Absorption and Transport of Phosphorus in Nodulated Soybean Plants and Diagnosis of Phosphorus Status Using Xylem Sap Analysis

Abstract

Phosphorus (P) is an essential major element for plants. The absorption and transport of P are important for soybean growth and yield, including nodule growth and N₂ fixation. Through an analysis of xylem sap, we investigated how nodulated soybean plants absorb PO₄ via the roots and transport it to the shoot. The nodulated soybean plants were treated with 0, 50, and 250 μM PO₄ concentrations for 1, 3, 7, and 15 days. The PO₄ concentration in the xylem sap significantly decreased after 1 day of P deprivation, and then it gradually decreased for 15 days. The high-concentration (250 μM PO₄) treatment increased the PO₄ concentrations in the xylem sap at 7- and 15-day timepoints but not at the 1- or 3-day timepoints. The soybean plants were treated with 0, 25, 50, 100, 150, 250, and 500 μM PO₄ for 3 days. The PO₄ absorption rate increased consistently in conjunction with the increase in the PO₄ concentration; however, the PO₄ concentrations in the xylem sap increased only from 0 to 50 μM PO₄ but were constant under higher P concentrations. The soybean plants accumulated extra PO₄ in the roots. The PO₄ concentration in the xylem sap immediately reflected the P deficiency conditions; thus, this index may be used as an indicator for the diagnosis of P deficiency.

1. Introduction

Phosphorus (P) is an essential major element and plays key roles in photosynthesis, respiration, and energy transformation in plants. In addition, P is a component of nucleic acids and membranes and plays a regulatory role in enzymes [1]. P is sometimes a limiting factor in the growth and yield of crops because the available P levels in soils are generally low, and the P mobility in soil is slow [2,3]. Therefore, P fertilizers are applied to sustain modern agriculture; however, the crops use only 15–30% of the applied P fertilizers, and the rest is lost through fixation on the soil or via leaching into groundwater or rivers [4,5]. At natural pH conditions, PO₄ is present as a mixture of HPO₄²⁻ and H₂PO₄⁻ and is predominantly present in the latter form [3]. Plant roots absorb P mainly in the form of H₂PO₄⁻, and plants cannot directly absorb organic-P forms, which are relatively abundant in soils compared with inorganic P [1]. Plant roots depend on high-affinity phosphate transporters (PHTs) to absorb H₂PO₄⁻ in the soil solution, and the uptake of H₂PO₄⁻ is an energy-mediated process driven by a proton-motive force [1].

It has been reported that P is a limiting factor for soybean growth and seed yield, and P deficiency represses nodule formation and nitrogen fixation [6,7,8]. Therefore, it is necessary to understand the P absorption, P accumulation, and P transport processes to obtain optimum growth and high yield of soybean plants with the efficient use of P fertilizers.

Nutrients and water in the soil are absorbed by the epidermis and cortical cells in the plant’s roots and then transported through the endodermis by passing through the symplast to the stele. Then, P is transported to the shoot through the xylem vessels, forced via both transpiration through the stomata of the leaves and root pressure. The xylem sap flow from the cut surface of the stem depends on the root pressure due to the lack of transpiration by the shoot. The xylem sap composition obtained from the stem cut surface may reflect the original composition before cutting; however, the composition or concentration of nutrients may be changed by the shoot removal, especially a long time after cutting, for example, due to the lack of photoassimilate transport from the leaves. In previous studies, xylem sap analysis has been used to evaluate nitrogen fixation activities and N absorption rate in the roots of field-grown soybeans [9,10]. Additionally, Noguchi et al. proposed that the xylem sap method can be used for estimating nutrient availability in soil [11,12].

Although numerous studies have revealed the characteristics of P absorption in the roots and PO₄ transporters [1,3,4,5,13,14,15], the comprehensive processes of absorption and transport of P from roots to shoots in nodulated soybean plants have not yet been fully understood. Li et al. [13] demonstrated, using dual-root systems in which P was supplied to one side of the roots, that P was a priori transported to the nodules in the P-free side from the shoots. The high-affinity P transporter GmPT5 may contribute to the transport of PO₄ from the roots to the nodules [14]. On the other hand, Chen et al. reported that PO₄ in a soil solution is absorbed directly from the nodule surface mediated by GmPT7 [15]. It has also not been investigated how the shoot cutting affects the P uptake and translocation of soybean plants. In this study, the xylem sap compositions of nodulated soybean plants were analyzed to investigate the P uptake by the roots and translocation rate from roots to shoots.

