Introduction

Saline stress represents a soil condition where neutral salts (NaCl or Na2SO4) predominate, whereas alkaline stress is mainly related to the occurrence of alkaline salts (Na2CO3 or NaHCO3; Yang et al. 2007). Both salt stresses disturb more than 10% of the world’s cultivable area, hindering agricultural outputs (Läuchli and Lüttge 2002). Negative effects of saline stress include reduction of water uptake from soil (Munns 2002) and Na+ or Cl− build-up within cells, leading to enzymatic activity inhibition, all altering photosynthetic and energetic processes (Yeo 1998; Tester and Davenport 2003). Alkaline salts may cause deficiency of nutrients such as phosphorus, iron and zinc (Clark 1982; Clark et al. 1982; Marschner 1995), inhibiting photosynthesis and plant growth, besides altering balances of reactive oxygen species and causing cell dying (Kukavica et al. 2013). Fluctuating neutral to alkaline salt proportions often co-exist according to the soil (Shi and Wang 2005; Li et al. 2010). Several authors have reported that when mixed, saline and alkaline stresses present synergistic detrimental actions on plant development (e.g.: Shi and Sheng 2005; Paz et al. 2012, 2014; Vu et al. 2015; Li et al. 2017; Kumar et al. 2018; Jia et al. 2019).

Salt stress often limits the photosynthesis (Ashraf and Harris 2013). In the long term, salinity-induced reductions in the photosynthetic activity may result from decreases in the chlorophyll (Chl) and carotenoid contents (Parida and Das 2005; Munns and Tester 2008; Duarte et al. 2013), increased stomatal closure (Kyle et al. 1987) and/or non-stomatal restrictions, such as electron transport chain disruption (Parida and Das 2005; Chaves et al. 2009; Xiang et al. 2016), among other factors. Indeed, several stress sources may affect the photosystem II (PSII) function, which alters fluorescence emission. Therefore, changes in fluorescence are used to assess and analyze abiotic stress responses in plants (Demetriou et al. 2007; Kalaji et al. 2011; Mathur et al. 2013; Zushi and Matsuzoe 2017).

In plants, fluorescence is primarily produced by Chl a integrating the PSII antenna complexes. Its detection and analysis constitutes one of the most informative approaches to monitor the photosystem II (PSII) functioning. Changes in fluorescence are related to processes occurring within and around the PSII reaction centers, which reveal alterations in the acceptors redox balance and the energy quantum yield (Goltsev et al. 2016). After a dark-adapted plant has been illuminated, the intensity of Chl a fluorescence varies in a typical way. This variation in fluorescence intensity over time is acknowledged as fluorescence transient and may be represented by a polyphasic curve (or Kautsky curve, Papageorgiou 1975). This curve shows a phase of rapid increase presenting four steps symbolized as O–J–I–P (Strasser et al. 1995). The O–J–I–P test gives clues on the condition of quinone A (QA), quinone B (QB) and plastoquinone (PQ) pools (Strasser and Govindjee 1992). The rise of Chl a fluorescence from its minimal level “O” (the minimum value of fluorescence, F0) to a “J” level (or FJ) occurs at about 2 ms as result of QA reduction by PSII; this is followed by a fluorescence rise to the “I” level (or FI) at about 30 ms, by cause of the filling up of the quinone pool (QA and QB); finally, a rise from the “I” level to the “P” level occurs (the maximum value of the fluorescence, FM), due to congestion of electron traffic at the acceptor side of PSI.

