Rhizosphere Acidification Determines Phosphorus Availability in Calcareous Soil and Influences Faba Bean (Vicia faba) Tolerance to P Deficiency

Abstract

Calcareous soils are known for their alkaline pH-promoting insoluble forms of certain nutrients, including phosphorus (P). Rhizosphere acidification is one of the main physiological mechanisms of phosphorus mobilization by plants. However, specific and genotypic differences in response to P deficiency are often observed, giving some genotypes particular tolerance abilities. This genetic potential gives us a new opportunity to colonize unused lands, improve yield in problematic soils, and install sustainable agrosystems. To this end, a potted experiment was conducted on three faba bean genotypes (Seville, SEV; Aguadulce, AGUA; and Tunisian, TUN) cultivated on calcareous soil (CS), as compared to fertile soil (FS). Measurements are made on plant growth, the SPAD index, photosynthesis, P distribution, rhizosphere acidification, and related interrelationships. Calcareous soil induced specific symptoms of P deficiency, reduced P concentration and decreased SPAD index, net photosynthesis, and plant growth. Rhizosphere acidification was significantly stimulated in CS. This activity determines the genotypic differences in response to P deficiency in faba bean. The genotype TUN was more adapted to calcareous-induced P deficiency than AGUA and SEV by increasing acidification activity, decreasing pH by 0.6 units in the rhizosphere, and having higher biomass production, photosynthesis, P remobilization, and P accumulation. The key functional traits (plant growth, chlorophyll biosynthesis, and photosynthesis) are strictly dependent on P availability, which remains in close relationship with the acidification capacity (AC). The tolerant genotype (TUN) expressed a lower stress index (SI) but higher P use efficiency (PUE), H-ATPase activity, and P uptake and translocation to shoots (PT), allowing it to maintain better metabolic functioning. AC, PT, PUE, and SI are among the main traits of P management in calcareous soils that promote resilient crops.

1. Introduction

With the increased demographic growth, climate change, and malnutrition expansion, crop yield and quality have become global concerns. Deficiencies in minerals such as phosphorus (P) have an issue with food availability and malnutrition. In fact, P is one of the key nutrients that are necessary for plant metabolism, growth, and development. Although abundant in the soil, P availability represents a major constraint for agriculture due to P fixation and precipitation [1,2]. Roots can only absorb dissolved phosphates [3], whereas more than 99% of the total P in the soil is bound in organic P forms or is present as insoluble Ca, Fe, and Al phosphates [4]. In highly acidic soils (pH < 5), phosphorus easily forms complexes with oxides and hydroxides of iron and aluminum, while associating with calcium in alkaline soils (pH > 7). Calcareous soils, which represent more than 1/3rd earth's surface [5] and 45% of the cultivated area in Tunisia [6], are one of these problematic lands. The presence of a high bicarbonate concentration and an alkaline pH renders P in an insoluble form [7]. Hagin and Tucker [7] suggested that CaCO₃ concentration is among the significant problems related to agricultural activity in calcareous soils. The CaCO₃ mineral present at high levels in calcareous soil fixes P through mineral precipitation [8,9]. Khan et al. [10] explained the low P solubility in calcareous soils by its fixation either with lime or with clay surfaces and precipitation with calcium, aluminum, or iron, depending on the soil pH. Anter et al. [11] showed that a rapid decrease in P uptake occurs at 8% CaCO₃ in the soil. Even if added as fertilizer, P is fixed before it is used by the plant [12], and plants benefit only from a small part of the added P [13]. Generally, phosphorus is available in the pH range of 6.5 to 7, and its availability decreased gradually with increased pH [12], mainly due to the formation of insoluble calcium phosphate compounds [8]. Inorganic phosphate (Pi) is one of numerous essential nutrients (zinc and iron) [5,14], known by their low availability for plants and crops because of their relative immobilization in agricultural soils [15]. Thus, reported symptoms of improper mineral nutrition in calcareous soils are necrosis, yellowing, and red and brown discoloration with a specific distribution. Reddish discoloration of mature leaves commonly reported in calcareous soils is known as a P deficiency symptom [11,16]. However, P is known among the most important nutrients affecting plant growth and yield production. Playing a key role in various plant metabolic activities and biological pathways [17,18], P is a constituent of nucleic acids and ATP-producing energy, associated with lipids and proteins (phospholipids and phosphoproteins), and implied in carbon metabolism, including signaling and regulation [19]. Thus, P occupies the crossroads of all metabolic reactions in the plant. Hassan et al. [20] explained the low yield of maize plants by their high demand for phosphorus and the limited availability of this nutrient. Other studies demonstrated that phosphorus could improve the stress resistance of crops [21,22]. Phosphorus application effectively improves the mineral nutrition of plants, strengthens membrane stability, and alleviates dehydration and oxidative stress caused by abiotic stress [23,24]. In the vegetative stage, phosphorus improves plant growth [25], whereas, in the reproductive stage, senescent leaves become the primary source of P for metabolic balance [26].

