MONOPOTASSIUM PHOSPHATE (KH2PO4) AND SALICYLIC ACID (SA) AS SEED PRIMING IN Vicia faba L. AND Vicia sativa L. FOSFATO DE MONOPOTÁSSIO (KH2PO4) E ÁCIDO SALICÍLICO (SA) COMO PRIMAGEM DE SEMENTES EM Vicia faba L. E Vicia sativa L

The first experiment was conducted to evaluate the impact of seed priming on germination behavior and seedling establishment in Vicia faba and Vicia sativa, for that, seeds priming was done using SA (100 μM) and KH2PO4. In order to determine the optimal concentration of KH2PO4 for improving germination, different concentrations were used: 25 μM, 50 μM, and 100 μM. The best germination behavior and seedling establishment were obtained with 25 and 50 μM KH2PO4, respectively for Vicia faba and Vicia sativa. Moreover, data showed that 100 μM of SA improved seed germination as well as the seedling establishment for both species. The second experiment was carried out to investigate the influence of seed priming for improving phosphorous (P) deficiency tolerance. To do, seedling obtained from primed and nonprimed seeds were grown in a hydroponic culture system with three different treatments: control (C, medium containing sufficient P concentration: 360 μM KH2PO4), direct phosphorus-deficient (DD, medium containing only 10 μM KH2PO4), and induced P deficiency by bicarbonate (ID, medium containing sufficient P concentration: 360 μM KH2PO4 + 0.5 g L -1 CaCO3 + 10 mM NaHCO3). Furthermore, the role of exogenous SA applied to P deficiency tolerance enhancement was explored. Seed priming or the exogenous application of SA significantly reduced the severity effect of P deficiency. In fact, the pretreated plants were observed more tolerant to P deficiency as reflected from the significant increase in plant biomass, P uptake, and an efficient antioxidant system. Overall, this paper highlights the beneficial effect of seeds priming or the exogenous application of SA in the improvement of plant tolerance to phosphorus deficiency.


INTRODUCTION
Phosphorus (P) is among the most crucial macronutrient required for plant growth and development. However, owing to its limited abundance in the soil and by its adsorption into various soil minerals, P is frequently inaccessible and restricts plant growth (CABEZA et al., 2017). Consequently, P deficiency is one of the most abiotic stresses negatively influencing the productivity of crop legumes over the world mainly in developing countries (GRAHAM, 2003).
To solve this nutritional disturbance, the application of fertilizers and foliar sprays are necessary approaches. Nevertheless, these methods are too expensive to be practiced by farmers, particularly in developing countries. Alternatively, sustainable agricultural practices will be necessary to increase crop productivity and quality under stressful conditions. One promising technique is seed priming. Several works had tested the effectiveness of seed priming for improving plant nutrition in deficient soils. MUHAMMAD et al. (2017) demonstrated that nutrient seed priming (Zn and Mn) improve soybean seed quality for early seedling development under limited nutrient supply or availability. In addition, AJOURI et al. (2004) concluded that P and Zn application through seed priming enhanced barley seeds germination and early growth stage under low nutrient availability.
A various of signal molecules and hormones are being used as exogenous sources to improve plant tolerance to different stresses (JANDA et al., 2017;SALAHUDDIN et al., 2017). Among these chemical substances, salicylic acid (SA) has been identified as an important stress-signaling molecule in plant stress response. In this context, HAYAT et al. (2010) showed that SA was accumulated in plants confronted with various types of environmental stresses. Moreover, numerous researches demonstrated that seed priming with SA enhances plant defense against water stress (HOSSEIN et al., 2015), heat stress (KHAN et al., 2013), and copper accumulation (MOSTOFA;FUJITA, 2013). LI et al. (2017) revealed that the exogenous application of ABA, GABA, and SA enhances drought tolerance in Agrostis stolonifera by stimulating amino acids and carbohydrates accumulation. The promotive effects of seed priming with SA in abiotic stress tolerance were also reported by MIURA; TADA (2014), who found that exogenous application of SA could improve cold tolerance by regulating antioxidant enzyme activities.
As mentioned above, several researches have highlighted the beneficial role of seed priming technique on plant tolerance to various abiotic stresses. Nevertheless, the improvement of legumes tolerance to P deficiency by seed priming technique has not been well investigated. In addition, to our knowledge, there is no research that has been interested in the effect of SA on P deficiency tolerance on the plant as well as the comparative effects between KH 2 PO 4 and SA as priming agents on P deficiency tolerance. For this purpose, the assessment of the contribution of seed priming with KH 2 PO 4 and SA (seed priming or exogenous application) on seed germination behavior and P deficiency tolerance in two legumes Vicia faba and Vicia sativa has been tested in the present research.