Lotus japonicus (Leguminosae) has been broadly used as model plant to study many important physiological aspects during adaptation to salt stress, whose genome has been sequenced, providing numerous tools for genomic/genetic research (Handberg and Stougaard 1992; Sato and Tabata 2006). Several authors have compared the most widely employed genotypes of this species, Gifu B-129 and Miyakojima MG20 (MG20) using classic physiology and/or omic tools, regarding their tolerance and response to saline (Melchiorre et al. 2009; Sanchez et al. 2008, 2011, 2012) and alkaline stresses (Babuin et al. 2014; Campestre et al. 2016; Bordenave et al. 2017), showing that the first ecotype deploys a higher sensitivity level than the second ecotype to both stresses. In 2016, Campestre and collaborators registered a decline in PSII performance in plants of several L. japonicus ecotypes grown under alkaline condition. However, no study has been undertaken to compare Gifu B-129 and MG20 responses to combined saline–alkaline (S–A) stress, nor has any study been directed on those constituents of the PSII, which are substantially altered by saline, alkaline and S–A stresses in these ecotypes.

Our hypothesis are (1) PSII is vulnerable to salt stress in both ecotypes, (2) Gifu B-129 and MG20 deploy different responses to S–A stress, (3) the S–A stress causes higher damage levels than separated saline and alkaline stresses. To test this, we compared the impacts of three distinct classes of salt-induced stresses, alkaline, saline and S–A, on plant growth, net photosynthesis rate, stomatal conductance, Chl contents and specific Chl fluorescence parameters in the two model L. japonicus ecotypes.

Materials and methods

Plant material and growth conditions

L. japonicus seeds from MG20 and Gifu B-129 ecotypes were scarified with concentrated sulfuric acid (98%) 3 min, washed ten times with sterile distilled water and sown in Petri dishes containing water-agar (0.8%). Plates were incubated in a growth chamber at 30 °C in darkness until germination, 7 days after sown. Each resultant seedling was transferred to a 8 × 20-cm (diam × length) cylindrical pot containing washed sand mix (50% fine/50% coarse sand; pH 7.0; E.C. = 0.05 mS cm−1) and irrigated with 0.5 × Hoagland’s nutrient solution (Hoagland and Arnon 1950). Transferred plants were cultured in a growth chamber with a 16/8-h photoperiod at 24 °C/21 °C ± 2 °C (day/night) and 55/65 ± 5% relative humidity and 250 μmol photons m−2 s−1 light intensity provided by Grolux fluorescent lamps (F 40 W), until the end of all experiments. A drip irrigation system (9001 Digital Watering Timer Weekly Program, ELGO®, www.elgo.co.il; flow rate = 6.25 ml/h) was used according to Paz et al. (2012).

Experimental design and stress treatment

Experiments were performed according to a completely randomized design of one factor (stress) and four levels: control, saline, alkaline and S–A stresses.

The 0.5 × Hoagland solution was used as the nutrient source for all stressful and control conditions. The separate alkaline and saline conditions in a pot were created by adding, respectively, 10 mM NaHCO3 and 100 mM NaCl to the nutrient solution. For the S–A treatment, 90 mM NaCl + 10 mM NaHCO3 was added, thus obtaining a stress solution with the same Na+-derived EC but a higher alkalinity than that of the saline stress treatment. Control treatment consisted of plants irrigated with 0.5 × Hoagland solution without NaCl or NaHCO3. The pH and EC of irrigation solutions were monitored every 3 days with a combined pH meter/conductimeter (HI 255; Hanna Instruments, Padova, Italy) and maintained at 5.8/1.2, 5.8/11.0, 8.0/1.9 and 8.0/11.0 pH units/dS m−1, for control, saline, alkaline and S–A conditions, respectively. In the case of alkaline treatment, 8-day-old seedlings received the final salt concentration: 10 mM NaHCO3. To avoid any osmotic shock in the saline and S–A treatments, plants were first subjected to acclimation. For this, 8-day-old seedlings initially received 30 mM NaCl (saline), and 20 mM NaCl + 10 mM NaHCO3 (S–A). Then NaCl concentration was stepwise increased during 1 week until reaching the final treatments concentrations. After acclimation, 15-day-old plants were grown under their respective treatments for further 20 days. Gas exchange parameters, Chl a fluorescence transients and chlorophyll contents were measured in 1-month-old plants, on intact, fully expanded leaves, which were basal to the internode immediately below the apical bud. Gas exchange and Chl a fluorescence transients were measured at midday. Plants were harvested at the age of 35 days, split into shoots and roots and kept for further determinations.