Recent studies have demonstrated that plant species develop several mechanisms allowing them to improve P bioavailability in the rhizosphere, grow, and accomplish their life cycle in low-P soils [27,28,29]. These mechanisms imply the exudation of organic acids and the modification of root architecture and root properties (development of subapical root hairs, formation of cluster roots, etc.) [30,31,32]. Remobilization of P from senesced leaves and regulation of P reserves are also proposed as adaptive strategies [33]. However, these mechanisms alone cannot explain the process by which plants remobilize P. The involvement of very diversified and more complex processes is almost certain. The genotypic variability of responses to this nutritional constraint, previously highlighted in diverse crops [34,35,36,37], supports this hypothesis. On the other hand, to guarantee a sustainable food supply for the growing population, agriculture has witnessed a massive use of chemical fertilizers, including P enrichment, to improve crop yield and quality. Nevertheless, this strategy has adverse economic and ecological impacts. Alternative sustainable agricultural practices require crops with improved P nutrition in the widespread problematic soils. Thus, it becomes more critical to identify genotypes with high efficiency of nutrient remobilization, uptake, and use, particularly in problematic soils such as calcareous ones. This is why our interests have been oriented toward the rhizosphere acidification, which, if stimulated, will contribute to improving the availability of P for root uptake and thus the subsequent metabolic reactions and functions. To this end, three faba bean genotypes were cultivated on calcareous soil (alkaline P-problematic soil), as compared to fertile soil (the control). The genotypic differences allow us to identify the valuable traits of tolerance-promoting P management in calcareous soils. P remobilization and uptake, P translocation, P use efficiency, plant growth, and key metabolic reactions (chlorophyll biosynthesis and photosynthesis) were analyzed, and respectful relationships were established. A special interest was granted to rhizosphere acidification as the promotor of P remobilization and the genotypic differences in response to this mineral constraint.

2. Materials and Methods

2.1. Plant Materials and Experimental Design

Three faba bean genotypes (AGUA, SEV, and TUN) obtained from the Tunisian Ministry of Agriculture and Water Resources were used. The experiment was conducted in potted soil under natural light. Screened and disinfected seeds (2% hypochlorite calcium) were sown individually in 1 kg pots filled with soft and mixed fertile soil (FS) sampled in the region of Gatrana (Sidi Bouzid, 35°9′52.366″ N, 9°40′23.689″ E) or calcareous soil (CS) sampled in the region of Faiedh (Sidi Bouzid, 35°4′38.536″ N, 9°40′32.167″ E). The experiment was conducted in a completely randomized design (soil quality X genotypes) with twenty replicates each. No organic or mineral fertilizers were added. The main soil characteristics are given in

Table 1. Main characteristics of the used soils. FS: Fertile soil sampled in the region of Gatrana (Sidi Bouzid, Tunisia, 35°9′52.366″ N 9°40′23.689″ E), CS: calcareous soil tested in the region of Faiedh (Sidi Bouzid, Tunisia, 35°4′38.536″ N 9°40′32.167″ E).

