High External K+ Concentrations Impair Pi Nutrition, Induce the Phosphate Starvation Response, and Reduce Arsenic Toxicity in Arabidopsis Plants

Abstract

Potassium (K⁺) and phosphorous (Pi) are two of the most important nutrients required by plants and there is an interest in studying how they are acquired. Most studies have focused on the characterization of the mechanisms involved in K⁺ and Pi uptake and their distribution within the plants, as well as the regulatory mechanisms involved. Evidence is emerging which points to interactions in the nutrition of different nutrients and to the existence of crosstalk in the signaling cascades regulating their acquisition. However, the interaction between K⁺ and Pi has been scarcely studied. Here we show that high concentrations of K⁺ in the external solution inhibit Pi uptake and impair Pi nutrition in Arabidopsis plants, resulting in the induction of phosphate starvation response (PSR) and the upregulation of genes encoding root phosphate uptake systems. The high K⁺-induced PSR depends on the PHR1 and PHL1 transcription factors that are key pieces of Pi signaling in Arabidopsis. Importantly, high K⁺ reduces arsenic accumulation in plants and its toxic effects. The results presented may help to design strategies to reduce Pi deficiency as well as the accumulation of arsenic in crops.

Keywords: arsenic; phosphate starvation response; phosphorous; potassium; uptake.

Figure 1

Shoot and root dry weights of plants exposed to 1.4 KCl, 10 mM KCl, 10 mM NaCl, and a concentrated nutrient solution. Plants of WT (dark grey bars) and phr1phl1 mutant (light grey bars) were grown for 33 days in 1/5 Hoagland control solution and then for 7 days in the presence of 1.4 mM KCl, 10 mM KCl, 10 mM NaCl, and a nutrient concentrated solution (3/4 Hoagland). Plants were separated in shoot and roots, dried at 65 °C, and the dry weights were determined. Shown are average values of shoot (A) and roots (B) dry weights of three repetitions and errors bars denoted standard error. ANOVA was used to study the effects of the treatment (T), the genotype (G), and their interaction (TxG), resulting in: T = *, G = *** and TxG = ***. Bars with different letters within lines denote significant differences according to Tukey test (uppercase letters for WT and lowercase letters for phr1phl1 line). Differences between genotypes within the same treatment were analyzed according to ANOVA. ***, **, * indicate significant differences at p <0.001, 0.01 and 0.05 respectively and n.s. indicates non-significant at p >0.05. (C) Picture of plants exposed to the described treatments.

Figure 2

Shoot and root P concentrations of plants exposed to 1.4 mM KCl, 10 mM KCl, 10 mM NaCl, and a concentrated nutrient solution. Plants of WT (dark grey bars) and phr1phl1 mutant (light grey bars) were grown as described in Figure 1. Collected shoots and roots were dried, acid digested, and their Pi concentrations determined by Inductively Coupled Plasma (ICP) spectrometry analysis. Shown are average shoot (A) and root (B) Pi concentrations of three repetitions and errors bars denoted standard error. ANOVA was used to study the effects of the treatment (T), the genotype (G), and their interaction (TxG) resulting in: (A) T = ***, G = *** and TxG = ***. Bars with different letters within lines denote significant differences according to Tukey test (uppercase letters for WT and lowercase letters for phr1phl1 line). Differences between genotypes within the same treatment were analyzed according to ANOVA. ***, **, * indicate significant differences at p < 0.001, 0.01 and 0.05 respectively. (B) T = n.s, G = n.s. and TxG = n.s. n.s indicates non-significant at p > 0.05.

Figure 3

Shoot and root Pi concentrations of plants exposed to different K⁺ and Pi concentrations. Plants of WT and phr1phl1 mutant were grown for 33 days in a control 1/5 Hoagland solution and the transferred for 7 days to solutions with 0, 0.05, 0.2, or 1 mM Pi and 0.1, 1.4, 10, and 20 mM KCl. After these treatments WT shoots (A) and roots (C) and phr1phl1 shoots (B) and roots (D) were collected, dried, acid digested and their P concentrations determined by ICP spectrometry analysis. Shown are average P concentrations of three repetitions and errors bars denoted standard error. ANOVA was used to study the effects of the Pi treatment (P), the K treatment (K), and their interaction (PxK), resulting in: (A) P = ***, K =*** and PxK = *. Bars with different letters within each treatment denote significant differences according to Tukey test (uppercase letters for P treatments and lowercase letters for K treatments). Differences between K treatments within each P treatment were analyzed according to ANOVA. *** and * indicate significant differences at p < 0.001 and 0.05 respectively. n.s. indicates non-significant at p > 0.05. For (B), (C) and (D) ANOVA and Tukey tests results are indicated below the corresponding panel.

