Physiological Responses of Wild and Cultivated Barley to the Interactive Effect of Salinity and Iron Deficiency

Literature on the separate effects of salinity and inadequate Fe supply on plant growth and nutrient uptake, concentration, and distribution is abundant but little is known about the interactive effects of these two abiotic constraints. Here, we investigated the interactive effect of iron availability and salinity on physiological responses of cultivated and wild barley (Hordeum vulgare and H. maritimum resp.). Seedlings of both species were grown for 9 days, under complete nutrient solution with or without iron supply. Then, NaCl treatment was applied at different concentrations (0, 100, 200, and 300 mM) for 60 hours. After salt exposure, shoot water content of H. vulgare was significantly reduced as compared to H. maritimum. Furthermore, Na+ accumulation in shoots increased parallel to increasing NaCl concentration in the medium. However, the increase was significantly higher in H. vulgare than in H. maritimum. These responses were associated with lower Fe absorption efficiency photosynthetic parameters in both species. The reduction was significantly higher in cultivated than in wild barley. Moreover, phytosiderophore exudation was enhanced in both species by direct (iron free medium) or indirect iron limitation (salt-induced iron limitation). Such a stimulation of phytosiderophore release was genotype and salt level dependant.


Introduction
It is well documented that hindrance effect of high soil alkalinity on plant growth is related to nutrients availability limitation; particularly of iron [1].Under such tricky environmental conditions, higher plants induce special mechanisms for iron acquisition, involving a plasma membrane H + -ATPase to increase root proton release capacity and a plasma membrane Fe(III) chelate reductase to reduce Fe(III) chelates at root surface (strategy I) as well as an enhancement of phytosiderophore secretion to the rhizosphere, parallel to an induction of an Fe(III)-phytosiderophore complex transport system (strategy II) [2].Salinity also presents several challenges to plant growth, including the decrease of the osmotic potential of the growing medium, a specific ion toxicity [3], and nutrient deficiencies and disorders [4].
Iron limitation and high-salinity stress have thus far been regarded as separate growth-limiting factors.Yet, the interaction effect of both stresses is little known.However, in sodic soils where pH and Na + concentration is high, the solubility of micronutrients is low [5,6].According to Gupta et al. [7], in alkaline soils, the pH usually increases with an increase in salinity due to the presence of sodium-bicarbonate carbonates.Thus, plants growing in such soils encounter both Na + osmotic and toxicity effects and micronutrient deficiency.Besides, Jumberi et al. [5] found that the relative uptake of iron by barley and rye decreased with increasing soil sodicity.This result allows the suggestion of several hypotheses.It is thus possible that in the sodic soil, which has a high pH and Na + concentration, (i) the availability of iron is reduced either by the alkaline pH or by the formation of insoluble iron-phosphate complexes, (ii) the synthesis pathway of the iron chelators and uptake systems for iron was partially inhibited at high salinity, (iii) the effective complexation of iron by chelators could be negatively affected by the high ionic strength of the growth medium.
As far as we know that salinity stress inhibits the inducible mechanism of iron acquisition in some glycophytes.In fact, recently, it was found that salt had a depressive effect on root acidification in Medicago ciliaris plants [8] and on phytosiderophore release in barley [9].Interestingly, recent studies demonstrated that expression of some proteins involved in iron uptake was significantly declined under salinity stress in barley salt-sensitive cv but highly induced in barley salt-tolerant cv [10].Furthermore, it was previously demonstrated a high-salinity induction of genes encoding the synthesis pathway of chelators and uptake systems for iron in Bacillus subtilis [11] as consequence of iron limitation.
In the present study, our objective was to investigate a comparative study between two graminaceous species differing by their salinity tolerance: H. vulgare (cultivated barley) a glycophyte species and H. maritimum (wild barley) a halophyte native of salted soils where it significantly contributes to the annual biomass production in such ecosystems [12], in order to know the impact of salinity on iron uptake and on the inducible mechanism of its acquisition and to examine if the eventual impact is dependent on the tolerance of plant to salinity.with a modified method of Takagi [15].An aliquot of 2 mL of 1 mM Fe(OH) 3 and 100 µL of concentrated root exudates was added to a final volume of 10 mL.The resulting mixture was shaken for 1 h at room temperature and then filtrated (Blue ribbon No 5893, Schleicher and Schüll, Dassel, Germany).Mobilized iron was determined by atomic absorption spectrometry and phytosiderophore release concentrations were calculated as Fe equivalents.