Parameters	FS	CS
pH	7.03	10.05
Organic Matter (%)	1.33	0.59
Active lime (%)	5.0	11.3
Total carbonates (%)	6.48	28.3
Fe (%)	0.51	0.469
K (%)	1.125	0.663
Mg (%)	0.599	0.661
N (%)	0.71	0.45
C (%)	0.59	0.22
P (%)	0.153	0.212

. Plant irrigation was performed with tap water near the field capacity. At the beginning of flowering, 45 days after germination, non-destructive measurements (SPAD index and gas exchange) were made, then ten plants were harvested and separated into shoots and roots. Roots were soaked in a 0.01 M CaCl₂ solution and washed thoroughly and successively in 3 baths of ultra-pure water to avoid contamination with elements from the soil [38].

2.2. SPAD Index

Measurements on relative chlorophyll content were assessed using a SPAD-502 (Konica Minolta, Japan) before the gas exchange measurements on the median fully expanded leaves. Ten plants for each soil and genotype were used. Values are expressed as SPAD units.

2.3. Gas Exchange

In order to homogenize measurements and obtain significant results, gas exchange parameters were measured on the same fully expanded leaves (of the ten plants per genotype and per soil) using a portable photosynthesis system (CI-340, USA). The induction of photosynthesis was ensured by a saturating light of 1000 μmol m⁻² s⁻¹. The other parameters were maintained constant: sample pCO₂ at 362 mbar, flow rate at 500 μmol s⁻¹, and temperature at 25 °C [39].

2.4. Phosphorus Analysis

Samples of fresh material were dried at 70 °C for 72 h and ground to a fine powder. After extraction in 0.5% HNO₃, the extract was discolored using charcoal and filtered, then a nitro–vanado–molybdic mixture was added. Colorimetric measurements at 436 nm were made according to Fleury and Leclerc [40].

2.5. Rhizosphere Acidification Capacity

Plant acidification capacity (AC) was measured before the final harvest on intact plants removed from fertile and calcareous soils. Roots of ten plants from each genotype and each soil were transferred individually in 200 mL of a continuously aerated solution containing 10 mM KCl + 1 mM CaCl₂, adjusted to pH 6.0 with 0.1 N NaOH [32,41]. pH values were measured every 30 mn and the experiment was repeated twice.

2.6. Analysis Criteria

The following acronyms refer to the criteria used for the several analyses performed:

AC: acidification capacity;

$$\mathrm{AC}=\mathrm{pHFS}-\mathrm{pHCS};$$

(1)

PUE-An: P use efficiency for photosynthesis, calculated as the ratio of net photosynthesis (µmol CO₂ m⁻² s⁻¹) to P concentration in shoot (µg g⁻¹ DW)

$$\mathrm{PUE}-\mathrm{An}=\frac{\mathrm{An}}{\left[\mathrm{Pshoots}\right]};$$

(2)

PUE-DW: P use efficiency for plant growth, calculated as the ratio of plant dry weight (g plant⁻¹) to shoot P concentration (µg g⁻¹ DW)

$$\mathrm{PUE}-\mathrm{DW}=\frac{\mathrm{DW}}{\left[\mathrm{Pshoots}\right]};$$

(3)

QP: the total quantity of P calculated in the plant (µg P plant⁻¹)

$$\mathrm{P}=\left[\left[\mathrm{Pshoots}\right]\times \mathrm{DWshoots}\right]+\left[\left[\mathrm{Proots}\right]\times \mathrm{DWroots}\right];$$

(4)

SI An: stress index related to net photosynthesis

$$\mathrm{SI}-\mathrm{An}=100\times \frac{\left(\mathrm{AnCS}-\mathrm{AnFS}\right)}{\mathrm{AnFS}};$$

(5)

SI-DW: stress index related to plant growth

$$\mathrm{SI}-\mathrm{DW}=100\times \frac{\left(\mathrm{DWCS}-\mathrm{DWFS}\right)}{\mathrm{DWFS}};$$

(6)

