Introduction

Adzuki beans (Vigna angularis) are one of the most important legume crops in China. Great attention has been paid to the remarkable health benefits and functional components of the species, including starch, fat, protein, and phytochemicals, among other aspects (Shi et al. 2017). Adzuki beans are particularly suited for production in arid and semiarid regions due to their high adaptability to unfavorable environments.

Phosphorus (P) has been reported as one of the most limiting mineral nutrients for the growth of legume crops; further, almost 30% of the arable soils around the world are P-deficient because of P-conversion to unavailable forms, which cannot be easily utilized by plants (Wissuwa 2003; MacDonald et al. 2011). Low P availability in soils directly reduces photosynthetic ability and finally limits plant growth and development (Yan et al. 2006; Thuynsma et al. 2014; Sulieman and Tran 2015). A lower soluble P content in different tissues of legume crops grown under P-deficiency stress contrasts with the high corresponding content in plant tissues properly fertilized (Sulieman and Tran 2015). However, the use of P chemical fertilizers has been demonstrated unsustainable and a serious hazard to human health and to the environment (Li et al. 2011). Therefore, there is a growing need to develop more P-use-efficient (PUE) crops as well as more precise methods to monitor P-status in plant tissues.

Many studies have reported that plant growth regulators (PGRs) play a central role in controlling the development of the root system in response to P-deficiency or to localized low soil-nutrient patches (Casson and Lindsey 2003). Additionally, it is possible that phosphate-solubilizing microorganisms (PSM) utilize soil phosphate reserves in semiarid regions, thereby contributing to the enhancement of crop yields (Khan et al. 2007; Gupta et al. 2011).

Seed priming is a common, effective, low-cost practice for seed quality improvement, aimed to foster biotic/abiotic stress resistance, thus enhancing crop yield (Paparella et al. 2015; Szafrańska et al. 2016). The positive effects of priming are due to specific metabolic changes induced at the physiological level, such as activation of DNA repair and triggering of antioxidant mechanisms (Foyer and Noctor 2005). Previous studies have demonstrated that the application of a biostimulator as a seed primer at pre-sowing improves oxidative stress tolerance in growing seedlings (Szafrańska et al. 2016).

Rare earth elements (REE) are used as micro-fertilizers in Chinese agriculture owing to their ability to improve growth and productivity (Thomas et al. 2014). Indeed, beneficial effects of REE foliar sprays, seed treatments or addition to soil or liquid rooting media have been reported (Tyler 2004; El-Ramady 2010).

Lanthanum (La), a representative REE, reportedly improves plant resistance to environmental stress (Wang et al. 2011a, 2012). The lanthanum cation (La3+) plays an important role in plant growth, particularly by stimulating photosynthesis and increasing chlorophyll content (Goecke et al. 2015; Řezanka et al. 2016). La3+ has been used as a plasma membrane calcium channel antagonist to activate abscisic acid (ABA) signaling (Hagenbeek et al. 2000), and for altering reactive oxygen species (ROS) homeostasis (Wang et al. 2005). ROS are vital signaling components in many biological processes (Mittler et al. 2011) and responses to environmental stimuli (Baxter et al. 2014). Photosynthesis often entails the production of ROS (Foyer and Shigeoka 2011) and is inhibited by P-deficiency stress. Antioxidant enzymes function as a protective mechanism to eliminate ROS by-products and are the first line of defense in response to stress (Balabusta et al. 2016). However, very few studies have examined the physiological effects of La3+ in plants exposed to environmental stress conditions.

P-deficiency may result in altered root elongation in plants. However, a previous study showed that La3+ caused nutrient alterations, disrupted root swelling (Wang et al. 2011b), and seemingly regulated the architecture of the root system (Ruízherrera et al. 2012). Some evidence indicates that root hydraulic conductivity (Lp) influences the supply of certain mineral nutrients, such as nitrogen (N), P, and sulfur (S) (Clarkson et al. 2000). The enhancement of root Lp optimized the delivery of water from the soil to the shoot (Thompson et al. 2007). REEs show a strong affinity for phosphate ions in vitro (Ding et al. 2005). However, current understanding of a role of phosphate in directing La3+ to specific plant responses to P-deficiency is poor.