Nutritional disorders, such as nutrient deficiency or excess toxicity, can cause poor plant growth and crop yield [16]. The diagnosis of nutritional disorders is important to obtain optimum growth, yield, and quality of the crops with an adequate supply of nutrients, which can avoid the excess application of fertilizers, minimizing the fertilizer cost and environmental impacts. Usually, the diagnosis of nutritional disorders has been performed using visible symptoms, plant analysis, or soil analysis [16]. Sometimes, a nutrient deficiency or an excess of nutrients causes visible symptoms such as chlorosis, necrosis, or deformations in the leaves and specific toxic symptoms on fruits [17]. Generally, visible symptoms occur after a long-term deficiency or toxicity condition, and consequently, crops cannot recover when the visible symptoms have become evident. The plant analysis usually uses the mineral concentrations in leaves. The chemical analysis of the dried leaves requires grinding them into a powder and using chemical or heat digestion to decompose the organic materials in the plant samples, thus solubilizing the nutrient elements. These processes require a lot of labor and are time-consuming. The plant analysis that uses the squeezed juice of petioles or leaves and xylem sap is more rapid without any drying, grounding, or digestive processes required. Xylem sap from the cut end of stems or juice squeezed from petioles or leaf blades has been used for the diagnosis of nutrition of N, P, or micronutrients [18,19,20,21]. Roppongi [18] analyzed the NO₃⁻ concentrations in the petiole juice of cucumber and proposed the optimum range of NO₃⁻ concentrations. A similar NO₃⁻ diagnosis was reported for eggplants [21]. The xylem sap analysis of P from soybean cut stems indicated that the inorganic P (Pi) concentration in xylem sap following excess P fertilizer application was as much as that from the P-deficient plant without P fertilizer [20]. The concentrations of K, Ca, Mg, P, S, Zn, Fe, Mn, Cu, Mo, and Si rose initially and then fell during the reproductive stage [22]. To estimate the N derived from N₂ fixation in the root nodules and the N absorbed from the roots, a relative ureide method was applied for the field-grown soybean, in which the concentrations of ureides, amides, and NO₃⁻ in the xylem sap were analyzed. The N concentrations of ureides were considered to be the N derived from N₂ fixation, and the sum of N concentration of amides and NO₃⁻ was the N derived from N absorption [9,10]. It is possible that the concentration of PO₄ in the xylem sap can be used as an indicator of current P status.

Generally, the P criteria for optimal growth is in the range of 0.3–0.5% based on leaf dry weight (DW) in the vegetative growth stage and the possibility of toxicity increases at concentrations higher than 1% [2]. Many food legumes are more sensitive to high concentrations of P, such as a concentration of 0.3–0.4% reported in pigeon pea leaves [2]. A reduction in leaf expansion occurs in plants suffering from P deficiency. The chlorophyll concentration in leaves tended to increase under P deficiency, which differed from N deficiency [23]. P starvation sometimes induces an anthocyanin accumulation in the stems and leaves [24]. The root growth is less inhibited under P deficiency than the shoot growth, and, as a result, the shoot/root dry weight ratio decreases [25]. Soybean plants require relatively large amounts of P, especially at the pod-setting stage [26]. The symptoms of P deficiency are not well defined in soybeans; however, it generally causes retarded plant growth, the presence of spindly and small leaves, and sometimes leaves with dark green color [26]. In our previous paper [27], the PO₄ concentration in the culture solution was optimum at 50 and 100 μM, and higher PO₄ concentrations over 150 μM reduced the plant growth and led to yellowing in the lower leaves. Foote and Howell [28] investigated the P tolerance and sensitivity of soybean varieties, and Lincoln plants exhibited decreased growth caused by P toxicity at P levels over 0.72 mM. Gremaud and Harper [29] used a culture solution with 100 µM P.

In this study, we investigated the absorption, transport, and accumulation of PO₄ in nodulated soybean plants cultivated with the original culture solution without adding potassium bicarbonate to maintain pH [27]. In the first experiment of this study, the soybean plants were cultivated either in an N-free solution or in a 5 mM NO₃ solution, and the changes in xylem sap flow rate and the concentrations of major nutrients in the xylem sap after cutting the shoot were measured. We compared the changes in xylem sap composition between N-free plants and NO₃ supply plants because NO₃ in the culture solution is rapidly absorbed and transported to the shoots [30], and a high concentration of NO₃ may affect the xylem sap composition. In the second experiment, the long-term effects of PO₄ treatment deficiency (0 μM), adequate (50 μM), or excess (250 μM) PO₄ in the culture solution were investigated after 1, 3, 7, and 15 days of the P treatments. In the third experiment, soybean plants were grown with seven levels of PO₄ for three days, and the effects of PO₄ concentrations in the culture solution on the absorption, transport, and accumulation of P in each part were investigated. In addition, the concentrations of ureides and amides were measured to monitor the effects on N₂ fixation and transport. Here, we tried to evaluate whether the xylem sap PO₄ analysis can be used for judging the P status of soybean plants for diagnosis.