SI-spad: stress index related to SPAD index

$$\mathrm{SI}-\mathrm{Spad}=100\times \frac{\left(\mathrm{SpadCS}-\mathrm{SpadFS}\right)}{\mathrm{SpadFS}};$$

(7)

SI-P: stress index related to P uptake

$$\mathrm{SI}-\mathrm{P}=100\times \frac{\left(\mathrm{QPCS}-\mathrm{QPFS}\right)}{\mathrm{QPFS}};$$

(8)

PT: Phosphorus translocation calculated as the ratio of shoot P to plant total P quantities

$$\mathrm{PT}=\frac{\mathrm{QPshoots}}{\mathrm{QPplant}}.$$

(9)

2.7. Statistical Analyses

Data and statistical analyses were performed using the software StatPlus Pro. All data are presented as mean ± standard error. Analysis of variance (ANOVA) was performed to check whether the effects of soil quality (FS and CS) on the respective factor were significant. The significance of differences among treatments was determined by Fisher's least significant difference test (LSD) at 5%. Means were declared significantly different when the difference between any two treatments was more important than the LSD value generated from the ANOVA. They are marked by different letters in the figures.

3. Results

3.1. Phenotyping, Growth, and Photosynthesis

The daily monitoring of plant aspects revealed a slight yellow-reddish discoloration characteristic of P deficiency on mature leaves of SEV five weeks after sowing in calcareous soil. The same symptoms appeared consecutively in AGUA and then TUN. No symptoms of P deficiency were observed in fertile soil (Figure 1).

Measurements made on the SPAD index demonstrated a significant decrease in all genotypes, with some differences. The SPAD index decreased by 37% in SEV, 35% in AGUA, and 21% in TUN genotypes cultivated on calcareous compared to fertile soil (Figure 2). When considered in CS, the TUN genotype accumulated 35% and 27% more chlorophyll than SEV and AGUA, respectively.

The quantification of plant biomass demonstrated that cultivating plants on calcareous soil significantly decreased plant growth in all studied genotypes (Figure 3). Nevertheless, some genotypic differences were observed. Dry weight production decreased by 28%, 17%, and 9%, respectively, in SEV, AGUA, and TUN on calcareous soil, compared to fertile soil. The last genotype produced 34% and 24% more biomass than SEV and AGUA on calcareous soil.

To deeply explore these observed genotypic differences, we calculated the stress index based on the SPAD values (SI-Spad) and plant growth (SI-DW,

Table 2. Stress index related to spad (SI-spad), stress index related to plant growth (SI-DW), stress index related to net photosynthesis (SI-An), and stress index related to accumulated P (SI-P) in three faba bean genotypes cultivated on fertile and calcareous soils. According to Fisher’s Least Significant Difference, within columns, means with the same letter are not significantly different at α = 0.05. Standard errors of means of 10 replicates.

	SEV	AGUA	TUN
SI-spad	−37.5 a ± 2.8	−35.5 a ± 3.2	−21.1 b ± 1.9
SI-DW	−28.3 a ± 1.9	−16.8 b ± 1.3	−6.6 c ± 4.6
SI-An	−37.0 a ± 2.9	−24.9 b ± 2.1	−12.4 c ± 1.1
SI-P	−55.4 a ± 4.1	−42.1 b ± 3.2	−28.3 c ± 1.9

). Obtained results demonstrated that the cultivation of faba bean on calcareous soil subject plants to severe stress, which differs among genotypes. TUN, which maintained better chlorophyll accumulation and plant growth, expressed the lowest stress index; SEV was the most sensitive; and AGUA described an intermediate behavior (Table 2). Compared to TUN and based on the SPAD index, SEV was 1.8 times and AGUA was 1.7 times more stressed on calcareous soil. When calculated based on biomass production, SEV was 4.3 times and AGUA was 2.5 times more stressed on calcareous soil than TUN.

Figure 4, which illustrates the net photosynthesis (An), shows a significant decrease of this key function when faba bean plants are cultivated on calcareous soil, as compared to fertile soil. An decreased by 37%, 25% and 12%, respectively, in SEV, AGUA, and TUN genotypes cultivated on CS, as compared to FS. However, the TUN genotype remained the least affected by calcareous soil. The An was 1.5 times and 1.3 times higher in TUN than SEV and AGUA, respectively.