In this study, we evaluated the hypothesis that La3+ improves legume performance under conditions of P-deficiency. We also aimed to determine whether La3+ decreases ROS accumulation by activating the photosynthetic protection machinery and by modulating the antioxidant defense system. Further, we wished to clarify the relationship between La3+ and specific plant characteristics associated with P-deficiency, such as root acid phosphatase (APase) and root activity.

Materials and methods

Plant material

Vigna angularis c.v. ‘Baohong 8824-17’ plump seeds were selected and sterilized with 3% H2O2 for 25 min and then washed three times with distilled water. These seeds were primed by one of the following treatments: water, 50, 100, 150, or 200 mg L−1 La(NO3)3 /water solutions. They were then placed in closed glass bottles for about 7 h. Eight seeds were sown in each pot (10 cm in diameter) filled with sterilized universal sand in an artificial climate incubator set to 28 °C/18 °C and 12 h/12 h (light/dark regime). After incubation, seeds were air-dried to their initial water content. Seedlings were then allowed to grow until the first pair of true leaves was fully expanded.

P-deficiency treatment

Seedlings were transplanted to a plastic box (40 × 30 × 15 cm), and cotyledons were removed to minimize the effect of the seed P-reserve. After 2 days, treatment difference was set by cultivation in 1/2 Hoagland nutrient solution in the absence or presence of P. In addition, the concentration of P [potassium dihydrogen phosphate (KH2PO4)] was 10 mg L−1 in the control treatment and 0 mg L−1 in the P-deficiency treatment; KCl was added to the P-deficiency treatment to ensure sufficient potassium in the absence of KH2PO4. Thereafter, adzuki bean seedlings were treated with CK1 (seeds were primed with water and seedlings grew in normal Hoagland solution); CK2 (seeds were primed with water and seedlings grew in P-deficiency Hoagland solution); L50 (seeds were primed with 50 mg L−1 of La(NO3)3 and seedlings grew in P-deficiency Hoagland solution); L100 (seeds were primed with 100 mg L−1 of La(NO3)3 and seedlings grew in P-deficiency Hoagland solution); L150 (seeds were primed with 150 mg L−1 of La(NO3)3 and seedlings grew in P-deficiency Hoagland solution); and, lastly, L200 (seeds were primed with 200 mg L−1 of La(NO3)3 and seedlings grew in P-deficiency Hoagland solution). Seedlings from each of three replications were sampled at 20 days after treatment initiation for each treatment.

Morphological characteristics

Total leaf area was measured using a leaf area meter (model 3000A, Li-Cor Inc., Lincoln, NE, USA). Intact plants were then separated into roots and shoots after they were thoroughly washed with distilled water. Plant height was measured. Roots and shoots were then oven-dried, and the dry weights were measured. The ratios of root to shoot were calculated on a dry weight basis. Root length was measured using a root analysis instrument (WinRHIZO, Regent Instrument Inc., Quebec, Canada).

Photosynthetic ability

Net photosynthetic rate (Pn) and transpiration rate (Tr) were measured using an automatic photosynthesis measuring apparatus, Li-6400 (LI-COR Inc., USA). Maximum photochemical efficiency (Fv/Fm) was measured using the OS6-FL Modulated Chlorophyll Fluorometer (PAM 2500; Walz, Germany) after dark adaptation for 25 min. Total chlorophyll content was estimated in fresh leaves according to the methods of Lichtenthaler (1987).

Root-related enzyme assay

Fresh roots from each treatment were homogenized with a mortar and pestle in 0.05 M sodium phosphate buffer (pH 7.5). The homogenate was centrifuged at 10,000 rpm for 20 min; the supernatant was used to analyze enzyme activity of superoxide dismutase (SOD), peroxidase (POD), and MDA (malondialdehyde) content. These protocols were performed at 4 °C. SOD and POD enzyme activities were determined according to the method of Li et al. (2000). MDA content was measured after Shu et al. (2012).

The method of Tabatabai and Bremner (1969), as modified by Hedley et al. (1982), was used to determine plant root APase activity. Root samples (approximately 0.3 g) were treated with 4 mL of extraction buffer (0.2 mol L−1 NaOAc–HAc at pH 5.8, and 1.35 mol L−1 p-nitrophenol phosphate (p-NPP) as the substrate). After incubation at 37 °C for 30 min, the reaction was stopped by adding 1.5 mL of 6 mol L−1 NaOH; The absorbance at 405 nm using spectrophotometer (Beckman 640 D, USA).

