Nitrate : ammonium nutrition alleviates detrimental effects of salinity by enhancing photosystem II efficiency in sorghum plants 1

This study was carried out to evaluate if replacement of nitrate (NO3 -) by ammonium (NH4 +) in the growth medium prevents the damage in the photosystem II (PSII) efficiency and degradation of photosynthetic pigments caused by saline stress in sorghum plants. Sorghum bicolor plants were cultivated in nutrient solutions containing different NO3 :NH4 + ratios (100:0, 75:25, 50:50, 25:75 and 0:100) at a final concentration of 5.0 mM. Salinity was imposed by adding NaCl at 75 mM in the growth medium; and the analysis were performed after ten days of salt stress. The most striking effects of nitrogen regime were observed in plants supplied with equal amounts of NO3 -: NH4 + (50:50). Under salt stress, 50:50-grown plants displayed an increase in the photosystem II maximum efficiency (Fv/ Fm), which was associated with a lower non-photochemical quenching (NPQ). The superior performance of saltstressed plants grown in 50:50 ratio was indicated by a higher effective quantum yield of PSII (ΦPSII) and electron transport rate (ETR), as well as increased chlorophyll b and anthocyanins. In conclusion, nitrogen nutrition with same amounts of nitrate and ammonium avoids the degradation of photosynthetic pigments and allows higher PSII efficiency in salt-stressed sorghum plants. Nutrição com nitrato e amônio minimiza os efeitos prejudiciais da salinidade pelo aumento da eficiência do fotossistema II em plantas de sorgo


A B S T R A C T
This study was carried out to evaluate if replacement of nitrate (NO 3 -) by ammonium (NH 4 + ) in the growth medium prevents the damage in the photosystem II (PSII) efficiency and degradation of photosynthetic pigments caused by saline stress in sorghum plants.Sorghum bicolor plants were cultivated in nutrient solutions containing different NO 3 -:NH 4 + ratios (100:0, 75:25, 50:50, 25:75 and 0:100) at a final concentration of 5.0 mM.Salinity was imposed by adding NaCl at 75 mM in the growth medium; and the analysis were performed after ten days of salt stress.The most striking effects of nitrogen regime were observed in plants supplied with equal amounts of NO 3 -: NH 4 + (50:50).Under salt stress, 50:50-grown plants displayed an increase in the photosystem II maximum efficiency (Fv/ Fm), which was associated with a lower non-photochemical quenching (NPQ).The superior performance of saltstressed plants grown in 50:50 ratio was indicated by a higher effective quantum yield of PSII (ΦPSII) and electron transport rate (ETR), as well as increased chlorophyll b and anthocyanins.In conclusion, nitrogen nutrition with same amounts of nitrate and ammonium avoids the degradation of photosynthetic pigments and allows higher PSII efficiency in salt-stressed sorghum plants.

Introduction
Salinity is one of the most severe factors limiting plant growth and yield.Photosynthesis is a physiological process closely related to crop productivity.Decline of photosynthetic capacity commonly occurs in salt stressed plants, but the underlying mechanisms are complex and remain unclear (Saleem et al., 2011).Photosystem II (PSII) plays an important role in plant photosynthesis, and it often operates abnormally under environmental stress (Havaux, 1992;Baker, 2008).
Over-reduction because of electron excess on thylakoid membranes is frequently related under salinity (Szepesi et al., 2005;Aragão et al., 2012).In order to avoid or minimize photo-damage, photo-inhibition and oxidative stress, the plants can deploy several mechanisms such as photorespiration, heat dissipation and the xanthophyll cycle.Recently, evidence has emerged that N metabolism and respiration may also be involved in energy dissipation (Osmond & Foster, 2006;Aragão et al., 2012).
Nitrogen availability has been cited as important strategy to induce salt stress acclimation in plants (Kant et al., 2007;Fernández-Crespo et al., 2012;Miranda et al., 2013a).Nitrogen is required in large quantities by plants and is taken up in the form of ammonium (NH 4 + ) and nitrate (NO 3 -).High supply of NO 3 -mitigated the salinity effects through an enhancement in the efficiency of photosystem II and CO 2 assimilation in Jatropha curcas plants (Aragão et al., 2012).On the other hand, although NH 4 + assimilation is energetically more favorable than NO 3 -, it is usually toxic for some plant species (Britto & Kronzucker, 2002).However, a combined nutrition by using NO 3 -and NH 4 + induced a substantial reduction in the harmful effects of salinity compared with NO 3 -supply (Kant et al., 2007;Zhonghua et al., 2011).
This study aimed to investigate whether replacement of NO 3 -by NH 4 + in the growth medium prevents the damage in the photosystem II efficiency and degradation of photosynthetic pigments caused by saline stress in Sorghum bicolor plants.Therefore, the differences in PSII photochemistry between plants grown in several NO 3 -:NH 4 + ratios in response to salt stress was investigated, as well as the role of nitrogen nutrition in mitigating the possible damage in the photosynthetic pigments as affected by salinity was also analyzed.