In parallel to net photosynthesis, the stomatal conductance decreased in calcareous soil compared to fertile soil. This decrease was estimated to be 19%, 27%, and 15%, respectively, in SEV, AGUA, and TUN genotypes (Figure 5a). The evapotranspiration followed the same schema of variation with a decrease of 21%, 25%, and 10%, respectively, in SEV, AGUA, and TUN on calcareous soil, as compared to fertile soil (Figure 5b).

With respect to the same strategy of exploration of the genotypic differences in the response of faba bean to P deficiency, we calculated the stress index based on net photosynthesis (SI-An, Table 2). The results demonstrated that the genotype TUN remains less stressed than SEV (most stressed) and AGUA. On the other hand, SEV was three times and AGU was two times more stressed than TUN.

3.2. Phosphorus Uptake and Use

Plants cultivated on calcareous soil exhibited a severe decrease in P concentration in plant organs compared to those cultivated on fertile soil. The previously observed genotypic differences were also maintained at this level. Compared to fertile soil, P concentration decreased in shoots by 38%, 33%, and 21%, respectively, in SEV, AGUA, and TUN cultivated on calcareous soil (Figure 6a). In roots, this decrease was estimated to be 38%, 20%, and 25%, respectively, in SEV, AGUA, and TUN genotypes (Figure 6b). Calculating the total quantities of P accumulated in the plant shows a significant decrease in all genotypes cultivated on calcareous soil compared to fertile soil (

Table 3. Quantities of phosphorus accumulated in plants (QP, µg Plant⁻¹); P use efficiency for photosynthesis (PUE-An), P use efficiency for plant growth (PUE-DW) and phosphorus translocation (PT, µg) in three faba bean genotypes cultivated on fertile soil (FS) and calcareous soil (CS). According to Fisher’s Least Significant Difference, within columns, means with the same letter are not significantly different at α = 0.05. Standard errors of means of 10 replicates.

	SEV		AGUA		TUN
	FS	CS	FS	CS	FS	CS
QP	2181.96 a ± 121	972.11 f ± 67.7	1714.5 c ± 101.7	1036.2 e ± 89.2	2050.57 b ± 123.9	1470.66 d ± 99.6
PUE-An	6.31 d ± 0.51	6.37 d ± 0.57	6.89 bc ± 0.50	6.77 c ± 0.59	7.08 b ± 0.62	8.36 a ± 0.63
PUE-DW	7.55 f ± 0.53	8.83 e ± 0.66	9.67 d ± 0.79	11.23 b ± 0.95	10.23 c ± 0.84	11.80 a ± 0.94
PT	41 c ± 3.11	41 c ± 3.32	52 b ± 3.67	49 bc ± 4.02	59 a ± 3.98	59 a ± 4.14

). This decrease was estimated to be 55%, 35%, and 28%, respectively, in SEV, AGUA, and TUN genotypes. However, TUN accumulated 42% and 52% more P on calcareous soil than SEV and AGUA, respectively.

P use efficiency (PUE) is another trait that can discriminate among the studied genotypes concerning their interaction with soil quality. For that, we calculated PUE based on plant growth (PUE-DW) and net photosynthesis (PUE-An) in the present study. Obtained results demonstrated an apparent increase in PUE-DW and PUE-An in calcareous soil compared to fertile ones (Table 3). As for the previous results, the Tunisian genotype (TUN) showed the highest values on calcareous soil, as compared to the other ones, with 31% and 23% more photosynthetic efficiency than SEV and AGUA, respectively, and 5% and 34% more growth efficiency than SEV and AGUA, respectively. Otherwise, we calculated P translocation to shoots (PT, Table 3). This parameter reflects not only the P uptake by roots but also the capacity of its allocation to the photosynthetic organs. Table 3 shows that the fraction of shoot P represents 59% of the total plant P in the genotype TUN, 49% in AGUA, and 41% in SEV. Nevertheless, when comparing plants in calcareous soil, the allocated P to shoots was 2.2 times and 1.7 times higher in TUN than SEV and AGUA, respectively.