2. Materials and Methods

2.1. Plant Cultivation

Soybean (Glycine max [L.] Merr., cv. Williams) seeds were sterilized with 70% ethanol for 30 sec and 0.5% sodium hypochlorite solution for 5 min sequentially. Then, the seeds were thoroughly rinsed with tap water and inoculated with a suspension of Bradyrhizobium diazoefficience (strain USDA110, 10⁸ cells/mL) [31]. Plants were cultivated in a biophotochamber (LH-350S; Nippon Medical & Chemical instruments Co. Ltd., Osaka, Japan) under 28 °C-day/18 °C-night temperatures, 55% relative humidity, and under a photon flux density of 228 μmol m⁻² s⁻¹ with a 16 h photoperiod and an 8 h dark period. At 7 days after planting (DAP), a seedling was transplanted into an 800 mL nitrogen-free nutrient solution in a 900 mL glass bottle covered with aluminum foil to shade the root and culture solutions. The composition of the nutrient solution was the same as the original composition in Table 1A in [27]. The pH of the culture solution was adjusted to 6.0 ± 0.2 with 0.1 M NaOH and 0.1 M HCl. The culture solution was continuously aerated by an air pump, changed every 2 or 3 days until starting the P treatments, and changed every day during the P treatment period.

2.2. Changes in the Xylem Sap Flow Rate and Its Chemical Compositions after Shoot Removal

Soybean plants were cultivated with an N-free solution or with 5 mM NO₃⁻ to investigate the appropriate time for xylem sap sampling. At 28 DAP, the basal part of the stem at 5 cm above the roots was cut with a razor blade, and the xylem sap that exudated from the cut surface was collected. The cut surface of the stem was wiped with a paper towel for 5 min after cutting; then, the xylem sap was collected in a 1.5 mL plastic tube with glass wool inside [10]. The xylem sap was collected sequentially from the same plant during 5–35, 35–65, 65–125, 125–185, and 185–245 min treatment periods in different tubes. The weight of xylem sap was measured by subtracting the weight of the tube with glass wool from the weight of the tube after xylem sap collection. The xylem sap was extracted from the glass wool by sucking the liquid out with an automatic pipette and then stored at −80 °C until analysis.

2.3. Effect of Period of PO₄ Treatments on the P Absorption Rate and P Concentration in Xylem Sap

The soybean plants were cultivated with an N-free culture solution containing 50 μM PO₄ until 23 DAP. After 23 DAP, some plants were grown with a 0 μM PO₄ (P0), 50 μM PO₄ (P50), or 250 μM PO₄ (P250) treatment. The 1-day and 3-day treatments were conducted using the plants from 29 to 30 DAP and 27 to 30 DAP, respectively. Other plants were treated from 23 to 30 DAP (7-day treatment) or from 23 to 38 DAP (15-day treatment). The shoots and roots were separately dried in a ventilation dryer at 80 °C until reaching a constant weight. The shoots were separated into leaves and stems, including petioles and buds. The underground parts were separated into the roots and nodules. The dry weight of each part was measured; then, the dry sample was ground into a fine powder.

2.4. Effect of P Concentrations in Culture Solution on the P Absorption Rate and P Concentration in Xylem Sap and Each Part of Soybean Plants

Soybean plants were inoculated with B. japonicum and cultivated with an N-free solution containing 50 mM PO₄ until 27 DAP. At 27 DAP, the plants were treated with seven concentrations of PO₄, 0, 25, 50, 100, 150, 250, and 500 μM by changing the concentrations of K₂HPO₄, referred to as P0, P25, P50, P100, P150, P250, and P500, respectively. The K concentrations were kept constant by decreasing the same concentration of K₂SO₄ in the original solution. The culture solutions were changed at 29 DAP, and the PO₄ absorption was determined through analysis of the P contents in a bottle before and after cultivation. At 30 DAP, the xylem sap was collected for 30 min, and the plants were sampled.

2.5. Chemical Analysis

2.5.1. Measurement of PO₄ Concentration

The PO₄ concentrations in the culture solution and the xylem sap were analyzed by a modified ascorbic acid-molybdenum blue method [32,33]. The reagents were as follows: 0.2 mM potassium antimonyl tartrate; 0.8% (w/v) ammonium molybdate with 2% (v/v) H₂SO₄; and 5% (w/v) ascorbic acid. The procedure was as follows. A 500 μL of 0.2 mM potassium antimonyl tartrate solution was put in a 1.5 mL plastic tube, and 10 μL of sample solution was added. Next, 50 μL of ammonium molybdate solution was added, and the tube was vortexed. Then, 20 μL of the ascorbic solution was added and vortexed. A total of 200 μL of the reaction mixture was put into the well of the microplate and left to stand overnight. The absorbance at 880 nm was read by a microplate reader (SSH-1000; Corona Electric Co., Ltd., Ibaraki, Japan).

Extraction of PO₄ from the plant powder was carried out as follows. The 20 mg DW of plant powder was put into a 5 mL plastic tube and 1 mL pure water was added. The tube was heated in a boiling water bath for 15 min. Next, it was ultrasonicated for 5 min, and the supernatant was separated with a centrifuge at 10,000 rpm for 5 min. The residue was re-extracted twice with 1 mL of water, and the three supernatants were combined and filled up to 3 mL.