Tissue Iron and Sodium Analysis.
Directly after root exudates collection, roots were carefully washed with solution of EDTA (10 mM) for 10 min under contentious aeration in order to remove the apoplasmic iron then rinsed with distilled water.Thereafter, roots and shoots were separated and weighted for determination of biomass production and dried at 60 • C. Samples were ground with an agate grinder for nutrient extraction by digestion with nitric/H 2 O 2 (4 : 3, v/v).
Samples were analyzed for Fe on a Perkin Elmer Analyst 100 Atomic Absorption Spectrophotometer.Na + was determined in the same extract using flame spectrometry.

Fe Absorption Efficiency Calculation.
Fe absorption efficiency (FeAE) was determined as the ratio of total iron quantity (µmol) accumulated in each plant during the experimental period to its root dry weight (g).

Statistical Analysis.
All data were subjected to one-way ANOVA test, and means were compared using the SPSS and   1).However, salt application led to decrease this parameter in H. vulgare (salt sensitive species) whatever iron availability in the medium was, but, practically, did not have any effect on that of H. maritimum (salt tolerant species).

Shoot Water Status.
A significant difference in water status between wild and cultivated barley was found when salt was added to the medium (Figure 1).In fact, shoot water content of H. vulgare was significantly reduced by salinity.Thus, the differences between 0, 100, 200, and 300 mM were constantly significant and this manifestation was clear in iron deficient plants.However, in H. maritimum the differences between treatments, in most cases, remained marginal and they became statically significant only at the most severe salt treatment level (300 mM NaCl).Results showed that there was not a significant difference of shoot water contents between the both levels of iron treatment (0 and 100 µM Fe) in H. maritimum.

Sodium Status in Plant Tissues
. Whatever the iron availability in the medium was, salinity caused significant increases in both roots and shoots Na + contents in both species (Table 2).As a whole, shoots exhibited the higher Na + contents as compared to roots in both species.However, shoot cultivated barley had higher sodium concentrations in comparison with that of wild barley plants, whereas at the level of roots it was the opposite.Under iron deficiency conditions (−Fe, +salt treatment), tissue Na + concentrations in wild barley were, practically, maintained as compared to those of iron-fed plants (+Fe, +salt treatment).However, in cultivated barley, iron deficiency constraint led to a drastically increase of Na + concentrations especially in the level of shoots as compared to iron sufficient conditions.In fact, the increases were about 22 and 92% at the application of 200 and 300 mM NaCl, respectively (Table 2).Nevertheless, Na + distribution between organs was species dependent.Thus, shoots of cultivated barley plants exposed to 300 mM NaCl contained 97 and 93% of the total amount of sodium in    However, in wild one, the amount of Na + allocated to shoots did not exceed 75%, either in iron deficient or iron sufficient plants (Table 3).

Iron Status in Plant Tissues.
Our results indicated that salt stress generally provoked an iron limitation in both species (Table 4).It was incredibly clear that iron concentrations were constantly decreased with increasing salt treatment level.In fact, the decrease of iron concentrations in H. vulgare (+Fe, +salt treatment) was 35-48% and 18-88%, respectively, in shoots and roots as compared to control ones (+Fe treatment, −salt treatment).In H. maritimum, the reduction of root iron concentrations was about 56-81%, while those of shoots were not affected.Besides, iron absorption efficiency (FeAE) was reduced in both species with increasing salt treatment level.Indeed, 300 mM NaCl caused a reduction of 65 and 72% in iron absorption efficiency, respectively, in H. maritimum and H. vulgare fed with 100 µM Fe (Table 5).