3.3. Acidification Capacity

Rhizosphere acidification is a daily activity in dicotyledon plants that results from the root membrane H-ATPase functioning and organic acid exudation. This activity is stimulated under certain conditions. In this study, we analyzed this activity in faba bean plants removed from their soil to check its relationship with rhizosphere acidification and P remobilization. Figure 7 shows that plants issued from fertile soil do not show remarkable activity of this enzyme. In contrast, plants taken up from calcareous soil started immediately to decrease the solution’s pH, causing significant rhizosphere acidification. All genotypes reached their maximum acidification capacity (lowest pH value) after 90 mn. The genotype TUN, and to a lesser degree, AGUA, showed the highest activity of rhizosphere acidification, reaching the lowest pH value of 5.42. The calculation of the acidification capacity (AC), expressed as the difference between control pH (fertile soil) and stressed pH (calcareous soil), demonstrated a clear superiority of TUN, which expressed, after 180 mn, AC 2.1 and 1.6 times higher than SEV and AGUA, respectively (numbers in Figure 7).

3.4. Physiological Relationships and Traits Interdependence

To progress in expressing the valuable traits of faba bean response to calcareous-induced P deficiency and explore the previously observed genotypic variability, we established several correlations. Figure 8a, which connects plant growth with P accumulated in the plant, shows no relationship between these two parameters in fertile soil (R² = 0.09). However, a strict positive relationship was established in calcareous soil (R² = 0.95, Figure 8b). The TUN genotype was distinguished by its high biomass production and P accumulation in calcareous soil.

The correlation between the critical metabolic reaction in the plant, photosynthesis (An), and P accumulated in the plant shows the same schema of variation, a strict positive relationship in calcareous soil (R² = 0.95, Figure 9a), and a weak relationship in fertile soil (R² = 0.10, Figure 9b). TUN maintains its superiority by developing a higher capacity of P accumulation and better photosynthetic activity.

Our results showed an important activity of rhizosphere acidification in calcareous soil that depends on genotypes. To investigate the relationship between this activity and P remobilization, we correlated the acidification activity with the quantities of P accumulated by plants in calcareous soil. Figure 10 shows a high, strict, and close negative relationship between these two parameters. Decreasing rhizosphere pH (acidification) was usually associated with P uptake. In calcareous soil, the genotype TUN expressed the highest AC (2.1 and 1.6 times higher than SEV and AGUA, respectively) and P uptake (51% and 33% higher than SEV and AGUA, respectively).

4. Discussion

With climate change, the growing world population, and the increased global consumption of agricultural products, low-input farming systems that seek to minimize the use of off-farm inputs, escape their environmental and coast issues, overcome the low crop efficiency in problematic soils, and improve yield are strongly encouraged. The identification of new tolerant genotypes and related physiological, biochemical, and molecular traits is one of the most promising approaches to achieving this goal.