The total-P concentration in each part was determined after HNO₃ digestion of plant powder. The 25 mg DW of powder was put into a glass bottle, and 5 mL of concentrated HNO₃ was added and then digested by heating the bottle. The PO₄ concentration in the digested solution was determined by the modified molybdenum blue method, as described above. The concentration of organic-P was calculated by subtracting PO₄ concentration from total P concentration.

2.5.2. Measurement of Cation and Anion Concentrations

The concentrations of anions and cations in xylem sap were determined by ion chromatography (IC-2010; Tosoh Techno System, Inc., Tokyo, Japan) using a cation column (TSKgel superIC-Cation, Tosoh Techno System, Inc.) or anion column (TSKgel superIC-Anion, Tosoh Techno System, Inc.).

2.5.3. Measurement of Amide and Ureide Concentrations

The concentrations of nitrate, glutamine (Gln), asparagine (Asn), allantoin, and al lantoate in the xylem sap were analyzed using capillary electrophoresis (7100; Agilent Technologies, Inc., Santa Clara, CA, USA). A fused silica tube (inner diameter: 50 µm; length: 104 cm) and a commercial buffer solution (α-AFQ109; Ohtsuka Electronics Co., Ltd., Osaka, Japan) were used with an applied voltage of −25 kV. Signal peaks were detected with a signal wavelength of 400 nm and a reference wavelength of 265 nm.

2.6. Statistics

The experiments were conducted with four biological replications. The plants were cultivated using a random arrangement in a growth chamber. Statistical significance using Tukey’s test was determined using the statistical analysis program of Biomedical Statistics, Graduate School of Medicine, Osaka University [34].

3. Results

3.1. Changes in the Xylem Sap Flow Rate and the Concentrations of Nutrients after Shoot Removal

Changes in the xylem sap flow rate and the concentrations of nutrients were measured sequentially after the shoot removal of the nodulated soybean plant either cultivated in an N-free solution or a NO₃ solution containing 5 mM NO₃⁻ to determine the appropriate time of xylem sap collection. The xylem sap flow rate was highest at the initial sampling period from 5 to 35 min after the shoot decapitation, then it decreased to about half and kept constant until 185–245 min in both the N-free solution and NO₃ solution (Figure 1). The xylem sap flow rate during the initial 5–35 min in the plants in the NO₃ solution was 560 μL/h and higher than that in the N-free solution (175 μL/h). This is because the plants cultivated with 5 mM NO₃ grew bigger compared with the plants grown in an N-free culture solution in which plants only depended on nitrogen fixation.

Figure 2 shows the changes in the concentrations of major anions in the xylem sap of the plants cultivated with an N-free solution or NO₃ solution. The PO₄ concentrations in the xylem sap of the plants with an N-free solution are relatively constant at 1.2–1.6 mM during the sampling time. The PO₄ concentration significantly increased in the xylem sap of soybeans cultivated with the NO₃ solution after 125 min of sampling time (Figure 2A). The trend is different from the changes in PO₄ concentrations in the xylem sap of the plants cultivated with an N-free solution. The SO₄ concentration in the xylem sap from the N-free solution and the NO₃ solution show similar trends: the SO₄ concentration increased for an initial 65–125 min and then decreased (Figure 2B). The NO₃ concentration in the xylem sap from the NO₃ solution is 16.5 mM, the highest among the measured cations. The NO₃ concentration remained constant until the 65–125 min period after cutting but decreased thereafter (Figure 2C). NO₃ could not be detected in the xylem sap obtained from the N-free plants. The concentrations of Cl were different between the N-free and NO₃ grown plants. The Cl concentration was the highest at the first sampling of the N-free plants, but it increased gradually in the xylem sap collected from the NO₃ grown plants (Figure 2D).

Figure 3 shows the changes in the concentrations of principal cations in the xylem sap of the plants cultivated with N-free or NO₃ solutions. While the trends were different from the changes in the concentrations of anions (Figure 2), the trends in the concentrations of cations were relatively similar between the N-free and NO₃-grown plants. The K concentrations were the highest among the cations and decreased in line with the time after cutting (Figure 3A). The concentrations of Mg (Figure 3B) and Ca (Figure 3C) increased initially and then decreased, showing the maximum concentration during the 65–125 min period after cutting, irrespective of the presence or absence of NO₃ in the culture solution.

From these experiments, the xylem sap flow rate was highest from 5–35 min and decreased thereafter both in N-free and NO₃ cultivations (Figure 1). The concentrations of SO4, Cl, K, Mg, and Ca changed during sampling time after the shoot removal with N-free grown plants, and the concentrations of PO₄, SO₄, NO₃, Cl, K, Mg, and Ca changed when cultivated with the NO₃ solution. Therefore, we used the xylem sap collected from the initial 5–35 min period after shoot removal in the following experiments.

3.2. Effect of Period of P Treatments in Culture Solution on the P and Nutrient Concentrations in Xylem Sap of Soybean Plants Cultivated with an N-Free Culture Solution

The dry weight of roots, nodules, stems, and leaves are not affected by the P treatments at 1 day, 3 days, and 7 days (Figure 4A). The dry weight of nodules at 15-day treatment is significantly lower in the P0 treatment than in the P50 treatment (Figure 4B). The DW of roots, stems, and leaves tend to also be higher in the P50 and P250 treatments than the P0 treatment, although not statistically significant.

