions. Open symbols – controls; closed symbols – saline grown plants. Xylem sap was collected by using the Scholander-type pressure bomb and analysed on flame photometer. Mean ± SE (n = 6). Panels B and C show correlations (Pearson’s r-values) between leaf sap Na+ and salinity tolerance (estimated as relative biomass of salt-grown plants expressed as % of control) for saline and control conditions, respectively.
salinity tolerance and shoot Na+ content in plants grown under control conditions (Fig. 2C), and a similar, but weaker, correlation in plants in Cluster 2 under saline condition (r = 0.53; Fig. 2B). However, high concentrations of Na+ in vacuoles need to be balanced by equally high amounts of osmolytes in the cytosol, to prevent water movement between two compartments. This can be done by accumulating either organic or inorganic osmolytes. As the former is energetically an expensive option that will cause yield penalty (Raven, 1985; Chen et al., 2007), accumulation of K+ is much more preferred. In addition, hyperosmotic stress exerted by saline conditions results in a decrease in the xylem pressure (i.e. a build-up of tension; see Fig. 10 in Shabala et al., 2010). At pressures below vacuum, vessels may become dysfunctional by cavitation; the cavitation frequency strongly increases with xylem tension (Wegner and Zimmermann, 1998). The pressure drop can be counterbalanced, hence, cavitation can be prevented, by the accumulation of K+ or Na+ in the xylem sap. Control of xylem loading as component of salinity tolerance mechanism Molecular mechanisms of Na+ loading into xylem remain elusive, mainly due to uncertainty of thermodynamic requirements for this process (Tester and Davenport, 2003; Shabala and Mackay, 2011). SOS1 transporters are known to be expressed in xylem parenchyma tissue (Shi et al., 2002, 2003), and it was suggested that the xylem Na+ loading in halophytic plants is an active process that requires an enhanced operation of SOS1 Na+ /H+ antiporters at the xylem parenchyma interface (Shabala and Mackay, 2011). It was argued that rapid Na+ loading into the xylem during the initial stages of salinity stress may be the preferred mechanism,
beneficial for shoot osmotic adjustment (Shabala et al., 2010), assuming this process is tightly controlled. Recent studies on barley (relatively salt tolerant crop) showed that transgenic plants overexpressing HvHKT2;1 transporters had higher Na+ concentrations in the xylem, enhanced translocation of Na+ to the leaves, and superior salt tolerance compared with the wild type (Mian et al., 2011). In this work, a strong negative correlation (r = −0.73; significant at P < 0.005) between xylem Na+ content and plant salinity tolerance was found (Fig. 5). This explains clustering between two groups of genotypes reported in Fig. 2 and suggests that control of xylem Na+ is more important for salinity tolerance in quinoa compared with root Na+ exclusion from uptake. It should be commented, however, that reported data represents one particular “snapshot” taken 8 weeks after stress onset. By that time, most varieties have finished their growth and were in reproductive stage of development. It has been reported before that plant sensitivity to salinity is increased after transition from vegetative to reproductive stage (Rao et al., 2008; Samineni et al., 2011). Thus, prevention of Na+ delivery to the shoot at that stage may be crucial for plant metabolism. This explains a strong positive correlation between quinoa ability to control xylem Na+ content and salinity stress tolerance in plants (Fig. 5B). The situation may be rather different, however, for early stages of plant growth. In barley, tolerant varieties load more Na+ into the xylem during the first weeks, presumably for quickly reaching an osmotic adjustment, but then they restrict Na+ translocation to the shoot (Shabala et al., 2010). Sensitive barley varieties have lower rate of Na+ loading but do it “non-stop”. Hence, after some time (e.g. weeks) they accumulate more Na+ in the shoot. This may be also the case for quinoa. Hence, importance of timing of measurements is emphasised.
Please cite this article in press as: Shabala S, et al. Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na+ loading and stomatal density. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.01.014
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Biomass, % control Fig. 6. Xylem K+ concentrations in quinoa plants grown under control and saline conditions. Open symbols – controls; closed symbols – saline grown plants. Xylem sap was collected by using the Scholander-type pressure bomb and analysed on flame photometer. Mean ± SE (n = 6). Panels B and C show correlations (Pearson’s r-values) between leaf sap K+ and salinity tolerance (estimated as relative biomass of salt-grown plants expressed as % of control) for saline and control conditions, respectively.