Materials and Methods
Seeds of forage sorghum [Sorghum bicolor (L.) Moench] of the genotype CSF20, provided by Instituto Agronômico de Pernambuco (IPA), Brazil, were sterilized with 2% sodium hypochlorite for 5 min and then washed with distilled water.Thereafter, the seeds were sown in vermiculite moistened with distilled water.Four days after sowing (DAS), the seedlings were transferred to a tray containing Hoagland's nutrient solutions formulated to contain inorganic nitrogen at 5.0 mM, supplied as NO 3 -and/or NH 4 + .The nitrogen treatments were as follows: percentage of NO 3 -in relation to NH 4 + of 100:0, 75:25, 50:50, 25:75 and 0:100.Twelve DAS, the plants were subjected to salt stress with NaCl at 0 (control) and 75 mM, which was applied in doses of 37.5 mM per day to avoid osmotic shock.
Nutrient solutions were renewed every three days and the plants were harvested ten days after the last salt addition.The experiment was carried out under greenhouse conditions, where the midday photosynthetic photon flux density was approximately 1,200 μmol m -2 s -1 , the mean air temperatures were 31.5 ± 2 °C during the day and 27.4 ± 2 °C at night, and a mean relative humidity was 65.6 ± 10%.
Photosynthetic pigments were extracted from fresh leaf material.Discs from leaves were kept in the dark for 48 h at room temperature in CaCO 3 saturated in dimethyl sulfoxide (DMSO).Absorbance of extracts was measured with a UV/ visible spectrophotometer for chlorophyll a, b and total, and carotenoids at A 480 , A 649 and A 665 , and concentrations were calculated using equations outlined by Wellburn (1994).Anthocyanins were extracted by agitating gently leaf discs in the dark for 48 h at 4 °C in 3 M HCl: H 2 O:MeOH (1:3:16 v:v:v).Anthocyanin content in the methanolic extracts was estimated as A 530 -0.24A 653 , as described by M'rah et al. (2010).
Experimental design was completely randomized with a 5 x 2 factorial scheme, which was composed by five nitrogen sources (percentage of NO 3 -in relation to NH 4 + of 100:0, 75:25, 50:50, 25:75 and 0:100) and two salinity levels (0 and 75 mM NaCl).The analyses were performed using five repetitions per treatment.The data were submitted to analysis of variance (ANOVA) and the means were compared using Tukey's test (p ≤ 0.05) by the Sisvar program (São Paulo, Brazil).

