SPX1 is a phosphate-dependent inhibitor of Phosphate Starvation Response 1 in Arabidopsis

Abstract

To cope with growth in low-phosphate (Pi) soils, plants have evolved adaptive responses that involve both developmental and metabolic changes. Phosphate Starvation Response 1 (PHR1) and related transcription factors play a central role in the control of Pi starvation responses (PSRs). How Pi levels control PHR1 activity, and thus PSRs, remains to be elucidated. Here, we identify a direct Pi-dependent inhibitor of PHR1 in Arabidopsis, SPX1, a nuclear protein that shares the SPX domain with yeast Pi sensors and with several Pi starvation signaling proteins from plants. Double mutation of SPX1 and of a related gene, SPX2, resulted in molecular and physiological changes indicative of increased PHR1 activity in plants grown in Pi-sufficient conditions or after Pi refeeding of Pi-starved plants but had only a limited effect on PHR1 activity in Pi-starved plants. These data indicate that SPX1 and SPX2 have a cellular Pi-dependent inhibitory effect on PHR1. Coimmunoprecipitation assays showed that the SPX1/PHR1 interaction in planta is highly Pi-dependent. DNA-binding and pull-down assays with bacterially expressed, affinity-purified tagged SPX1 and ΔPHR1 proteins showed that SPX1 is a competitive inhibitor of PHR1 binding to its recognition sequence, and that its efficiency is highly dependent on the presence of Pi or phosphite, a nonmetabolizable Pi analog that can repress PSRs. The relative strength of the SPX1/PHR1 interaction is thus directly influenced by Pi, providing a link between Pi perception and signaling.

Keywords: phosphate sensor; phosphate starvation signaling.

Fig. 1.

SPX1 interacts with PHR1 in planta. (A) Co-IP of GFP-SPX1 and HA-PHR1. N. benthamiana leaves agroinfiltrated with HA-PHR1 and GFP-SPX1 or GFP-expressing constructs were treated with formaldehyde after harvest; protein extracts were immunoprecipitated with anti-HA antibody and detected in Western blots with anti-HA and anti-GFP antibodies. (B) Analysis of SPX1 and PHR1 interaction by BiFC. Confocal images of N. benthamiana epidermal cells expressing different construct combinations as indicated are shown. The interaction between SPX1 and PHR1 in the nucleus leads to reconstitution of YFP fluorescence in the nucleus of cells that coexpress the YFP^N-SPX1 and YFP^C-PHR1 constructs. (Scale bar: 10 μm.)

Fig. 2.

Physiological and molecular effects of altering SPX1 and SPX2 activity, and the influence of the Pi growth regimen. (A) Pi levels in WT, spx1 and spx2 single-mutant plants, spx1spx2 double-mutant plants, and two independent transgenic lines overexpressing GFP-SPX1 (OxSPX1-1, OxSPX1-2), all grown in four Pi regimens (2,000, 100, and 30 μM, and −Pi) for 10 d. Data show mean ± SD (n = 3). Shared or different letters above bars indicate nonsignificant and significant differences between groups (P < 0.05) according to Student t tests. (B) Diagram showing transcriptomic analysis of the effect of Pi growth conditions on gene expression in WT and spx1spx2 plants grown for 8 d in +Pi, in −Pi, or after brief Pi refeeding (4 h). The total number of genes whose expression is induced or repressed by Pi starvation in WT plants or is higher (Refeeding > −Pi) or lower (Refeeding < −Pi) in Pi-refed vs. Pi-starved WT plants is shown above bars (2× cutoff; false discovery rate is ≤0.05). The number of genes whose expression is higher [mutation (mut) > WT] or lower (mut < WT) in spx1spx2 plants than in WT plants in each growth condition is also shown. The percentage of Pi starvation-responsive genes (−Pi-induced and −Pi-repressed) is indicated, as well as the percentage of PHR1 direct targets [as described by Bustos et al. (9)]. Three biological replicates were analyzed.

Fig. 3.