There were six plants per treatment for each measured parameter (n = 6), and the experiment was performed twice. Only most representative results are shown.

Evaluation of Gifu B-129 and MG20 tolerance

The tolerance of MG20 and Gifu B-129 genotypes to the different stress sources was evaluated on the base of the plant growth, in terms of total dry matter production. For this, roots and shoots were dried at 60 °C until constant weight.

Measurement of gas exchange parameters

Net photosynthesis rate (Pn), stomatal conductance (Gs), transpiration rate (E) and internal CO2 (Ci) were measured under light saturation (1500 µmol photons m−2 s−1 illumination, LED light (peak wavelength: 625 nm), ambient carbon dioxide (400 ppm, average) at 24 °C, using a portable photosynthesis system (TPS-2 Portable Photosynthesis System, MA, USA). Data were collected at midday, 2 min to average steady state.

Measurement of Chl a fluorescence transient kinetic and OJIP parameters

Non-invasive Chl fluorescence fast-transient test (OJIP test) was conducted with a portable Chl fluorometer (PocketPEA v.1.1, Hansatech Instruments, Ltd., UK) at room temperature according to Babuin et al. (2014). Blade sections of intact leaves were covered with a leaf clip to adapt them to darkness for 20 min and then exposed for 3 s to 3500 µmol photons m−2 s−1 (637 nm peak wavelength) and Chl a fluorescence was recorded. The fluorescence data were processed by PEA plus software (Hansatech Instruments, U.K.) to obtain the OJIP parameters. Measurements were performed on six plants per treatment for each ecotype. A summary of OJIP parameters used in this study is shown in Table 1.

Table 1 Summary of parameters, formulae and their description using data extracted from chlorophyll a fluorescence (OJIP) transient (Strasser et al. 1999; Tsimilli-Michael and Strasser 2008)
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Chlorophyll a and b determination

Leaves were harvested and stored at − 80 °C until use. For pigment extraction, 40 mg of plant material grounded in liquid nitrogen was shaken in 100% acetone (4 °C, overnight). The extract was cold centrifuged and the supernatant removed. Measurements were made at wavelengths 663 nm (Chl a) and 647 nm (Chl b) in a spectrophotometer (Perkin Elmer Lambda 25 UV/VIS spectrometer), and pigment concentration was calculated according to Lichtenthaler (1987).

K+, Na+ and Cl− determinations

The K+ and Na+ contents were extracted from shoots with 100 mM HCl and estimated by standard flame photometry according to Chen et al. (2001). Briefly, chloride was determined by a thiocyanate–Hg-based colorimetric reaction. For this, 12.5 mg of powdered dry plant material was extracted in 0.5 ml of a solution containing H2O2 (30%):concentrated HNO3:isoamyl alcohol:H2O at 1:1:0.08:7.9 (v/v), incubated at room temperature for 15 min, diluted to 5 ml with Milli-Q water, and vigorously agitated in a vortex. Then 1.5 ml of the extraction mixture was centrifuged (10,000 rpm, 5 min) and the supernatant transferred to another Eppendorf tube. The colorimetric reaction solution contained polyethylene glycol dodecyl ether–water (Brij 35®, 4%):mercuric thiocyanate (4.17 g/l methanol):(NO3)3Fe (202 g/l Milli-Q water plus 21 ml concentrated HNO3):Milli-Q water at 0.05:15:15:70 (v/v). One milliliter of this reaction was added to 320 µl of the supernatant (control treatment). In the case of saline treatments, 50 µl of the supernatant was previously diluted to 320 µl with the extraction solution. Sample absorbance was determined at 450 nm with a spectrophotometer (Hitachi U-1100) and interpolated into a KCl calibration curve (0, 5, 10, 15, 20 ppm) to calculate Cl− concentration.