Root activity measurement was performed according to the triphenyl tetrazolium chloride (TTC) method (Wasaki et al. 2003). Roots were collected from the adzuki beans. Each sample (0.5 g) was immersed in a solution (10 mL) containing 0.4% TTC and 66 mmol L−1 phosphate buffer (pH 7.0); samples were stored at 37 °C for 3 h, and the reaction was terminated by the addition of sulfuric acid (1 mol L−1). Roots were homogenized using a mortar and pestle in 3–5 mL of ethyl acetate and a small amount of quartz sand, the red solutions were transferred to 10-ml volumetric flasks, and the residues were washed 2 or 3 times with ethyl acetate. Samples were placed in volumetric flasks, ethyl acetate was added, and OD at 485 nm was determined spectrophotometrically.

Root hydraulic conductivity (L p)

Root Lp was determined according to the method of Trubat et al. (2012) with slight modifications. Each shoot was cut off at the base of the root system, leaving 4 cm of mesocotyl. Next, the whole root was placed in a pressure chamber (Soil Moisture Equipment Crop., Santa Barbara, USA), ensuring that the incision was above the seal ring. The pressure in the chamber was raised in steps of 0.1 MPa up to 0.5 MPa. Exuded sap was collected with absorbent cotton and weighed. The root surface area was measured using a root analysis instrument (WinRHIZO) and the root Lp was calculated.

P content and PUE

A total of 0.1 g of dried plant sample (root, stem, and leaf) was placed in a digestive tube, supplemented with 5 mL of H2SO4, and left overnight. Samples were then placed in the digestion furnace and heated to 360 °C. Three drops of 30% H2O2 were added every 30 min during the digestion period until the solution was colorless and clear. Cooled solutions were removed and added to a tube (100 mL) for determination of P content (Bao 2000). Next, 5 mL of diluted test solutions were pipetted into 50-mL volumetric flasks, diluted with water to 30 mL, and supplemented with two drops of a dinitrophenol indicator in 4 mol L−1 NaOH solution, until solutions turned yellow. Then, the solution faded significantly upon addition of 2 mol L−1 H2SO4. Molybdenum antimony reagent (5 mL) was added and distilled water was also added to a final volume of 50 mL; sample solutions thus processed were maintained for 30 min with slight shaking. Finally, OD was determined spectrophotometrically at 880 nm.

The determination of PUE was performed according to previously reported methods (Wang et al. 2014), with modifications. PUE was calculated according to the following equation: PUE = (UL − U0)/F, where UL is the P absorbed by plants treated with La3+, U0 is P absorbed by plants without P application (P-deficiency treatment), and F is the total amount of P applied.

Membership function value method to assess the ability of La to induce a plant response to P-deficiency

P-deficiency resistance involving multiple traits was evaluated by the Membership function value method (MFV), using the membership functions based on the modified theory of fuzzy mathematics (Zadeh 1965; Liu et al. 2015).

According to the phosphorus-deficiency coefficient (PC), the MFVP was calculated using the following the equations:

$$ Uij=\frac{PCij- PCj\min }{PCj\max - PCj\min } Ui=\frac{1}{n}\sum {\displaystyle \begin{array}{l}n\\ {}j=1\end{array}} Ui j $$

where Uij is the membership function value of the trait (j) for the treatment (i) for phosphorus-deficiency resistance; PCjmax and PCjmin are the maximum and minimum values for the phosphorus-deficiency resistance coefficient for the trait (j) of all the treatments, respectively; Ui is the average value of the membership function values of all traits measured for the treatment (i).

Experimental design and statistical analysis

The experiment had a completely randomized design. Analysis of variance (Duncan’s multiple range test) was conducted using SPSS software (Chicago, IL, USA). Results shown are means ± standard errors (SEs) of three replicates for each treatment. p ≤ 0.05 was considered significant.