Changes in stomatal density as a mechanism contributing to improved water use efficiency in quinoa Another interesting observation in this study was the phenomenon of salinity-induced reduction in stomatal density observed in quinoa leaves in all 14 varieties (Fig. 8). It is normally expected that salinity reduces cell growth rate (Munns and Tester, 2008), and this results in the larger number of cells per surface area (i.e. increased cell density). This is obviously not the case for quinoa (Fig. 8). These findings confirm our previous observations on a few selected quinoa genotypes (Adolf et al., 2012; Shabala et al., 2012) and are consistent with reports from other cultivars of quinoa (Orsini et al., 2011; Adolf et al., 2013). Also consistent with these findings, Omami et al. (2006) have reported that in
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Genotype Fig. 7. Chlorophyll fluorescence characteristics (Fv/Fm ratio) of quinoa plants grown under control (open symbols) and saline (closed symbols) conditions. Mean ± SE (n = 10).
a related species amaranth (Amaranthus sp.), an accession grown under salinity and not surviving 200 mM NaCl, had thinner leaves, more stomata per unit leaf area, and larger stomatal apertures compared with other, more tolerant amaranth species. A freshwater species of Spartina had high stomatal densities, while salt marsh species had significantly lower stomatal densities (Maricle et al., 2009). These observations emphasise the importance of reducing stomatal density as an important adaptive tool under saline conditions. It should be added that, while all varieties have reduced their stomata density as a part of their adaptation to saline environment, tolerant varieties still had higher stomata density compared with sensitive ones (Fig. 8). This may be explained by the fact that positive correlation between stomata density and salinity tolerance is also observed in genotypes under control (non-saline) conditions (Fig. 8C). When salinity stress is imposed, all varieties reduce their stomata density by 20–30%, to deal with the reduced water availability. However, as tolerant varieties had much higher stomata density at beginning (prior to stress onset), this difference is preserved under saline conditions as well. It may be suggested that these varieties are better in their ability to control guard cell functioning and thus can afford having more stomata, both under control and saline conditions. We believe that changes in stomata density may represent a fundamental mechanism by which plant may optimise water productivity under saline conditions. A substantial amount of water evaporated from the leaf surface may bypass stomata and occur through the cuticle. Depending on conditions, this non-stomatal component can be as high as 28% of the water transpired through stomata (Boyer et al., 1997). Cuticular transpiration is usually concentrated in the area surrounding stomata, where there are
Please cite this article in press as: Shabala S, et al. Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na+ loading and stomatal density. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.01.014
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Biomass, % control Fig. 8. Stomata density (number of cells per filed of view) in quinoa plants grown under control and saline conditions. Open symbols – controls; closed symbols – saline grown plants. Mean ± SE (n = 15). Panels B and C show correlations (Pearson’s r-values) between stomatal density and salinity tolerance (estimated as relative biomass of salt-grown plants expressed as % of control) for saline and control conditions, respectively.
more and larger cuticular pores (Marschner, 1995). While stomatal conductance is a dynamic process that can be rapidly regulated by ion fluxes into and out of guard cells, cuticular transpiration relies almost exclusively on existing (passive) hydraulic permeability of the leaf surface and, hence, cannot rapidly adjust to changing conditions. Thus, even at much more modest rates of cuticular transpiration, reducing the density of stomata, and the more numerous cuticular pores associated with them, would be of benefit to osmotically stressed plants (Shabala et al., 2012). However, in quinoa when tolerant varieties had higher stomata density, it may be a compensatory mechanism, e.g. more capacity to fix CO2 , with less yield penalties.
Conclusion Responses to salinity differed greatly among the varieties. Least affected were varieties from the Bolivian altiplano whose natural habitat is highly saline. All varieties reduced stomatal density and increased leaf sap K+ concentrations when exposed to high salinity. Tolerant varieties were more efficient in controlling the amounts of Na+ loading into xylem.
Acknowledgements This project was supported by the ARC Discovery and ARC Linkage grants to Prof. Sergey Shabala. We are grateful for the financial support from the EU 7th Framework Programme through the project “Sustainable water use securing food production in dry areas of the Mediterranean region”. Dr. Yuda Hariadi was a recipient of the Australian Endeavour Fellowship.
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Please cite this article in press as: Shabala S, et al. Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na+ loading and stomatal density. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.01.014