Statistical analysis

The data of all parametric measurements were tested within each genotype through to one-way analysis of variance (ANOVA) and to multiple comparisons by Duncan’s test. Statistical analyses were performed with the InfoStat package (Di Renzo et al. 2008).

Results

Impact of salinity, alkalinity and saline–alkaline treatments on plant growth and photosynthesis

Neutral, alkaline and mixed NaCl and NaHCO3 salts significantly reduced total dry weight of Gifu B-129 and MG20 plants with respect to the control treatment (Fig. 1). The magnitude of the induced reductions due to salinity or mixed S–A stress tended to be higher in Gifu B-129 than in MG20 (respectively, 53% and 83% in the first, versus 34% and 61% in the second ecotype), and similar under alkalinity (Gifu B-129 = 61%; MG20 = 64%). Pn was reduced in both ecotypes by the mixed S–A stress, and by the alkaline stress in Gifu B-129 plants, whereas Gs and E were strongly reduced by the three types of stress in both ecotypes and Ci did not significantly vary (Table 2).

Fig. 1
figure 1

Total dry weight of L. japonicus plants. Same letter within each ecotype means no statistical difference (Duncan’s test, P < 0.05). Empty bars = MG20. Gray bars = Gifu B-129

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Table 2 Gas exchange parameters: net photosynthesis rate (Pn, µmol CO2 m−2 s−1), stomatal conductance (Gs, mmol H2O m−2 s−1), transpiration rate (E, mmol H2O m−2 s−1) and internal CO2 (Ci, µmol mol−1) in leaves of L. japonicus Gifu B-129 and MG-20 plants
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Effect of alkaline, saline and saline–alkaline treatments on Chl a fluorescence kinetics and behavior of PSII photosynthetic machine, determined by OJIP parameters

Chlorophyll fluorescence rise transients obtained from control leaves of both genotypes showed a typical OJIP shape (Fig. 2). The “spider plot” diagrams (Fig. 3) showed that the behavior of the different parameters characterizing PSII functioning varied according with the ecotype and the stress type. Overall, most significant variations with respect to the control situation were found in the S–A stress, those variations being more evident in Gifu B-129 than in MG20. Following paragraphs describing variations in fluorescence parameters were statistically supported by Duncan’s test at P < 0.05.

Fig. 2
figure 2

Chlorophyll a fluorescence OJIP transient curves obtained from leaves MG20 and Gifu B-129

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Fig. 3
figure 3

Spider plots of the parameters measured and deduced from Chl a fluorescence OJIP transient curves in MG20 (a, c, e) and Gifu B-129 (b, d, f)

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No significant salt-induced changes in the OJIP curves were observed, except in Gifu B-129 plants treated with S–A stress, which showed a much lower fluorescence rise. Despite the fact that no salt-induced changes in F0 and Fm were observed in Gifu B-129 (neither in MG20), some OJIP parameters derived from their relationship were affected by the S–A stress in this ecotype (Fig. 3a, b; Table 3): FV/FM (φP0) and FV/F0 decreased (− 5 and − 10%, respectively), whereas F0/FM increased (20%).

Table 3 Significant variations (%) observed in the different OJIP parameters in MG20 and Gifu B-129 plants under the three stressful conditions in comparison with the control (Duncan’s test, P < 0.05)
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Salt treatment also affected parameters normalized to FV, although differently, according to the ecotype (Fig. 3a, b; Table 3). dV/dto, or M0, reflects the accumulation rate of the active reaction center (RC) fraction that are closed, thus providing information about the acceptor side of PSII (Strasser et al. 1995, 2004; Strauss et al. 2006; Xia et al. 2004; Mehta et al. 2010a, b). M0 was increased 68% by S–A stress in Gifu B-129 and reduced (21%) in MG20 treated with NaCl.