The daily transpiration rates were measured for the 15-day plants during the P treatments (Figure 5). The transpiration rates are higher with the P50 and P250 treatments than those with the P0 treatment.

As shown in Figure 6, only after 1 day of P0 treatment is the concentration of PO₄ in xylem sap significantly lower (2 mM) than those of the control P50 treatment (3.7 mM) and P250 treatment (4.2 mM), suggesting that the P deficiency rapidly reflects the concentration of PO₄ in xylem sap (Figure 6A). At 15 days, the PO₄ concentrations in the xylem sap of P0 treatment further decreased to 0.3 mM. At 1 day and 3 days of treatment, the PO₄ concentrations are not significantly different between the P50 and P250 treatments. However, it is significantly different at the 7-day and 15-day timepoints.

The SO₄ concentrations are almost the same at 1 day and 3 days among the PO₄ treatments, but the P0 treatment at the 7-day and 15-day timepoints are significantly lower than those of the P50 and P250 treatments (Figure 6B).

In relation to the cation concentrations, the K concentrations in the xylem sap in the P0 treatment are significantly lower than those in the P50 and P250 treatments at 7 days and 15 days of PO₄ treatment (Figure 7A). Similar results are observed for the Mg concentration at 7 days (Figure 7B). Different from K and Mg, the Ca concentration is significantly higher in the P250 treatment than those in the P0 and P50 treatments from 1 day to 15 days of treatment (Figure 7C).

The concentrations of Asn (Figure 8A) and Gln (Figure 8B) are not significantly different at 1 day and 3 days of P0, P50, and P250 treatments. However, the Asn concentration in xylem sap significantly decreased at 7 days and 15 days with the P0 treatment compared with the control P50 treatment. The Gln concentration also decreased at 15 days with P0 treatment but not at 7 days of treatment.

The concentrations of allantoin (Figure 8C) and allantoate (Figure 8D) are not affected by the P treatments, including long-term P deficiency (P0) during the 15-day period. These results suggest that the P0 treatment did not bring about a depression in N₂ fixation, N assimilation, and N transport from nodules.

3.3. Effect of P Concentrations in Culture Solution on the P Concentrations in Xylem Sap and Plant Parts of Soybean Plants Cultivated with N-Free Culture Solution

Figure 9 shows the dry weight of each organ of the plants treated with various P concentrations for 3 days (Figure 9A) and the distribution of dry matter (Figure 9B). The average dry weight of the roots (0.54 g), nodules (0.18 g), stems (0.23 g), and leaves (0.87 g) across the seven treatments, and the dry weight in all organs is not significantly different among P treatments for 3 days (Figure 9A). The percentage distribution of the roots (30%), nodules (10%), stems (13%), and leaves (47%) is similar among the P treatments (Figure 9B).

Figure 10A shows the xylem sap flow rate. The average xylem sap flow rate among all P concentrations is 0.21 mL/h. The xylem sap flow rate tends to be lower in P0, P250, and P500 than in P100, although the flow rates are not statistically significant among P treatments. The transpiration rate is also not significantly different among P treatments, where the average is 49 mL/d (Figure 10B). The average transpiration rate is estimated to be 3.06 mL/h, to calculate which, the daily transpiration rate was divided by 16 because transpiration mainly occurs during the daytime for 16 h per day. The xylem sap flow rate (0.21 mL/h) is much slower than the transpiration rate (3.06 mL/h), which accounts for only about 7% of the transpiration rate. This result may be due to shoot removal in which the evapotranspiration from leaves ceased, and the exudation of xylem sap was only dependent on the root pressure.

Figure 11A shows the PO₄ absorption rates estimated from the decrease in PO₄ content in the culture medium from 27 to 29 DAP. The higher the P concentration in the culture medium, the PO₄ absorption rate increases consistently.

The PO₄ concentration in xylem sap is shown in Figure 11B. The PO₄ concentration in the xylem sap of the P0 treatment is 1.28 mM, about half of the P50 treatment (2.58 mM), and therefore significantly lower. However, the xylem sap collected from soybean plants with higher concentrations of PO₄ in the medium P100, P150, P250, and P500 are almost the same as that in P50. Interestingly, the PO₄ absorption is higher in the higher concentration of PO₄ in the culture solution (Figure 11A); meanwhile, the PO₄ concentration in xylem sap reached the maximum of about 2.5–3.0 mM after P50 treatment with 50 μM PO₄ (Figure 11B). The PO₄ concentration in xylem sap may be a usable indicator of P deficiency, but it does not reflect the excess P conditions.