Seed materials and priming
Common vetch Vicia sativa L (commercial variety introduced from France many years ago to be used by farmers for forage and hay purpose) and Vicia faba L (minor variety) were immersed for 2 min in a 30% CaCl 2 solution (commercial substance). After that, the seeds were rinsed 10 times with demineralized sterile water in order to ensure the safe removal of any sterilizing agent. Seeds were then blotted on sterile Whatman filter paper sheets. The seeds of each specie were subjected to seed priming, the first group was nutrient primed (soaked in 25, 50, and 100 µM KH 2 PO 4 for 24 h), while the second group was hormonal primed (soaked in 100 µM salicylic acid (SA) for 24 h). Following priming, seeds were washed in distilled water. Whereas the non-primed seeds (soaked in distilled H 2 O for 24 h) were used as the controls.
Experiment 1: seed priming effect on germination behavior and early growth stage Seeds were then placed to germinate in Petri dishes (90 mm diameter) containing a sheet of filter paper, saturated with distilled at 25 °C in the dark.
In each treatment, three replicates (each one contained 15 seeds) were used. Radical emergence was checked daily, and germination was defined as radical emergence of ≥2 mm (SMITH; COBB, 1991), and primary root length and shoot were measured for 5 days.
The measurements of physiological characteristics such as total seed germination (TG), germination index (GI), mean germination time (MGT), and vigor index (VI) were determined using those formulas: where, n is the total germinated seeds and N is the total seeds sowed.
Germination index (GI) was calculated as explained by MAGUIRE (1962): GI= where Gt= number of germinated seeds on Day t, Tt= time corresponding to Gt in days.

MGT=
where n i = the number of germinated seeds on the i th day and Ti= the rank order of day i (number of days counted from the beginning of germination).
Germinated seeds were transferred to square plates filled with sterile sand and irrigated with deionized water. After 15 days of growth, shoot, and root lengths of six randomly selected seedlings were measured. The dried shoots and roots (at 70 °C) were then weighted.
For the conductivity test, ten seeds of each treatment were placed in 10 mL distilled H 2 O for 24 h at 25 °C after which the initial conductance (C 0 ) was measured with a conductivity meter. Seeds were then exposed to 80 °C for 30 min then returned to 25 °C for 30 min before measuring the total conductance (Ct). Percent of electrolyte leakage was expressed as (C 0 /Ct) x 100 (BECWAR et al., 1982).
Experiment 2: effects of seed priming with KH 2 PO 4 , SA and the exogenous application of SA on P deficiency tolerance in Vicia faba and Vicia sativa
The experiments were achieved in a glasshouse under controlled conditions (the temperature varies between 24 °C during the day and 16 ºC overnight, a 14 h photoperiod and with the relative humidity of 70 ± 5%). The nutrient solution was continuously aerated and was changed every 5 days. The treatment lasted 27 days. At the end of the experiment, leaves and roots were separated, rinsed with distilled water, and dried in a stove for 48 h at 60 °C. Afterward, the dry matter weights were determined. Moreover, leaves and roots were frozen in liquid nitrogen and kept at −80 °C to be used for enzyme activity assays.

Determination of phosphorous concentrations and acid phosphatase activity
Phosphorus concentration was assayed following FLEURY; LECLERC (1943) method using vanado-molybdate. Roots acid phosphatase activity was measured spectrophotometrically by monitoring the p-nitrophenol released following the protocol described by TALBI ZRIBI et al. (2015).

Lipid peroxidation (MDA) assay
To assay leaves and roots MDA content, the protocol described by CAKMAK; HORST (1991) was adopted.