Figure 4

Pi fluxes in Pi-sufficient and Pi-starved plants from a 30 µM external Pi in the absence or the presence of 1.4 mM KCl, 10 mM KCl or NaCl. Plants of WT (dark grey bars) and phr1phl1 mutant (light grey bars) were grown for 33 days in a control 1/5 Hoagland solution containing 0.2 mM Pi and then transferred for 7 days to the same solution (+Pi) or a solution without Pi (−Pi). Then, plants were incubated for 8 h in nutrient solution with 30 µM Pi in the absence or in the presence of 10 mM KCl or 10 mM NaCl. Samples of this external solution were taken at different time points to determine their Pi concentration. Flux rates of Pi were calculated from the variation in external Pi per h and per g of root dry weight. Shown are average Pi fluxes of three repetitions and errors bars denoted standard error. ANOVA was used to study the effects of the treatment (T), the genotype (G), and their interaction (TxG) resulting in: (A) T = ***, G = *** and TxG = *. Bars with different letters within lines denote significant differences according to Tukey test (uppercase letters for WT and lowercase letters for phr1phl1 line). Differences between genotypes within the same treatment were analyzed according to ANOVA., **, * indicate significant differences at p < 0.01 and 0.05 respectively, n.s. indicates non-significant at p > 0.05.

Figure 5

Effects of external K⁺ and Na⁺ on the expression of the AtIPS1, AtPHT1.4, AtPHT1.8, AtPHO2_5, AtPHR,1 and AtPHL1 genes. Plants of WT (dark grey bars) and phr1phl1 mutant (light grey bars) were grown for 33 days in a control 1/5 Hoagland solution and then transferred for 7 days to a solution containing 0.05 mM Pi in the presence of 0.1, 1.4, 20 mM KCl, or 0.1 mM KCl + 20 mM NaCl. Then roots were harvested and their total RNA extracted to synthesize cDNA that was used for real time PCR determinations of gene expression by the ∆∆Ct method. (A) Expression of AtIPS1 in WT plants, (B) expression of AtIPS1 in phr1phl1 plants, (C) expression of AtPHT1;4 in WT plants, (D) expression of AtPHT1;4 in phr1phl1 plants, (E) expression of AtPHT1;8 in WT plants, (F) expression of AtPHT1;8 in phr1phl1 plants, (G) expression of AtIPHO2_5 in WT plants and (H) expression of AtIPHO2_5 in phr1phl1 plants. Shown are the averages of three repetitions for the Fold-Change of gene expression with respect to the calibrator sample corresponding to the 0.1 mM KCl treatment.

Figure 6

Effects of external K⁺ and Na⁺ on the expression of the AtPHR1 (A) and AtPHL1 (B) genes. Plants of WT were grown and processed as described in Figure 5. Then roots were harvested and their total RNA extracted to synthesize cDNA that was used for real time PCR determinations of gene expression by the ∆∆Ct method. Shown are the averages of three repetitions for the Fold-Change of gene expression with respect to the calibrator sample corresponding to the 0.1 mM KCl treatment.

Figure 7

As(V) fluxes and shoots and roots dry weights of plants exposed to different concentrations of As(V) and K⁺. Plants of WT (dark grey bars) and phr1phl1 mutant (light grey bars) were grown for 33 days in a control 1/5 Hoagland solution containing 0.05 mM Pi and then transferred for 1 day to solutions containing 0.05 mM Pi with 0.05 or 0.1 mM As(V) in the presence of 1.4 or 20 mM K⁺. Then shoots and roots were harvested, dried at 65 °C, acid digested, and their arsenic concentrations determined by ICP spectrometry. The arsenic fluxes (A) were calculated from the arsenic accumulation in the plant per day and gram of root dry weight. The average shoot (B) and root (C) dry weights of these plants are shown. Shown are the averages of three repetitions and error bars denote standard error. ANOVA was used to study the effects of the treatment (T), the genotype (G) and their interaction (TxG), resulting in: (A) T = ***, G = * and TxG = *. Bars with different letters within lines denote significant differences according to Tukey test (uppercase letters for WT and lowercase letters for phr1phl1 line). Differences between genotypes within the same treatment were analyzed according to ANOVA. * indicate significant differences at p < 0.05 and n.s. indicates non-significant at p > 0.05. (B,C). T= ***, G = *** and TxG = ***. Bars with different letters within lines denote significant differences according to Tukey test (uppercase letters for WT and lowercase letters for phr1phl1 line). Differences between genotypes within the same treatment were analyzed according to ANOVA. ***, **, * indicate significant differences at p < 0.001, 0.01 and 0.05 respectively and n.s. indicates non-significant at p > 0.05.

Figure 8

Induction of Phosphate Starvation Respones (PSR) by high external K⁺. The figure shows a simplified proposed model for the mechanism involved in the induction of the PSR by high external K⁺. Shown are key elements of the PSR that have been characterized in the present study. (A) Under low K⁺, a non-depolarized membrane potential drives the uptake of H₂PO4⁻ through the PHT symporter, the cell does not detect Pi deficient, the PHR1 and PHL1 transcription factors are not activated, the transcription of Phosphate Transporters (PHT) is not induced, and the PHO2 conjugase also reduces their activity. (B) In the presence of high external K⁺ (1, red upward arrow), K⁺ uptake depolarizes the plasma membrane potential (2, red arrow), reducing the driving force for H₂PO4⁻ and its accumulation in the cell (3). This partial Pi deficiency activates (green arrow) the PHR1 and PHL1 transcription factors (4) that induce the transcription of the PHT genes encoding Pi uptake systems (5, green arrow) and of the non-coding RNA IPS1 (6, green arrow) that indirectly reduces the accumulation of the PHO2 transcript (7), releasing the negative regulation of PHO2 on PHT transporters. The PHT and IPS1 genes are represented by red wide arrows when not induced and by green wide arrows when induced.