A rise in VJ suggests an increase of the proportions of closed RC and reduced QA at J step, whereas a rise in VI indicates accumulations of reduced QA and plastoquinone, which cannot transfer electrons to the dark reactions (Pan et al. 2010). VJ was increased (40%) in Gifu B-129, and showed no change in MG20, whereas VI showed mild increases in MG20 and Gifu B-129 under S–A stress, and in MG20 treated with NaHCO3 alone. VJ and M0 also defined the OJIP parameters Ss = VJ/M0 and Sm = Area/(FM − F0) that represent the normalized total complementary areas corresponding to the O–J phase and OP phase of the kinetic Chl fluorescence, respectively. Ss and Sm reflect the single-turnover QA reduction events and the multiple-turnover QA reduction events, respectively. Ss and Sm were reduced by S–A stress in Gifu B-129 (− 25% and − 39%, respectively). Conversely, MG20 increased the Ss values under salinity and S–A stress (22 and 24%, respectively) and showed a slight fall of Sm (− 8%) under S–A stress.

In the S–A treatment, the specific energy fluxes ABS/RC (or also, the absorption flux (of antenna chlorophylls) per RC, DI0/RC and TR0/RC showed contrasting behaviors between genotypes since they decreased (15% average) in NaCl-treated MG20 plants, but increased (27% average) in Gifu B-129 ones (Fig. 3c, d; Table 3). On other hand, ET0/RC was 15% average reduced by NaCl in both ecotypes, regardless alkalinity.

Under NaCl treatment, the density of RC or amount of RC per excited cross section (RC/CS0) and the active Chl associated with RC (ɣRC) of MG20 leaves were increased in a 20% and a notorious 130%, respectively, while these parameters showed no salt-induced change in Gifu B-129 (Fig. 3c, d; Table 3).

In Gifu B-129 plants, S–A stress induced 4%, 30% and 30% decreases of φP0, and ψE0 and φE0, respectively (Fig. 3e, f; Table 3), whereas no changes in these parameters were registered in the remaining treatments.

PIABS is a multiparametric expression elaborated through independent components, all contributing to photosynthesis. PIABS was increased (29%) by NaCl as a sole stress source in MG20, whereas it was dramatically decreased by the S–A stress in Gifu B-129 (− 63%). In line, PIABS components behaved markedly different according to the ecotype and the type of salt (Fig. 3e, f; Table 3).

Effect of alkaline, saline and saline–alkaline treatments on the chlorophyll and ion contents

In Gifu B-129 plants, the S–A treatment led to decreased Chl a and total Chl contents, and reduced Chl a/b ratio, whereas no effect was observed on these parameters with the other two stresses (Fig. 4). In MG20, Chl b was reduced by the three types of salt stress, whereas Chl a was not affected. As result, total Chl content was reduced by the S–A stress, and no salt-induced change of the Chl a/b ratio was observed in this ecotype.

Fig. 4
figure 4

Contents of Chl a, b, Chl a/b ratio and total Chl content in apical leaves of Gifu B-129 and MG20 plants. Asterisks mean statistical difference from control at P < 0.05, according to Duncan’s Test

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Higher shoot Na+ and Cl− levels were found in plants of both ecotypes when confronted with saline or S–A treatments, compared with respective controls (Supplementary Table 1). In contrast, a non-significant change was registered in these levels for plants treated with the alkaline salt alone. Regardless alkalinity, NaCl reduced K+ contents and K+/Na+ ratios in plants of both ecotypes, whereas the alkaline salt added as a sole stress source affected these parameters only in Gifu B-129 plants.

Discussion

Salt stress impact on growth, gas exchange and ion contents

Although the main objective of this work was to gain insight into the salt-induced behavior of the L. japonicus photosynthetic apparatus, plant growth, gas exchange and ion accumulation were also analyzed to improve our knowledge on L. japonicus responses to the three stresses.