Enzyme assays
Roots and leaves (200 mg) were homogenized with 10% (v/v) polyvinyl-polypyrrolidone and 1 ml phosphate buffer (50 mM; pH=7.8) containing 0,1% (v/v) triton x-100, 1 mM phenylmethylsulphonyl fluoride. After that, the homogenate was centrifuged at 12 000 g for 30 min at 4 °C. The supernatant was used to investigate enzyme activities. SOD and GPOX activities were determined as previously described by MHADHBI et al. (2005). The protein content of each sample was measured by adopting the method of BRADFORD (1976).
Total phenolic compounds analysis Roots and leaves (1 g) were extracted using 10 ml pure methanol as solvent (M'SEHLI et al., 2008). For the total phenolic compounds' determination method of (M'SEHLI et al., 2008) was followed using Folin-Ciocalteu as a reagent. Total flavonoids analysis was carried out following DEWANTO et al. (2002) method.

Statistical analysis
In experiment 1, all studied parameters for primed seeds were statistically compared with those from non-primed seeds using one-way ANOVA. A two-way analysis of variance (ANOVA) was performed for the whole data in experiment 2 using the STATI-CF statistical software. Means were compared using the Newman Keuls test at P<0.05 when significant differences were found.

Experiment 1. Seed priming effect on germination process and seedling growth
The analysis of the data presented in Table 1 shows that, in Vicia sativa, seed priming treatments significantly improved total seed germination. Hormonal priming (100 µM SA) enhanced germination percentage as compared to KH 2 PO 4 priming treatment.
Seeds primed with 50 and 100 µM KH 2 PO 4 resulted in lower MGT and GI than that of control. Furthermore, SA and 25 µM KH 2 PO 4 had a positive effect on the vigor index (VI). But seed priming treatment with 50 and 100 µM KH 2 PO 4 had not positive effects on VI and conductivity tests. Overall, seed priming with 100 µM SA was suitable compared to others (Table 2). Seed priming significantly influenced seedling shoot and root related parameters (length and biomass) in both studied species (Figure 1). For Vicia sativa, the maximum length and biomass were observed in seedlings from seeds primed with 100 µM SA and 50 µM KH 2 PO 4 . However, 100 µM KH 2 PO 4 significantly decreased seedlings length (-40%) and biomass (-60%) as compared to the control seedlings (from non-primed seeds) ( Figure  1A, B and Figure 2). Data presented in Figure 1C and D indicates that, in Vicia faba, the biomass was increased up to 44% in seedlings primed with 100 µM SA and 25 µM KH 2 PO 4 as compared to controls. However, seedling growth parameters were inhibited by a high concentration of KH 2 PO 4 (50 and 100 µM) ( Figure  2).  Experiment 2: seed priming effect on P deficiency tolerance improvement Considering the main results found in experiment 1, 50 µM KH 2 PO 4 and 25 µM KH 2 PO 4 were defined as the optimal concentration for seed priming in Vicia sativa and Vicia faba, respectively. Additionally, in this part of the experiment, the impact of the direct addition of SA in the nutrient solution (expressed as SA. B) in P deficiency tolerance enhancement was considered.
Plant growth: it can be noticed from Figure  3 (A) and (B) that P deficiency (DD or ID) reduced significantly the total dry weight in both species. The seed priming with KH 2 PO 4 and SA showed to be helpful in alleviating the depressive effect of P deficiency on plant growth. The results presented in Figure 3(A) and (B) demonstrated that the addition of 100 µM of SA directly in the hydroponic solution (SA. B) improve plant response to P deficiency in both species. For example, under ID treatment, we can see that the decrease in dry biomass which can reach up to 31% in control plants was less than 10% in plants received an exogenous application of SA. Plant phosphorus status and root acid phosphatase activity: the data presented in Figure 4 (A) and (B) showed that under phosphorus deficiency, a noticeable reduction in P content was showed in both species. The observed decrease became evident in plants from non-primed seeds; while it was less pronounced in P-deficient plants from seeds primed with KH 2 PO 4 , SA, or received an exogenous SA in hydroponic box. The analysis of Figure 5 (A) and (B) showed that P deficiency led to a significant enhancement of acid phosphatase activity. The observed increase was more spectacular in plants from primed seeds with KH 2 PO 4 and SA or those received an exogenous application of SA.  Leaves and roots MDA concentration: in Vicia sativa, P deficiency significantly induced MDA accumulation by 43% in leaves, as well as 40% in roots under DD treatment; and by 40% in leaves and 32% in roots under ID treatment ( Figure  6 A and C). Both KH 2 PO 4 and SA have a significant inhibitory effect on MDA accumulation under P deficiency conditions. The same applies to the exogenous application of SA. Similarly, in Pdeficient plants of Vicia faba, the same inhibitor effect of seed primed with KH 2 PO 4 and SA or the exogenous application of SA on MDA accumulation was illustrated (Figure 6 B and D).
Antioxidant defense system: the results presented in Table 3 and Table 4 revealed that P deficiency causes a significant increase of all antioxidant enzyme activities (SOD, GPOX, and CAT) in leaves and roots of P-deficient plants. This stimulator effect was significantly more pronounced in P-deficient plants from seeds primed with SA or those received an exogenous application of SA. Biosci The same applies to the secondary metabolites; seed priming significantly influenced the average of secondary metabolites accumulation (polyphenols and flavonoids) in P-deficient plants.
The highest concentration was detected in plants treated by SA (seed primed or exogenous application) whereas the lowest was observed in plants from non-primed seeds (Tables 3 and 4). Table 3. Effect of seed priming with 50 µM KH 2 PO 4 , 100 µM SA or its exogenous application (SA.B) on SOD (USOD mg -1 Proteins), CAT (µmol H 2 O 2 min -1 mg -1 Proteins), GPOX (µmol H 2 O 2 min -1 mg -1 Proteins) activities and secondary metabolites production (polyphenols: mg GAE g -1 DW, flavonoids: mg CE g -1 DW) in leaves (L) and roots (R) of Vicia sativa under P deficiency conditions. Untreated (immersed in H 2 O) seeds were used as the control. Values followed by different letters are significantly different at P<0.05 according to the Newman-Keuls test.