The total P contents in each part of the plants and the distribution are shown in Figure 12. After 3 days of P treatment, the P content in the roots and leaves increased significantly for the P250 and P500 treatments compared with the P0, P25, and P50 treatments; meanwhile, the P content in nodules and stems is not different among all P treatments (Figure 12A). The results indicate that the excess PO₄ absorbed in the roots is mainly accumulated in the roots and leaves but not in the nodules and stems. The total-P distribution among organs (Figure 12B) shows that the percentage distribution of total-P in the roots increases from 37% (P0) to 45% (P250), but that in the leaves decreases from 44% (P0) to 41% (P500). This result indicates that the roots are the primary organ that accumulates extra-P from the culture solution under a high P concentration, and some PO₄ may be transported to the leaves and accumulated there.

The PO₄ and organic-P (total-P minus PO₄) contents in each part of soybean plants after 3 days of P treatments are shown in Figure 13. The organic-P fraction consists of nucleic acids, phospholipids, phosphosugars, phosphoproteins, etc., which are fundamentally essential for maintaining biological components and physiological processes in the cells. In the alternative, low levels of inorganic PO₄ are necessary to maintain energy, such as ATP, or regulatory processes, such as the phosphorylation of enzymes. In every organ examined, the inorganic PO₄ was the predominant P form compared with the organic-P. Even in the P0 treatment, a large amount of PO₄ remained after the 3-day period of P deficiency. The higher the P concentration of the culture solution, the more significant the increase in the PO₄ content of roots and leaves, although the PO₄ content was constant among P treatments in nodules and stems. The content of the organic-P was constant in stems, leaves, and nodules but significantly increased in the roots. These results showed that the extra PO₄ absorbed mainly accumulated in the roots in the form of PO₄ and was supplemented in the leaves. Some extra P absorbed in the roots assimilated into organic forms, but those were not remarkable in the leaves, stems, and nodules. However, after 3 days of P deficiency at P0 treatment, a high accumulation of PO₄ compared with organic-P was observed. The rapid response of PO₄ concentration in the xylem sap in P0 treatment may be related to the PO₄ localization in the roots, where a PO₄ pool at the transport pathway might be separated from the PO₄ storage pool, possibly in the vacuoles.

The concentrations of amides (Asn, Gln) and ureides (allantoin, allantoate) are related to the N₂ fixation activity and N metabolism in nodules and roots. As shown in Figure 14, the concentrations of Asn, Gln, allantoin, and allantoate between P0 and P25 treatments are not significantly different from the P50 control treatment. This suggests that P deficiency for 3 days did not affect the N₂ fixation, N metabolism, and transport. On the other hand, the concentrations of N compounds in P250 and P500 tend to be higher than those in P0 and P25, although not statistically significant.

4. Discussion

4.1. Changes in the Xylem Sap Flow Rate and the Concentration of Nutrients after Shoot Removal of the Plants Cultivated with N-Free or NO₃ Culture Solution

After shoot removal, the xylem sap flow rates and the concentrations of nutrients in xylem sap may be changed mainly due to the lack of photosynthate supply and evapotranspiration. So, we first measured the xylem sap flow rates and concentrations of major cations and anions in the xylem sap of soybean plants cultivated with N-free culture solution and 5 mM NO₃ solution. In the previous report [30], the nodulated or non-nodulated soybean plants were supplied with an N-free solution or a solution with only NO₃, NH₄, urea, or NO₃ + NH₄ for 1 day. In the xylem sap of the nodulated plants with an N-free solution, ureides were the major N metabolites, followed by Asn and Gln, whereas, in the NO₃ treatment, the ureide concentrations in the xylem sap decreased compared with the control plants.

In this experiment, the xylem sap flow rates were highest at the initial 5–35 min period, then decreased but continued until the 185–245 min period in the plants cultivated with an N-free solution and NO₃ solution. The PO₄ concentrations in the xylem sap were relatively constant when the plants were cultivated with an N-free solution. On the other hand, when the plants were grown with a NO₃ solution, the PO₄ concentration increased, while the NO₃ concentrations decreased in line with the sampling period. The increase in PO₄ and Cl concentrations in xylem sap collected from the plants with NO₃ solution might be due to the compensation for the decrease in NO₃ concentration. At any rate, the xylem sap flow rates and the chemical compositions were not constant after the shoot removal, so we used the xylem sap collected during the initial 30 min period from 5 to 35 min after the shoot removal.

Sato et al. [31] reported the concentrations of cations and anions in the xylem sap of the hypernodulation mutant lines and their parent type, cv. Williams, grown in a sandy dune field. The results showed that nitrate was the major anion (1.75 mM: average of all lines and sampling date at 50, 70, 90, 120 DAP), followed by phosphate (1.32 mM), sulfate (0.85 mM), and chloride (0.50 mM). Potassium was the predominant anion (5.72 mM), followed by magnesium (1.82 mM), calcium (1.77 mM), and sodium (0.45 mM). The results obtained here and shown in Figure 2 and Figure 3 are relatively the same as those obtained by Sato et al., while the NO₃ concentration in xylem sap is much higher in this experiment (Figure 2) because 5 mM NO₃⁻ was supplied in the culture solution. Noodén and Mauk [22] also reported the mineral element concentrations in the xylem sap of the nodulated soybean during the reproductive stage cultivated in soil. The ranges of each element were similar to those obtained by Sato et al. [31]; however, the concentration of P decreased from the full-pod extension stage (2.5 mM) to the early leaf yellowing stage (0.4 mM) reported by Noodén and Mauk [22].