Results

La3+ treatment alleviated P-deficiency-induced growth inhibition in adzuki bean seedlings

Compared to controls, P-deficiency stress inhibited growth of adzuki bean seedlings, resulting in reductions in plant height (Fig. 1a, b) and leaf area (Fig. 1e, f), as well as a dramatic increase in root length (Fig. 1c, d) and root:shoot ratio (Fig. 1g). However, seed priming with 150 mg L−1 La3+ affected seedling response to P-deficiency, resulting in significant reduction in plant height (20.7%) and leaf area (14.61%), as well as decreased root length (39.21%) and root:shoot ratio (19.76%), compared to the corresponding measurements on seedlings subjected to P-deficiency alone (Fig. 1).

Fig. 1
figure 1

Plants after 20 days under P-deficiency stress. a Phenotype of above ground. b Plant height. c Phenotype of roots. d Root length. e Phenotype of true leaves. f Leaf area. g Root-shoot ratio. CK1, control; CK2, P-deficiency; L50, P-deficiency plus 50 mg L−1 La3+; L100, P-deficiency plus 100 mg L−1 La3+; L150, P-deficiency plus 150 mg L−1 La3+; L200, P-deficiency plus 200 mg L−1 La3+. Data are means ± SE for three biological replicates. Statistical differences (p < 0.05) among La3+ concentrations are indicated by different letters

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La3+ treatment reduced P-deficiency-induced ROS accumulation

We examined the effect of P-deficiency on membrane damage and antioxidant enzyme activity under P-deficiency stress (Fig. 2). P-deficiency significantly increased MDA content by 2.24-fold compared with MDA content in controls. However, La3+ treatment reduced MDA accumulation, and 100 mg L−1 La3+ was associated to a decrease of 45.19% in MDA content, compared to MDA content in plants subjected to the P-deficiency stress treatment alone. Concomitantly, SOD and POD activities differed similarly depending on exogenous La3+ treatment. Namely, SOD and POD activity levels were improved after treatment with La3+ by 77.57% and 18.74% in the 100 mg L−1 La3+ treatment, and by 77.72% and 11.77% in the 150 mg L−1 La3+ treatment, respectively, compared with the corresponding activity levels measured in plants grown under P-deficiency alone.

Fig. 2
figure 2

Malondialdehyde and antioxidant enzyme activities in roots of adzuki bean seedlings grown under P-deficiency. a MDA content. b SOD activity. c POD activity. Data are means ± SE of three replicates. Different letters above bars indicate significant difference at p < 0.05

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La3+ treatment activated root physiological traits associated with P absorption

The effect of seed priming with La3+ on APase activity and root activity was estimated (Fig. 3). APase activity improved, while root activity was weakened P-deficient, compared to control seedlings. APase activity in seedlings initially increased with La3+ concentration but did not increase any further at La3+ concentrations higher than 150 mg L−1. However, the opposite results were recorded for root activity. APase and root activity differed substantially by 72% and 224%, between seeds treated with La3+ at 150 mg L−1 and seeds subjected to P-deficiency stress respectively.

Fig. 3
figure 3

Root activity and acid phosphatase activity in adzuki bean seedlings were measured after a 20-day exposure to P-deficiency. a TTC activity. b Root APase activity. Data are means ± SE for three biological replicates. Statistical differences (P < 0.05) among La3+ concentrations are indicated by different letters

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La3+ treatment reduced P-deficiency-induced photosynthetic damage mechanism

P-deficiency stress significantly decreased Fv/Fm, Pn, Tr, and chlorophyll content compared with controls. La3+ seed priming alleviated this P-deficiency-induced leaf photosynthesis damage. As shown in Fig. 4, several leaf photosynthesis-related symptoms in the third leaves were investigated. Pn decreased after 20 days of P-deficiency, but La3+ significantly alleviated this decrease (Fig. 4a). Similarly, Fv/Fm, Tr, and chlorophyll content also decreased after 20 days in the P-deficiency treatment, but La3+ treatment significantly alleviated these P-deficiency-induced changes (Fig. 4b–d).