Effect of Salt Stress on Phytosiderophore Exudation.
Under iron deficiency, we obtained an induction of root exudation in both species.The amount of phytosiderophore (PS)  secreted by iron deficient plants (−Fe treatment) was at least 10 times higher than those released by iron sufficient plants (+Fe treatment) (Figure 2).Thus, the PS exudation amount of iron deficient plants (−Fe treatment) reached, at least, 250 µmol/g root FW 4 h −1 , but that of iron sufficient ones did not exceeded 32 µmol/g root FW 4 h.H. vulgare had the highest PS exudation amount as compared to H. maritimum.Salt application had an effect on PS exudation, which clearly depended on salt-concentration-applied species and iron availability in the medium (Figure 2).In fact, the application of 100 mM NaCl to iron deficient plants (−Fe, +100 mM NaCl treatment) enhanced PS root exudation in both species without exception as compared to nonsaltstressed plants (−Fe treatment, +0 mM NaCl treatment).However, up to 100 mM NaCl, the responses of two species became slightly complicated.Thus, at 200 mM NaCl, wild barley iron-deficient plants (−Fe, +200 mM NaCl treatment) still released practically the same amount of PS which detected at −Fe treatment and showed a PS inhibition only at 300 mM NaCl, while that of cultivated ones (−Fe, +salt treatment) it started to be drastically decreased from 200 mM NaCl.In the presence of 100 µM Fe, stimulation of PS release under salt stress occurred in both species despite the fact that the growth medium contained enough iron (100 µM) to repress PS exudation.Increasing the salinity of the growth medium that contained 100 µM Fe (+Fe, +salt treatment) triggered an increase in PS levels, resulting in a linear relationship between the salt concentration and the amount of PS exuded (Figure 3).It was only at the highest salinity level (300 mM NaCl) that the two species exhibited different variations in PS exudations; a sharp decline was observed in H. vulgare (+Fe, 300 mM NaCl treatment) restoring the value of control plants (+Fe treatment) versus a strong improvement in H. maritimum (+Fe, 300 mM NaCl treatment).

Discussion
It is clear that as salinity increase, iron uptake was constantly decreased in both species.Besides, iron absorption efficiency (FeAE) was reduced in both species with increasing salt level treatment.Indeed, 300 mM NaCl caused a reduction of 65 and 72% in iron absorption efficiency, respectively, in H. maritimum and H. vulgare fed with 100 µM Fe (Table 5).
The magnitude of salt effect on different organ iron contents was genetically dependent.Thus, iron contents were constantly reduced in roots of both species with increasing salinity level, while that of shoots were drastically reduced    in H. vulgare andremained invariable (+Fe, +salt treatment) or slightly reduced (−Fe, +salt treatment) in H. maritimum as compared to control (+Fe or −Fe treatment, resp.).This is consistent with literature reports.In fact, several studies proved that salinity reduces Fe content in shoots of barley, [9], medicago [8], wheat, and faba bean [17].Nevertheless, high Fe contents were found in shoots of tomato, soybean, squash [18], and rye [5] plants grown on saline soil.Grattan and Grieve [19] attributed these contrasting results to plant and tissue type, salinity level and composition, iron concentration in the medium, growing conditions, and the duration of treatment.
Iron limitation resulting either from direct iron deficiency or salt stress (inducible iron deficiency) was concomitant with an induction of phytosiderophore (PS) release (Figure 2).Such induction was significantly related to the genotype and the salt stress level.In fact, as it was previously proved that under iron deficiency, H. vulgare released an amount of PS higher than H. maritimum [20].The correlation between shoot Na + concentrations and the amount of PS released from root system was investigated (Figure 3).As a result, in plants fed with iron (+Fe, +salt treatment), a positive correlation was found between shoot Na + concentrations and PS exudation in both species but was more significant in wild barley than in cultivated one (R 2 = 0, 831 and R 2 = 0, 565, resp.)(Figure 3(a)).However, in iron deficient plants (−Fe, +salt treatment), a strong negative correlation was found between shoot Na + concentrations and PS exudation in cultivated barley (R 2 = 0, 780), while no relation was observed in wild one (R 2 = 0, 113) (Figure 3(b)).These findings allow speculating that H. maritimum was more efficient than H. vulgare to safeguard its inducible mechanism of iron acquisition under salt stress.Besides, studies proved an intra-and interspecific variability in the protection or stimulation of the synthesis pathway of iron chelators and iron uptake systems [10][11][12][13][14][15][16][17][18][19][20][21].
For each species there is a threshold of Na + concentration beyond which PS exudation capacity was inhibited either in presence or absence of iron in the medium.In H. vulgare, such threshold was 0.56 and 1.33 mmol Na + g −1 shoot DW under iron starvation and iron supply, respectively, while in H. maritimum it was 1.0 mmol Na + g −1 shoot DW under iron starvation and more than 1.34 mmol Na + g −1 shoot DW under iron supply.The thresholds of Na + concentration beyond which phytosiderophore release was reduced were higher in H. maritimum than in H. vulgare.Along with this finding, one can assume that wild barley was more efficient in the sequestration of Na + as compared to cultivated one.This could be justified by the slight reduction of shoot water contents in H. maritimum as compared to H. vulgare (Figure 1).Indeed, as it was mentioned in literature that the bad compartmentalization of salt ions (Na + and Cl − ) increases the risk of tissues deshydratation [22].Furthermore, at high salinity levels (200 and 300 mM), H. maritimum accumulated relatively lesser amount of Na + in shoots than did H. vulgare.Thus, it appears that the former was also more efficient than the latter in "controlling" the total amount of Na + reaching the leaves [23].
Our data clearly demonstrated that due to the osmotic effect, which was dramatic in cultivated barley as compared to wild one, interrelated photosynthetic parameters (transpiration rate, stomatal conductance, and assimilation rate) were significantly reduced in H. vulgare than in H. maritimum.Thus, controlling Na + transport to photosynthetic organs and its sequestration, which were critically appeared in wild barley, avoided the osmotic effect and its repercussion on photosynthetic activity, which might be indirectly involved in PS biosyntheses and secretion pathway [15][16][17][18][19][20][21][22][23][24][25].In addition, we found that the maintaining of PS release under salt stress was closely related to plant iron status, which is the key of photosynthetic activities.In fact, the threshold of Na + shoot concentration which inhibited PS release was higher under iron supply than under iron starvation.Furthermore, the correlation between shoot Na + concentration and PS release was positive in Fe-sufficient plants, whereas it was negative in Fe-deficient ones.