4.2. Long-Term Effect of Three Levels of P Treatment on the P and Nutrient Concentrations in Xylem Sap of Soybean Plants

Soybean plants were cultivated with 0 μM PO₄ (P0), 50 μM PO₄ (P50), or 250 μM PO₄ (P250) for 1, 3, 7, and 15 days to investigate the long-term effects of P deficiency, adequate, and excess P conditions.

As shown in Figure 6A, the concentration of PO₄ in the xylem sap with P0 treatment significantly decreased at 1 day compared with the control P50 treatment and further decreased at 15 days. Based on this result, it can be postulated that the PO₄ concentration in xylem sap reflects the PO₄ availability in the culture medium. The PO₄ concentration in the xylem sap may be usable as an indicator for diagnosis of P deficiency. The PO₄ concentrations of P250 were not different from the control P50 treatment at the 1-day and 3-day treatment periods, but those at the 7-day and 15-day treatment periods were significantly higher than the control treatment. The long-term excess conditions of P might increase the P absorption, accumulation, and P transport from roots to the shoots.

It has been established that ureides (allantoate, allantoin) are the principal N compounds with a small fraction of amides (Asn, Gln) from fixed N₂ in the root nodules, while on the other hand, the N absorbed from roots is mainly transported to the shoots in the form of amides and NO₃⁻ with a small number of ureides [30]. However, the effects of P deficiency or P excess on the transport of ureides and amides have not been investigated. The concentrations of amides and ureides in the xylem sap were not significantly different at any period of P treatments (Figure 8). This suggested that P-deficient conditions did not inhibit N₂ fixation in nodules until 15 days of P starvation. This indicated that the P already stored in the plants at the start of the P0 treatment could support the amount of P necessary for nodule growth and N₂ fixation for 15 days at this stage. In addition, P-excess treatment (P250) did not inhibit the N transport in the xylem sap.

4.3. Effects of Seven Levels of P Concentration on the P Concentration in Xylem Sap and in Each Plant Part of Soybean Plants

The effects of seven levels of P concentration in culture solution for 3 days on the PO₄ absorption in the roots and transport in the xylem sap were investigated. During the PO₄ treatments, the dry weight of each part (Figure 9), xylem sap flow rate (Figure 10A), and transpiration rate (Figure 10B) were not significantly affected by PO₄ levels. The PO₄ absorption rate was increased consistently with increasing PO₄ concentrations in the medium (Figure 11A), while the PO₄ concentrations in the xylem sap increased from P0 to P50, and those reached a constant level among the P100, P150, P250, and P500 environments (Figure 11B). These results suggested that excess PO₄ was absorbed in the roots when the PO₄ concentration in the solution was high, but the bulk of them was not readily transported to the shoot through the xylem. Instead, the extra PO₄ might be accumulated in the roots, possibly in the vacuoles of root cells [2]. With an adequate phosphorus supply to the plants, 85–95% of the total PO₄ was reported to be located in the vacuoles [2].

PO₄ is absorbed into root cells mediated by a high-affinity phosphate transporter (PHT1) located in the plasma membrane of root epidermal cells [35,36]. Shin et al. [37] demonstrated that Arabidopsis: PHT1;1 and PHT1;4 play a major role in P acquisition from both low (2 μM) and high (500 μM) PO₄ environments. Members of the phosphate 1 (PHO1) transporter families were found to mediate root-to-shoot translocation by loading PO₄ into xylem vessels [37,38]. Pratt et al. [39] reported that the cytosolic Pi concentration is very low (60–80 μM) and that it dropped very rapidly following the onset of Pi starvation by in vivo ³¹P-NMR analysis. The vacuole is a main storage pool of PO₄, and vacuolar phosphate transporters (VPTs) facilitate Pi transport across the tonoplast [35,39]. However, the Pi efflux from the vacuole was insufficient to compensate for the absence of an external PO₄ supply [39]. PHT1;1 is a key determinant of phosphorus acquisition in Arabidopsis. Plant vacuoles serve as the primary intracellular compartments for inorganic phosphate (Pi) storage. The passage of Pi across vacuolar membranes plays a critical role in buffering the cytoplasmic Pi level against fluctuations in external Pi and metabolic activities [38]. Liu et al. demonstrated that PHT5 functions as vacuolar Pi transporters [38], and it has further been shown that there are at least 14 PTH1 family genes in soybean [14,40].