DISCUSSION
Influence of salicylic acid on germination behavior, early growth stage and P deficiency tolerance The obtained results depicted that Vicia sativa and Vicia faba seeds primed with 100 µM SA exhibited a higher germination percentage, germination index (GI), and vigor index (VI) than the non-primed ones. In addition, SA-primed seeds  (2010) stated that SA could stimulate or inhibit seed germination as a function of the concentration used. In fact, those researchers found that higher concentrations of SA (SA > 100 µM) inhibited Arabidopsis seeds germination. Most previous reports investigated the effect of SA concentrations in seed germination. They deduced that the restrain effects of high SA concentrations may be caused by the toxic effects (RAO et al., 1997;ALONSO-RAMIREZ et al., 2009;LEE et al., 2010). Several reports have described the beneficial effects of seed priming with SA on early seedling growth. In the present investigation, results showed that seeds primed with 100 µM SA caused considerable enhancement of early growth of seedlings (dry weight, length) compared to controls, in both species. The findings were corroborative of the early reports of SAKHABUTDINOVA et al. (2003) in wheat and REHMAN et al. (2015) in maize. The better seed germination/ seedling growth depicted in seed primed with SA might be attributed to higher α-amylase activity and total soluble sugar contents in primed seeds (WANG et al., 2016).
To date, the effectiveness of seed priming with SA on P deficiency tolerance improvement has not been thoroughly investigated. This part of the present work was conducted to check the influence of SA-seed priming on P deficiency tolerance improvement in Vicia faba and Vicia sativa.
The present study clearly indicates that plant growth was reduced in both species when they were grown under P deficiency conditions. Our findings pointed out also that SA treatment (seed priming or exogenous application) could be quenched this dramatic effect of stressful conditions on plant biomass. The observations are consistent with earlier findings indicating the ameliorative effect exerted by SA treatment on plant growth potential confronted with different abiotic stresses (KHAN et al., 2015;NOREEN et al., 2017). Interesting research found that P, K, Mg, and Mn concentrations of SA-treated plants were increased under stressed conditions and these findings proposed that SA could be used to improve plant growth and mineral status under stress conditions (PER et al., 2017). Additionally, KONG et al., (2014) reported that low concentrations of SA could alleviate chlorosis by improving Fe absorption and increasing chlorophyll concentrations.
In this experiment, P deficiency significantly decreased P concentration compared with control treatment. This inhibitory effect was significantly alleviated by SA treatments (seed priming or its exogenous application in nutrient solution). The role of SA treatment in plant P status promotion under stressful conditions could be explained by the higher stimulation of acid phosphatases activity (APase) in SA-treated plants. These enzymes are induced by P deficiency and they are involved in Pi acquisition in plants (MEHRA et al., 2017).
Various researchers suggested the implication of SA in the modulation of antioxidant metabolism to stimulate plant-tolerance to abiotic stresses (HASANUZZAMAN et al., 2014). In a recent study, KOHLI et al. (2017) have reported that salicylic acid enhanced the level of plant tolerance to heavy metal by up-regulating the antioxidative system defense.
Data from this study indicated that exogenous applications of SA or its use in seed priming were effective in decreasing MDA concentration under P deficiency treatments in both species. This fact was positively correlated with the stimulation of antioxidant system defense (antioxidant enzymes and accumulation of secondary metabolites) that increased membrane stability and tolerance for Vicia faba and Vicia sativa to P deficiency. The results corroborated well the early findings of HUSSAIN et al. (2016) who observed that oxidative stress caused by abiotic stress including P deprivation was effectively mitigated with selenium-or salicylic acid-priming in rice. Summarizing the findings, it might be concluded that SA treatment whether by its exogenous application or by seed priming may enhance plant tolerance to P deficiency.