Results and Discussion
In general, in control, contents of chlorophyll a and carotenoids were higher in plants supplied with NO 3 -(100:0 ratio) than in the ones grown with NH 4 + (0:100 ratio) (Table 1).Salinity reduced the contents of chlorophyll total and carotenoids only in plants from treatment 100:0.On the other hand, in plants grown with the same amounts of NO 3 -and NH 4 + (50:50 ratio), salt stress promoted a 32% increase in the chlorophyll b content.In presence of salinity, the highest values of chlorophyll b content were observed in plants from treatment 50:50 and the lowest in the ones grown in the ratios 100:0 and 75:25, respectively; whereas the opposite, at least in part, was observed for carotenoids contents (Table 1).Under control conditions, the highest values of anthocyanins were found in plants in the ratios 0:100 and 25:75.The anthocyanins content was reduced by salinity in plants under treatments 100:0, 25:75 and 0:100, whereas it increased in plants grown with ratio 50:50.Additionally, stressed plants under 50:50 treatment displayed the highest accumulation of anthocyanins in the leaves (Table 1).
Several studies have cited reductions in photosynthetic pigments by salinity (Sudhir & Murthy, 2004;Miranda et al., 2013b).In this study, S. bicolor plants supplied by ratio 100:0 showed decreases in chlorophyll total and carotenoids (Table 1); conversely, nitrogen nutrition with ratio 50:50 was able to increase the chlorophyll b content under NaCl stress.The higher chlorophyll b content may be attributed to an enhanced oxidative protection displayed by anthocyanins (Table 1).Anthocyanins are known as potent antioxidants and their accumulation possibly prevented photo-oxidative damage to leaf cells, reducing the harmful effects of salt stress in 50:50-grown sorghum plants (Been Saad et al., 2012).
Salinity did not promote photoinhibition of PSII photochemistry, as provided by Fv/Fm ratio (Figure 1A); furthermore, plants grown with equal amounts of NO  (50:50 ratio) displayed an increase in the PSII maximum efficiency (Fv/Fm) when subjected to salt stress.In absence of NaCl, effective quantum yield of PSII (ΦPSII) and electron transport rate (ETR) were higher and lower in plants grown in the ratios 25:75 and 50:50, respectively (Figure 1B and 1C).Under salinity conditions, plants cultivated in the ratio 50:50 strongly enhanced ΦPSII and ETR compared to control.In general, energy excess at the PSII level (EXC) and photochemical quenching (qP) were little or not altered by both NO 3 -:NH 4 + ratio and salinity stress (Figure 1D and  1E).Non-photochemical quenching (NPQ) was higher in non-stressed plants treated with ratio 50:50 (Figure 1F).Salt stress severely decreased the NPQ of plants from treatments 100:0, 75:25 and 50:50.Additionally, under salinity conditions, 0:100-grown plants had higher NPQ values than the plants from other nitrogen treatments.
ΦPSII estimates the efficiency at which light absorbed by PSII is used for quinone A (Q A ) reduction (Baker, 2008).It provides an estimate of the quantum yield of linear electron flux through PSII.ETR estimates the noncyclic electron transport rate through PSII.Another component of PSII efficiency is the qP, which results from using excitation energy within PSII to drive electron transport from P680 to Q A (Baker, 2008).Herein, in control condition, it is plausible that 50:50-grown plants raised an excess of reducing power, as evidenced by the marked reduction in ΦPSII, ETR and qP (Figure 1B and 1C), and thus they displayed a large thermal dissipation, as denoted by the improved NPQ (Figure 1F).
Fv/Fm ratio provides an estimate of the maximum efficiency of PSII photochemistry, which is the PSII operating if all the PSII centers are open (Q A oxidized) (Baker, 2008).A decay in this ratio provides an indicator of photo-inhibitory damage induced by the incident photon flux density when plants are subjected to a wide range of environmental stresses (Bjorkman & Demming, 1987;Aragão et al., 2012).In this study, as revealed by the high values of Fv/Fm, the significant photochemical alterations induced in the salt-stressed sorghum plants and cultivated with equal levels of NO 3 -and NH 4 + were able to avoid the photoinhibition and photo-damage in PSII, thus allowing a favorable electron transport (ETR) and a higher ΦPSII (Figure 1A, 1B and 1C).As a result, these favorable processes might allow a higher electron-transport rate from PSII to CO 2 assimilation under conditions of restrictions in the stomatal opening caused by salinity, as well as reduce the heat dissipation (NPQ).Additionally, the masking of chlorophyll by anthocyanins that prevent photo-oxidative damage is particularly important since it allows higher PSII efficiency (Been Saad, 2012).
Superior performance of 50:50-grown stressed plants may be associated with the greater accumulation of activated PSII reaction centers, as well as high capability for dissipation and transfer of the excitation energy.Nevertheless, it can be assigned to a mechanism to up-regulate photosynthetic electron transport so that production of ATP and NADPH would be in equilibrium with an improved photosynthetic capacity (Figure 1) (Lu & Zhang, 2000).Moreover, up-regulating the linear electron transport among the two photosystems to match the demand of NADPH generation could decrease the electron transport to O 2 on the acceptor side of PSII (i.e., in the Mehler reaction), thus minimizing the production of reactive oxygen species (Miller et al., 2010).
Radar plot evidences that the nitrogen nutrition in S. bicolor plants implies a differential pattern of responses to salt stress, demonstrating a key role of inorganic nitrogen source in ameliorating plant's sensibility to NaCl (Figure 2).

Conclusions
1. Inorganic nitrogen source directly affects the photosynthetic pigments of Sorghum bicolor plants under salinity.
2. Nitrogen supply with same amounts of nitrate and ammonium avoids the degradation of photosynthetic pigments and allows high PSII efficiency in salt-stressed Sorghum bicolor plants.

#Figure 1 .
Figure 1.Photosystem II maximum efficiency -Fv/Fm (A), effective quantum yield of PSII -ΦPSII (B), electron transport rate -ETR (C), energy excess at the PSII level -EXC (D) photochemical quenching -qP (E) and non-photochemical quenching -NPQ (F) of sorghum plants cultivated in five combinations of nitrogen nutrition supplied as NO 3 -:NH 4 + , under control (white bar) and salt stress (grey bar) conditions with NaCl at 0 and 75 mMMeasurements were performed ten days after salinity imposition.In the same nitrogen source, different capital letters represent significant differences due to salt stress and; in the same NaCl level, different lowercase letters represent significant differences due to the nitrogen source tested using Tukey's test (p ≤ 0.05)

Figure 2 .
Figure 2. Overall representation of Sorghum bicolor physiological responses to salinity stress (red line).Nitrogen treatments supplied as NO 3 -:NH 4 + ratios, in which the plants were grown, are individually presented in a radar plot.The reference plants were grown in absence of NaCl stress (blue line)

Table 1 .
Concentrations of chlorophyll (Chl), carotenoids and anthocyanins of Sorghum bicolor plants cultivated in five combinations of nitrogen nutrition, supplied as NO 3 -: NH 4 + , in presence of NaCl at 0 (control) and 75 mM (salt stress) #