Our results from total biomass (Fig. 1) showed that the two ecotypes diverged in their sensitivity level to salinity, in agreement with earlier works (Sanchez et al. 2008, 2010), and that such divergence also applies to the S–A stress. In addition, we showed that both ecotypes were less tolerant to the last stress than to separate alkaline and saline stresses (in that order). A comparable result was previously reported by Paz et al. (2012) on plants of a different Lotus species, L. tenuis, subjected to the same three stress sources. Previous studies on other plant species have revealed that, when compared at the same concentration level, alkaline salts exert a more detrimental action on plant growth than neutral ones (Shi and Yin 1993; Tang and Turner 1999; Yang et al. 2008, 2009; Gong et al. 2013). It is worthy to note that the alkaline treatment here used had a much lower strength (10 mM of salt) than the saline one (100 mM of salt), what prevents us of confirming former statement. On the other hand, the S–A stress solution here employed had equal electrical conductivity to that of the saline treatment (neutral salt) and equal pH to that of the alkaline one. Therefore, the worse growth performance registered in the S–A treatment could be a result of a possible additive effect of high saline strength and high pH.

In this work, some expected cause–effect relationships involving biomass and Gs parameters were not observed. Specifically, the negative effect of salt stress on plant biomass was not always accompanied by lower Pn values, suggesting that factors such as a limited nutrient condition (Munns 2005; Tsai et al. 2004; Hong et al. 2009) or toxicity (Marschner 1995) may have intervened in such decreases. Indeed, this could be the case in our work: the reduced shoot K+ content and K+/Na+ ratio, or the increased shoot Na+ and Cl− accumulation registered in some saline and alkaline treatments (Supplementary Table 1) to levels possibly toxic to diverse metabolic pathways, could have contributed to the reduction of the total biomass in those treatments. In fact, the higher ability of MG20, compared to Gifu B-129 to prevent Na+ reaching the shoot was the physiological parameter better explaining the more satisfactory response to salt stress of the first ecotype, in terms of plant biomass. Likewise, significant stress-induced stomatal closures were registered in both genotypes, in the absence of photosynthetic performance variation. The last phenomenon has been previously reported by other authors (Munns and Tester 2008; James et al. 2002) and may be explained through salt-induced changes in leaf anatomy leading to the improvement in CO2 diffusion (Acosta-Motos et al. 2015a, b; Gómez-Bellot et al. 2015). In this regard, it is worth to mention that alkaline and S–A salts caused a replacement of palisade by spongy parenchyma in leaves of L. tenuis (Paz et al. 2014). Further anatomical studies performed on L. japonicus ecotypes MG20 and Gifu B-129, testing the hypothesis of a salt-induced reorganization of leaf mesophyll would be valuable for a better understanding of present gas exchange results.

Salt stress impact on PSII photosynthetic performance

Data pertaining to the Chl fluorescence analysis showed that the most significant effects induced by salt addition concerned Gifu B-129 plants treated with combined neutral–alkaline salts, and MG20 plants treated with NaCl. The two photosynthesis-related indexes, φP0 and PIABS, were found to be notably altered by diverse environmental constraints in several plant species (Pietrini et al. 2005; Gazquez et al. 2015, 2018). As φP0 may provide clues of photoinhibition occurrence (Yamane et al. 2008; Goh et al. 2012), our data showing no significant salt-induced variation in this parameter (or a − 4% decrease in Gifu under S–A) suggest the absence of photoinhibition events due to salt treatment. The φP0 also reflects the PSII capacity to reduce the primary acceptor QA (Calatayud and Barreno 2001). The PIABS is regulated by the three main functional steps of photosynthetic activity by the PSII, which are absorption of light energy (ABS), trapping of excitation energy (TR) and conversion of excitation energy (ET; Strasser et al. 2000). The fact that both parameters were reduced by combined NaCl and NaHCO3 salts in Gifu B-129 plants (Fig. 3), but not in MG20, supports the notion extracted from biomass data of a higher tolerance to the S–A stress in the last ecotype, compared with Gifu B-129. Interestingly, NaCl alone increased PIABS in MG20 plants, indicating an improvement of the PSII functioning in these plants. However, last result was not reflected by Pn values. This lack of parallelism between net photosynthesis and the mentioned indices may be explained by the electron flux being not used in carbon metabolism, but re-routed to other biochemical pathways such as Mehler reaction, or photorespiration (which may sum 36% of photosynthetic electrons dissipated, e.g., in tomato under water stress; Haupt-Herting and Fock 2002).