Fig. 4
figure 4

Net photosynthesis rate (Pn), transpiration rate (Tr), maximum photochemical efficiency (Fv/Fm), and chlorophyll content in adzuki bean seedlings grown under P-deficiency. a Pn. b Tr. cFv/Fm. d Chlorophyll. Data are means ± SE of three replicates. Different letters above bars indicate significant difference at P < 0.05

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La3+ treatment ameliorated P-deficiency effects on root hydraulic conductivity

P-deficiency stress significantly decreased root Lp of seedlings after 20 days, which was 64.16% of the Lp observed in controls (Fig. 5). La3+ treatment improved root Lp, but for low concentrations of La3+, there was no significant difference in root Lp from that of seedlings subjected to P-deficiency alone. Root Lp increased as La3+ increased to a maximum of 150 mg L−1 but decreased rapidly for La3+ concentrations above 150 mg L−1. Root Lp at 150 mg L−1 La3+ significantly increased by 116.27% with respect to the P-deficiency stress treatment.

Fig. 5
figure 5

Root hydraulic conductivity of adzuki bean seedlings grown under P-deficiency. Data are means ± SE of three replicates. Different letters above bars indicate significant difference at P < 0.05

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La3+ treatment countered P-deficiency-induced reduction in P content and improved PUE

P content in the root, shoot, and leaf were evaluated (Table 1). P-deficiency decreased P content in all plant parts studied. Under P-deficiency—and in the presence of La3+—there was a significant increase in root P content, but no significant differences in P content in other plant parts were observed. Finally, extensive variation in P content was observed after treatment with 150 mg L−1 La3+, i.e., differences of 5.22% (root), 54.09% (stem), and 29.11% (leaf), compared with the corresponding values for the P-deficiency treatment. PUE in the root, stem, and leaf improved by 1.25%, 1.96%, and 2.09% after treatment with 150 mg L−1 La3+, respectively (Fig. 6).

Table 1 Phosphorus content in roots, stems, and leaves of adzuki bean seedlings under P-deficiency
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Fig. 6
figure 6

Phosphorus use efficiency in roots, stems, and leaves of adzuki bean seedlings grown under P-deficiency

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Discussion

Enhanced yield and improved quality of crops and vegetables can be attained by using REE-microfertilizers due to their specific properties (Mcdowell et al. 2015; Ma et al. 2014). La and other REEs have been demonstrated to play a role in alleviating stress-induced oxidative damage in plants caused by a variety of agents, including cadmium (Wang et al. 2012), salt (Liu et al. 2016; Huang and Shan 2018), drought (Zhang et al. 2006), acid rain (Liang et al. 2017), ultraviolet-B radiation (Huang et al. 2013), and others. However, thus far, there had been no reports on La inducing the antioxidant plant response to improve plant resistance to P-deficiency stress, especially in adzuki beans. In addition, here we addressed the relationship between PUE and La-treatment concentration.

Excess ROS accumulation results in lipid peroxidation damage and cell death under various environmental stress conditions (Shi et al. 2013). Hu et al. (2016) suggested that MDA can be used as an indicator of free radical damage to cell membranes. In the present study, root MDA content increased dramatically (Fig. 2) under P-deficiency because of ROS production. However, this adverse effect was reversed by La3+ treatment, a finding which is consistent with observations in other studies, such as those that have shown La treatment to decrease MDA content and to increase the activity of the plant antioxidative defense system under cold stress (Wang et al. 2011a). Thus, it was revealed that La was clearly involved in the regulation of antioxidant systems (D’Aquino et al. 2009). Moreover, the antioxidant defense ability is an important mechanism in plant adaptation to P-deficiency (Chen et al. 2015). Similarly, alterations in the antioxidant activities of SOD, POD, and CAT enzymes are induced by oxidative stress in many plant species upon REE treatment. In this study, both SOD and POD activities decreased under P-deficiency, while La3+ treatment induced the activation of antioxidative enzymes to reduce ROS-induced oxidative stress by P-deficiency. These results are consistent with the findings of Pang et al. (2002) indicating the potential ability of La for the enhancement of scavenging of excess oxygen radicals and, eventually, for improving wheat resistance to lead stress.

The fact that photosynthesis can provide immediate and direct evidence of P-starvation effects on plants was systemically demonstrated by Hammond and White (2008). In turn, our own work confirmed the benefit of La3+ treatment on two aspects (stomatal and non-stomatal limitations) to maintain photosynthesis activity of adzuki bean under P-deficiency (Fig. 4). In the present study, Fv/Fm, Pn, Tr, and chlorophyll content of adzuki bean leaves were significantly reduced under P-deficiency compared to controls. These results were reversed in the presence of the proper concentration of La. Similar results suggested that a low concentration of La3+ could increase the photosynthetic rate by promoting the activation of RuBPcase (Chen and Zhao 2000; Chen et al. 2001). We also found that the highest concentration La3+ tested (200 mg L−1) did not have any significant effect.