In the present study, we investigated the morphophysiological behavior of three faba bean genotypes cultivated on calcareous and fertile soils. The main results emerging from this study are the stressful conditions of calcareous soil regarding P availability, the genotypic differences in faba bean behavior, and the identified physiological traits promoting P nutrition. Symptoms of P deficiency appeared, plant biomass was decreased, net photosynthesis was hampered, and phosphorus uptake disturbed in parallel with a significant stimulation of rhizosphere acidification in CS as compared to FS. Regarding all these parameters, the genotype TUN showed great plasticity, allowing it to express higher capacities of tolerance on CS as compared to AGUA and particularly SEV. Accordingly, other authors (Hagin and Tucker [7], Ferhi et al. [42], Wahba et al. [16], Roriz et al. [43], and Vasconcelos and Grusak [44]) demonstrated that calcareous soils represent the main hamper for agricultural production in the world. Anter et al. [11] reported a sudden decrease in P uptake at only 8% CaCO₃. Marschner [5] announced that P and Fe deficiency are the main factors limiting plant growth in calcareous soil. Netzer et al. [19] observed apparent symptoms of P deficiency in poplar through decreased photosynthesis. Other authors explained phosphorus deficiency in the soil by its low content or low availability [45]. Yang et al. [46] demonstrated that P bioavailability is often limited by a number of rhizospheric factors, including soil texture, moisture, organic matter, and pH. In calcareous soils, P interacts with calcium and carbonate to form a complex of calcium carbonate-P that affects P solubility [47]. Weyers et al. [8] reported that calcareous soils contain high Ca and CaCO₃ levels that fix P via adsorption or precipitation. Accordingly, Mortvedt et al. [48] reported that phosphate reacts with Ca and Mg at high pH to form phosphate compounds of limited solubility. P adsorption is regulated by Fe/Al oxides in acidic soils [49] and by CaCO₃ in calcareous soils [30]. Wahba et al. [16] demonstrated that P availability in calcareous soils is usually restricted and reaches its maximum in the pH range of 6.0 to 7.5. López-Bucio et al. [50] reported that P deficiency hampers the activity of meristematic root cells and primary root growth.

Analysis made on the P concentration in used soils showed significant concentrations, even higher in CS than FS. However, having higher P concentrations does not mean that P is a non-limiting factor. In fact, these concentrations represent the total P, which is mainly insoluble and not available to plants. As previously demonstrated, P in the soil is fixed via adsorption or precipitation with calcium/magnesium carbonates and Fe/Al oxides [51,52,53]. Otherwise, P nutrition decreased significantly in all genotypes cultivated in calcareous soil. Nevertheless, TUN remains the least affected. TUN accumulated 1.5 and 1.4 times more P on calcareous soil than SEV and AGUA, respectively. This specific capacity of P uptake can explain the relative tolerance of TUN. In fact, by correlating plant growth and net photosynthesis with plant P content, we revealed a strict and close relationship. TUN accumulated more P, expressed higher photosynthetic activity, and produced more biomass than SEV and AGUA in calcareous soil. In fact, P is an integral component of the photosynthetic metabolism, having a crucial role in ATP reactions which require inorganic phosphate to be associated with ADP [54]. Unrestricted P availability is thus a critical factor in cell metabolism since P levels affect photophosphorylation, rubisco activity, and the Calvin cycle reactions. The impaired photosynthetic activity in the sensitive genotypes SEV and AGUA, which results from low chlorophyll concentration caused by low P availability, is, therefore, the cause of the total biomass reduction in calcareous soil. The calculated stress index based on biomass production (SI-DW), photosynthesis (SI-An) or plant P content (SI-P) discriminated the used genotypes and confirmed these genotypic differences. Thus, we suggest that SI is a useful trait of tolerance, and the response of faba bean to calcareous-induced P deficiency depends closely on its ability for P remobilization and uptake. The preferential allocation of this nutrient to shoots to support chlorophyll production and photosynthesis remains the main promoter of plant growth. This is confirmed by the higher PT calculated in TUN, as compared to the other genotypes (Table 3). Furthermore, the calculated PUE-An and PUE-DW support the genotypic differences previously observed. TUN genotype turned out as the most efficient, expressing PUE-DW 1.3 times more important than SEV and AGUA, and PUE-An 1.1 and 1.3 times more important than SEV and AGUA, respectively, on calcareous soil. PUE is thus another trait of tolerance useful for screening programs. The calculated AC and PT do not escape this rule. TUN expresses the greatest potentialities of AC and PT, as compared to AGUA and SEV in CS. However, the mechanism by which faba bean remobilize P and give a special performance to the tolerant genotype, TUN, remains the main question to be answered. As previously discussed, P exists but in an unavailable form at alkaline pH. Therefore, our hypothesis was that a genotype able to acidify its rhizosphere will certainly have significant capacities for P remobilization. Previous studies demonstrated that soil pH plays a critical role in soil P retention by altering its adsorption capacity, changing the solubility of secondary minerals (Fe, Al, Ca-P), and affecting the organic P mineralization [55,56]. Other authors suggested that plant roots promote H⁺ extrusion to solubilize mineral P either directly through proton release or indirectly through organic acid exudation [57,58]. Thus, the adaptation to P-deficiency and the P acquisition ability vary between plant species and cultivars. The present findings do not escape this hypothesis and confirm the close relationship between soil pH and soil-P solubility, giving us a new trait of tolerance to induced P deficiency in calcareous soils. The revealed tolerant genotype, TUN, expressed the capacity of rhizosphere acidification 2.1 times and 1.6 times more important than that of SEV and AGUA and accumulates 51% and 33% more P than SEV and AGUA, respectively, in calcareous soil. In fact, Figure 10 shows the close relationship between P accumulation in the plant and rhizosphere acidification. Thus, the relative tolerance of TUN can be explained, in addition to the previous particularities, by its high activity of rhizosphere acidification, which creates an adequate environment for the transformation of P from an insoluble to a bioavailable form. Recently, Ma et al. [4] reported that Lupine plants acidified the rhizosphere more intensively than maize when subjected to P deficiency. The pH was 1.5 units lower for lupine than for maize. In fact, good plant growth is very important for photosynthesis and all metabolic functions, which ultimately affect yield. The genotype TUN showed higher biomass production (Figure 3), photosynthesis (Figure 4), and P uptake and translocation to shoots (Table 3) compared to other genotypes. This might be due to improved P remobilization and use efficiency with the significantly improved rhizosphere acidification. These results are in line with Ahmed et al. [59], who reported that P levels enhanced plant height and the dry weight of maize. Atkinson et al. [60] reported that rhizosphere acidification influenced P interaction with other cations in the soil and enhanced anion exchange, which increased P availability. The high decrease in soil pH was recorded in all genotypes cultivated on calcareous soil, with a clear superiority in TUN. The findings of the current study suggested that this reduction might be due to the excessive release of protons (H⁺) due to H-ATPase activity, without neglecting the production of organic acid that might also have contributed to reduced pH.