Influence of KH 2 PO 4 on germination behavior, early growth stage and P deficiency tolerance Our findings showed that KH 2 PO 4 , depending on its concentration, decreases or enhances germination processes, seed quality, and early seedlings growth of Vicia sativa and Vicia faba. Out of three different concentrations used for KH 2 PO 4 as a priming agent in the present experiment, 25 µM, and 50 µM KH 2 PO 4 were found to have the best results in Vicia faba and Vicia sativa, respectively. These concentrations were selected to examine the possibility of using KH 2 PO 4 as a priming agent to improve P deficiency tolerance in Vicia sativa and Vicia faba.
Previously, it was been reported that seed priming with the limited nutrient element was more effective in overcoming the nutrient deficiencies problem and improving plant growth on deficient soil, comparing to soil or foliar applications (FAROOQ et al., 2012). To date, the contribution of KH 2 PO 4 to improve plant tolerance to phosphorus deficiency was not well explored. To our knowledge, AJOURI et al. (2004) are the only researchers who have investigated the implication of seed priming with KH 2 PO 4 in the enhancement of germination performance and seedling growth in barley under P deficiency and they found that this technique can improve the germination of barley and increase the seed nutrient content. The present work provided further confirmation of their conclusion. The analysis of our results showed that, in both species, KH 2 PO 4 -treated seedlings have higher growth compared to control ones under P deficiency conditions. Besides, the same pattern was observed for plant P nutrition illustrating that the highest P content was detected in seedling treated with KH 2 PO 4 by stimulating acid phosphatases activity (APase).
Taken together, the maintenance of plant biomass and P uptake improvement made seedlings treated by KH 2 PO 4 more efficient to overcome P deficiency conditions. Data on lipid peroxidation showed that MDA production was intensified under P deficiency in both species. However, our results revealed that P-deficient plants that emerged from KH 2 PO 4primed seeds manifested significantly lower MDA contents.
The observed reduction in MDA content by KH 2 PO 4 -priming suggests an effective antioxidative mechanism. In the present research, the highest activities of antioxidant enzymes and secondary metabolites (polyphenols and flavonoids) contents were recorded in KH 2 PO 4 -treated plants. In this paper, we highlight the first time the ameliorative effect of seed priming with KH 2 PO 4 on P-deficiency tolerance by alleviating oxidative stress in Vicia sativa and Vicia faba.

CONCLUSION
Results showed that seed priming with KH 2 PO 4 , SA, or the exogenous application of SA could enhance P deficiency tolerance in Vicia sativa and Vicia faba without affecting the performance of seeds germination. This mainly results from the enhancement of plant growth, P nutrition, and alleviates oxidative stress in treated plants under P deprivation conditions. Surprisingly, the comparison of KH 2 PO 4 and SA effects on P deficiency tolerance lets us deduce that KH 2 PO 4 aided the plants to overcome P deficiency by supporting a suitable P acquisition, whereas, SA by stimulating the antioxidant system defense to scavenging ROS. Overall, we propose the potential application of seed priming procedure by KH 2 PO 4 and SA for improving plant tolerance to P deficient soils.

ACKNOWLEDGMENTS
This work was supported by the Tunisian Ministry of Higher Education and Scientific Research.