Salt stress-induced modifications of the photosynthetic apparatus structures

RC/ABS and RC/CS are measures of the density of RC per ABS and cross section or CS, respectively. The most commonly observed RC response to salinity in glycophytes is their reduction, (e.g., Scenedesmus obliquus, Demetriou et al. 2007; Cucumis melo, Xiang et al. 2016; Triticum sp, Mehta et al. 2010a, b; Wolffia arrhiza, Wang et al. 2011 and Solanum lycopersicum, Zushi and Matsuzoe 2017). In this study, the RC pool size was another parameter differently altered by salt stress according to the ecotype. RC reduction was registered in Gifu B-129 plants exposed to combined NaCl and NaHCO3 salts, confirming the glycophytic L. japonicus status (Sanchez et al. 2008, 2011). In contrast, both RC densities (per both cross section and absorption energy flux) increased in NaCl-treated MG20 plants, in line with higher values of ɣRC, registered in this ecotype. Having in mind that salt-treated plants of the halophyte Artemisia anethifolia presented higher RC/CS than corresponding controls (Wen et al. 2005), former result could be interpreted as an adaptation, through improvement of RC stability, of the photosystem machinery to NaCl in this ecotype. Interestingly, the MG20 ecotype was considered as a moderate halophyte by Melchiorre and collaborators (2009) when confronted with 50 mM NaCl. Thus, the opposite S–A-induced responses observed in the RC/ABS between genotypes, an increase in MG20 and a decrease in Gifu B-129, could have also contributed with the contrasting behavior of PIABS in these ecotypes.

Variations in the effective antenna size of PSII may be inferred from changes in ABS/RC, chlorophyll content and Chl a/b ratio. These parameters varied concomitantly in Gifu B-129 plants under the mixed S–A treatment, supporting the notion that mixed NaCl and NaHCO3 salts induced a higher effective antenna size in this last ecotype, with preferential loss of reactions center complexes (DI0/RC, Table 3; Figs. 3, 4). In parallel, the chlorophyll contents and chl a/b data support specific loss of LHC in MG-20.