Although the impact of rhizosphere acidification on the bioavailability of phosphorus in alkaline calcareous soils is not well established, our findings revealed that decreasing soil pH improved the chemical properties of the soil, enhanced P uptake and translocation, and supported chlorophyll biosynthesis, photosynthesis, and plant growth. The key functional traits (plant growth, chlorophyll biosynthesis, and photosynthesis) are strictly dependent on P availability. The latter is demonstrated in relation to the activity of rhizosphere acidification. In addition to P use efficiency for photosynthesis (PUE- An) and plant growth (PUE-DW), AC determines the genotypic differences in the response of faba bean to P deficiency in calcareous soils. A common thread that links the different metabolic reactions involved in the response to calcareous-induced P deficiency was highlighted. Rhizosphere acidification, either through root H-ATPase that release protons or through organic acid exudation, produces a favorable environment for the solubilization of P into a form available for roots. Thus, P uptake and allocation to shoots increased, and all P-dependent metabolic reactions (chlorophyll biosynthesis, photosynthesis, etc.) improved (Figure 11).

5. Conclusions

Calcareous soils, which account for more than 1/3 of land surface, are seriously problematic for crop production because of their high pH and bicarbonate content, which limit the availability of P for plants. The establishment of sustainable agriculture necessarily requires the adoption of an efficient approach based on the valorization of biological resources through the selection of adapted genotypes and the identification of useful traits of tolerance for further screening programs. The present study allowed us to identify the faba bean genotype having effective P management in these soils. The main modulator of this efficiency is the rhizosphere’s acidification capacity (AC). Thus, the tolerance or sensitivity of any genotype or species is primarily determined by the effectiveness of the system (rhizosphere acidification—P remobilization and uptake), which determines the key metabolic reactions (chlorophyll biosynthesis, photosynthesis, plant growth, and associated P-dependent metabolic reactions). The physiological traits investigated in this study, AC, PT, PUE, SI, are useful traits of tolerance that can be used for further programs of tolerant genotype screening.