Various physiological and biochemical traits are involved in crop PUE (Wang et al. 2010). Roots play a critical role in plant tolerance to nutrient deficiency. Altered root architecture and activation of gene expression related to signal transduction and transcriptional regulation in response to P starvation has been reported (Rubio et al. 2001; Wu et al. 2003; Misson et al. 2005). Richardson et al. (2009) showed that enhancement of root elongation is the most pronounced plant adaptation strategy under low-P conditions. Additionally, we found that root length of adzuki bean seedlings increased significantly under P-deficiency stress (Fig. 1d). However, La3+ treatment could shorten root length. These findings are consistent with previous results indicating that La3+ inhibits primary root growth (Ruízherrera et al. 2012).

Enhanced synthesis and excretion of root APase has been reported in numerous plant species under P-deficiency (Liu et al. 2004; Richardson et al. 2009). Root activity could reflect root absorption, synthesis, oxidation, and reduction ability (Sechenbater 2001; Dresbøll and Kristian 2012). In this study, we found that root activity decreased and APase activity increased under P-deficiency (Fig. 3a, b). However, these effects were alleviated in seedlings treated with 150 mg L−1 La3+.

Modification of root water-uptake capacity is important for avoiding stress-induced growth reduction (Matsuo et al. 2009; Aroca et al. 2012). Moreover, water absorption is required for nutrient absorption by roots; therefore, root hydraulic conductivity (Lp) may be involved in plant adaptation to diverse environments, such as drought, salinity, low temperature, and nutrient availability (Steudle 2000). In this study, Lp decreased significantly under P-deficiency (Fig. 5), in agreement with the reduction in root Lp by N, P, and S deficiencies observed by Clarkson et al. (2000); however, La3+ treatment restored root Lp, thereby increasing P content and PUE in roots, stems, and leaves (Table 1 and Fig. 6). Xu and Wang (2007) showed that P uptake could be regulated by the application of REE-containing fertilizers. Similar results were obtained for the influence of La on nutrient uptake and distribution (Xie et al. 2002).

In summary, when adzuki bean seedlings are pretreated with La3+ they showed greater tolerance and adaptability to subsequent P-deficiency stress. La3+ reduced MDA content by inducing high levels of antioxidant enzyme activities under P-deficiency. Further, our results prove that La3+ improves P absorption in various plant tissues, possibly as a consequence of changes to root system architecture and to the activation of physiological traits associated with responses to P-deficiency, such as APase activity. Root Lp results indicate that La3+ played an important role in regulating root water uptake and in facilitating nutrient transport to the leaves. La3+ treatment effectively alleviated the photosynthetic damage induced by P-deficiency stress. Therefore, clearly our results have widened our understanding of the mechanisms by which La3+ mediates plant tolerance to P-deficiency.

Conclusions

REEs are widely used chemical soil fertilizers throughout China. Our results showed that La3+ as a novel seed priming agent associated with the physiological mechanisms that protect the plant against P-deficiency stress. La3+-primed adzuki bean seeds exhibited significant amelioration of the adverse effects of P-deficiency stress. La3+ priming treatment of the seeds effectively activated SOD and POD protective activities which resulted in prevention of membrane lipid peroxidation, as demonstrated by the low levels of MDA observed. Acting as a protective molecule, La3+ triggered the P-deficiency stress response which induced the photosynthesis protective mechanisms and maintained pigment content. La3+ also improved PUE in roots, stems, and leaves of adzuki bean seedlings by promoting root elongation and modulating activity associated with root responses to P-deficiency. In addition, root hydraulic conductivity was involved in the process. Our results also indicate that the usefulness of the MFV method to compare the relative ability of different concentrations of La3+ to alleviate P-deficiency effects depends on the agronomic trait in question, the photosynthetic parameters, and on the root physiological characters for different treatments (Table 2). The fact that a high dose of La3+ (i.e., 150 mg L−2) actually improved the tolerance and PUE of adzuki bean seedlings under P-deficiency stress.

Table 2 MFVP method to access the difference of different concentration of La under P-deficiency
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