Salt effects on electron transport chain and carriers

Changes in several obtained and calculated parameters support the notion that, in Gifu B-129 plants confronting mixed (neutral and alkaline) salts, photoinhibition took place on both sides of PSII (acceptor and donor). On the one hand, the reduction in Fv/F0 reflects a decrease in the water-splitting complex activity, at the donor PSII site (Schreiber et al. 1995; Sayed 1998; Kalaji et al. 2011). Also, the decrease of Ss values in Gifu B-129 under S–A stress indicated a detriment of the processes associated with the primary photochemistry. Interestingly, higher Ss values in MG20 under saline and S–A stresses would indicate an improvement of the primary photochemistry. On the other hand, the decrease in ψE0 and the rise in VI, VJ and M0 in Gifu B-129 plants treated with combined NaCl and NaHCO3 salts conform to the idea of a slowdown of electron transfer from QA to the secondary acceptor QB, on the acceptor PSII side. Also, the decrease in Sm registered in Gifu B-129 plants under S–A stress suggests a decline in the pool of electron carriers between PSII and PSI, as Sm reflects the total electron carriers per RC (Jiang et al. 2008). Last results point to a reduction of the total electron acceptor capacity of S–A-treated Gifu B-129 leaves. Besides (unlike MG20), Gifu B-129 plants under S–A treatment presented increased TR0/RC although there was no significant change in the RC level. According to Wen et al. (2005), last situation indicates a lower QA re-oxidation. In fact, our data showing a reduction in the electron transport per reaction center (ET0/RC) in these plants are in line with the last phenomenon, and support the decreased ψE0, and the increased DI0/RC (non-photochemical quenching of energy) registered. Indeed, this scenery of electron transport blockage, in addition to a higher effective antenna size as it was pictured above for Gifu B-129, explains the higher energy dissipation of the excess excitation energy trapped in the non-RC (DI0/RC, Fig. 3d, Table 3). A rise in non-photochemical quenching may be the outcome of changes in either photoinhibition or protective high-energy-state quenching. As we stated above, our results from φP0 suggested the absence of photoinhibition events due to salt treatment. In tomato, increasing non-photochemical quenching, associated with lower φP0, was suggested to intervene in excess energy dissipation to keep photosynthetic apparatus from being dismantled (Gong et al. 2013). Energy dissipation prevents the reduction of QA to Q −A and, therefore, it does not contribute to the variable fluorescence (see Strasser and Strasser 1995; Strasser et al. 2000; Krüger et al. 1997; Appenroth et al. 2001; Jafarinia and Shariati 2012). That is why the OJIP curves obtained from Gifu B-129 plants treated with combined NaCl and NaHCO3 salts presented lower fluorescence levels than those from control plants (Fig. 2). Taken as a whole, these results explain the observed PIABS and φP0 decreases in the Gifu B-129 ecotype.

Conclusions

Figure 5 summarizes the effects of different stresses on the photosynthetic apparatus of both genotypes, explained on the base of their energy fluxes, quantum yields and efficiencies, and pools of each photosynthetic machine component. Our results allow us to accept the proposed hypothesis. Here we conclude that NaCl-derived stress sources affect the photochemical events in PSII in both ecotypes, being this effect more evident under higher pH condition, whereas alkalinity per se has a mild or no effect on these events. Our study also allowed us to determine quantitative and qualitative differences between both ecotypes regarding Chl a fluorescence response to salt stresses. The S–A treatment induced a more severe effect on Gifu B-129, compared with MG20, particularly on those parameters related to the functioning of a section spanning the donor (water-splitting complex) and the first acceptor (QA) sides of the electron transport chain. In contrast, NaCl improved primary photochemistry in MG20, although the step between QB and PSI was functionally compromised in this ecotype. The fact that some components or sequential steps of the PSII photosynthetic activity in Gifu B-129 and MG20 had diverged during their salt-induced responses indicates that both model ecotypes would not be equivalents for performing experiments addressing more specific hypothesis on physicochemical or structural aspects of the L. japonicus photosynthetic machinery. Therefore, we consider the information here obtained is useful and should be taken into account in future research on structural and biochemical aspects of the photosynthetic apparatus, aimed at elucidating the mechanisms that make possible the tolerance to these stressful conditions.

Fig. 5
figure 5

Schematic representation of OJIP test results. Central block represents a control condition for both ecotypes. Right and left blocks depict the sets of salt-induced events revealed by Chl fluorescence data in MG20 and Gifu B-129. Black boxes with A, S or S–A mean alkaline, saline or S–A treatments, respectively. RC active reaction center, RC with dashed line means “non-RC”; ChlRC active Chl in RC, QA and Q −A quinone A oxidized and reduced, respectively. Smaller and bigger arrows and letters with respect to the central block mean negative and positive changes regarding quantum efficiencies and the activity of the water-splitting complex, compared with the untreated control

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Author contribution statement

ABM: conceptualization; RR, CDB and MPC: methodology; ABM and AAR: formal analysis; SJM and OAR: funding acquisition; ABM: writing original draft; ABM, AAR and CDB: writing review and editing.