Cellular Pi-dependent interaction between SPX1 and PHR1 in planta. (A) Co-IP assay of the in planta interaction between GFP-SPX1 and HA-PHR1 in plants grown in +Pi (2 mM) and −Pi conditions. Arabidopsis plants constitutively expressing GFP-SPX1 and HA-PHR1 were grown for 8 d in +Pi or −Pi conditions and prefixed with formaldehyde after harvest to preserve the in planta protein interaction status (44). Protein extracts were immunoprecipitated with anti-HA and detected by Western blotting using anti-GFP antibody. (B) Confocal microscopy images showing that GFP-SPX1 is located in the nucleus, irrespective of the Pi growth regimen of the plant. (Scale bar: 50 μm.)

Fig. 4.

Cellular Pi-dependent interaction between PHR1 and its targets in planta. ChIP and promoter PCR amplification analysis of PHR1 targets in plants grown in +Pi (2 mM), in −Pi, and after Pi refeeding (Ref.). Control Columbia (Col) and transgenic PHRI promoter (PHR1_pro)::PHR1-MYC plants were used in the experiment, in which three PHR1 targets (SPX1, IPS1, and PHT1) and one control [ACT8 (Act)] were analyzed by quantitative PCR. Recovery of target by co-IP with anti-MYC antibody was compared with recovery of a nonbound control (Act) in the same immunoprecipitation. The Pi levels in plants used in the experiment are shown (Upper Right). Data show mean ± SD (n = 2). Shared or different letters above bars indicate nonsignificant and significant differences between groups (P < 0.05), respectively, according to Student t tests. FW, fresh weight.

Fig. 5.

Direct Pi effect on the SPX1/PHR1 interaction. (A) EMSA of the interaction between MBP-ΔPHR1 and P1BS, showing Pi-dependent inhibition of the MBP-ΔPHR1/P1BS interaction by GST-SPX1. The experiment was performed with 0.1 pmol of 4× P1BS; 0.3 pmol of MBP-ΔPHR1; and 0, 0.6, 1.2, 2.5, and 5 pmol of GST-SPX1. (B) Pull-down assays showing that the MBP-ΔPHR1/GST-SPX1 interaction is displaced by P1BS only when Pi is lacking in the incubation buffer. The experiment was performed with 1.5 pmol of MBP-ΔPHR1 or MBP; 12.5 pmol of GST-SPX1; and 0, 0.2, 0.5, 1.25, or 3 pmol of 4× P1BS probe. (C) Pull-down assays showing that only Phi can replace the Pi effect on the SPX1/PHR1 interaction. All reactions included fixed amounts of MBP-ΔPHR1, GST-SPX1, and P1BS (1.5, 12.5, and 3 pmol, respectively). The control (Ct) reaction contained 50 mM NaCl in pull-down buffer; in other cases, 45 mM NaCl was replaced by 15 mM NaH₂PO₄ (+Pi), 15 mM NaH₂PO₃ (+Phi), 45 mM NaNO₃ (+N), and 22.5 mM Na₂SO₄ (+S). Proteins were pulled down with dextrin Sepharose resin and detected in immunoblotting with anti-GST antibody. The tagged ΔPHR1 and SPX1 proteins used in these experiments were bacterially expressed and affinity-purified.

Fig. 6.

Model for the negative regulatory loop between SPX1 and PHR1, and its Pi dependence. SPX1 is a target of PHR1. In the presence of Pi, SPX1 displays high binding affinity to and sequesters PHR1; thus, binding of PHR1 to its PSI targets via P1BS is inhibited, and their transcription, including that of SPX1, is just basal. In the absence of Pi, the affinity of the SPX1/PHR1 interaction is reduced and PHR1 interacts with its targets, resulting in their transcriptional induction. As a consequence, in −Pi-grown plants, there is increased SPX1 expression and protein accumulation, although these plants lack inhibitory activity; however, high SPX1 protein levels allow rapid shutdown of PHR1 target gene expression after Pi refeeding. AAA, Poly A tail of mRNA.