Conclusion
The controlling Na + transport to photosynthetic organs and its sequestration, which were more appeared in wild barley than in cultivated one, avoided the osmotic effect and its repercussion on Fe absorption efficiency and photosynthetic activity.Iron limitation resulting either from direct iron deficiency or salt stress (inducible iron deficiency) was concomitant with an induction of phytosiderophore release in both species.However, phytosiderophore exudation capacity was more maintained under salt stress in wild barley than in cultivated one.

Figure 1 :
Figure 1: Effect of salinity (0, 100, 200, and 300 mM NaCl) and iron availability (+Fe = 100 µM and −Fe = 0 µM) in the culture solution on shoot water content of H. maritimum (a) and H. vulgare (b).Means of three replicates.Means followed by the same letters are not significantly different at 5% according to Duncan's test.

Figure 3 :
Figure 3: Phytosiderophore (PS) exudation rates (mmol/g root FW• 4 h −1 ) as a function of shoot Na + concentration (mmol/g DW) in the presence (a) or the absence of Fe (b).White symbols stand for H. vulgare while black symbols stand for H. maritimum.

Table 1 :
Interrelated photosynthetic parameters of total chlorophyll (TChl), stomatal conductance (g s ), transpiration rate (E), CO 2 assimilation rate (A), and relative intercellular CO 2 concentration (C i /C a , given as intercellular CO 2 concentration/ambient CO 2 concentration) in H. maritimum and H. vulgare plants hydroponically grown under iron supply (+Fe) or iron starvation (−Fe) at different salt levels (0, 100, 200 mM) over 60 h.Means of five replicates ± SE.For each line, means followed by the same letters are not significantly different according to Duncan's test at P ≤ 0.05.

Table 2 :
Na + concentrations (mmol Na + •g −1 DW) in shoots and roots of H. maritimum and H. vulgare plants hydroponically grown under iron supply (+Fe) or iron starvation (−Fe) at different salt levels (0, 100, 200, 300 mM) over 60 h.Means of three replicates.For each line, means followed by the same letters are not significantly different at 5% according to Duncan's test.

Table 4 :
Fe concentrations (µmol Fe•g −1 DW) in shoots and roots of H. maritimum and H. vulgare plants hydroponically grown under iron supply (+Fe) or iron starvation (−Fe) at different salt levels (0, 100, 200, 300 mM) over 60 h.Means of three replicates.For each line, means followed by the same letters are not significantly different at 5% according to Duncan's test.

Table 5 :
Salinity effect (0, 100, 200, and 300 mM NaCl) on Fe absorption efficiency (FeAE) in H. maritimum and H. vulgare grown on nutrient solution containing 100 µM Fe.Means of three replicates.For each line, means followed by the same letters are not significantly different at 5% according to Duncan's test.