Figure 12A shows the total-P contents in each part of soybean plants with seven levels of PO₄ concentrations. The total-P contents increased mainly in the roots with increasing PO₄ concentrations. In addition, total P contents in the leaves increased in line with PO₄ concentrations. Figure 13 shows the PO₄ and organic-P contents separately in each part of soybean plants with various PO₄ treatments. After 3 days of P0 treatment, the PO₄ accounted for a higher portion of the total P in all organs. This result suggests that the plants before the PO₄ treatments at 27 DAP already stored a high accumulation of PO₄ in each part. The organic-P content in the roots (Figure 13A) only increased with the increasing PO₄ concentrations in the culture solution. This suggests that some PO₄ was assimilated into organic forms of P in the roots. However, the organic-P content in the leaves, stems, and nodules did not significantly change with increasing PO₄ concentrations in the culture solution.

The concentrations of Asn, Gln, allantoin, and allantoate in the xylem sap were not significantly affected by PO₄ concentrations in the culture solution (Figure 14). This result supports the former speculation that the soybean plants with P0 treatment did not suffer physiologically from P deficiency because the PO₄ stored before the P treatments served as sufficient P to support plant growth and metabolism for 3 days.

4.4. Diagnosis of P in Soybean Plants by Xylem Sap Analysis

Based on this experiment using young nodulated soybean plants, the PO₄ concentration in the xylem may be used as an indicator of P deficiency because a lack of PO₄ for only one day induced a decrease in PO₄ concentration in the xylem sap (Figure 6A); additionally, the PO₄ concentration decreased with the treatment period until 15 days (Figure 6A). Although soybean plants accumulate PO₄ in all organs, when a high concentration of PO₄ was supplied, it was mainly stored in the roots and supplemented in the leaves (Figure 13). Concerning the high concentrations of PO₄ in the culture solution, the PO₄ concentration in the xylem sap did not respond to PO₄ concentrations in the solution over 100 μM PO₄ at 3 days of treatment (Figure 11). However, long-term P treatment increased the PO₄ concentration in xylem sap at 7 and 15 days (Figure 6A). Therefore, the PO₄ analysis in xylem sap can be applicable in excess or toxic levels of PO₄. In practical agriculture, excess P toxicity does not occur because PO₄ concentrations in the soil solution are lower than 11 μM [3]. HPO₄⁻ is readily adsorbed in the soil particles and is taken up by soil microbes. In the case of the solution culture, excess P may occur, so diagnosis using the xylem sap may be beneficial.

Concerning the diagnosis of nutrients using xylem sap, there are several advantages compared with plant analysis using chemical digestion and extraction. First, xylem sap can be easily obtained from many crops cultivated in the field. Second, xylem sap is generally transparent and does not disturb colorimetric analysis. Real-time in situ analysis may be applicable when a portable analyzer can be used for determining the nutrient concentrations. Noguchi et al. [11,12] reported on the mineral concentrations in the xylem sap of loofah (Luffa cylindrica Roem), and they concluded that the nutrient concentrations in the xylem sap were proportional to those of the nutrients in the culture solution. Furthermore, the nutrient concentrations in the xylem sap were proportional to those of the nutrient content accumulated in shoots. Xylem sap analysis has been applied for investigating various compounds such as nutrients and microelements [41,42], phytohormones [43], or signal compounds for plant–microbe interactions [44]. Further experiments are necessary to establish the diagnosis of P by xylem sap, especially to ascertain the deficient, adequate, and excess range of PO₄ in field-grown soybeans.

5. Conclusions

The flow rate and the concentration of anions and cations in the xylem sap collected from the basal cut stem of nodulated soybean plants cultivated with N-free solution or 5 mM NO₃ solution changed gradually; therefore, we used the xylem sap obtained from the initial 5–35 min period for further experiments. The soybean plants were cultivated with deficient (0 μM PO₄), adequate (50 μM PO₄), or excess PO₄ (250 μM PO₄) for 1, 3, 7, and 15 days, and the concentrations of PO₄ in the xylem sap was significantly lower in the P-deficient plants than in the control adequate plants after just 1 day. Therefore, the PO₄ concentration in xylem sap can be used for the diagnosis of P deficiency. However, the excess P conditions did not change the PO₄ concentration after 1 or 3 days of treatment, but it did become significantly higher after 7 and 15 days of treatment. The PO₄ absorption rates and the concentrations of PO₄ in the xylem sap were compared with those in plants cultivated with seven levels of PO₄ concentration from 0 to 500 μM PO_4. The PO₄ absorption rate increased with the increase in the concentration of PO₄, while the concentration of PO₄ in the xylem sap increased only from 0 to 50 μM PO₄, and it reached a constant over 100 μM PO₄. The nodulated soybean plants cultivated with the control solution containing 50 μM PO₄ accumulated relatively large amounts of PO₄ compared with organic-P in the roots, nodules, stems, and leaves. Under excess PO₄ conditions, the soybeans accumulated the extra PO₄ mainly in the roots supplemented with leaves for 3 days. Based on the results, the diagnosis of P deficiency through xylem sap analysis can be applicable when more data on PO₄ concentrations among various P concentrations, plant stages, varieties, and environmental conditions, and the range of adequate concentrations of PO₄ in xylem sap are accumulated.