Phosphite, an analog of phosphate, suppresses the coordinated expression of genes under phosphate starvation

Abstract

Phosphate (Pi) and its analog phosphite (Phi) are acquired by plants via Pi transporters. Although the uptake and mobility of Phi and Pi are similar, there is no evidence suggesting that plants can utilize Phi as a sole source of phosphorus. Phi is also known to interfere with many of the Pi starvation responses in plants and yeast (Saccharomyces cerevisiae). In this study, effects of Phi on plant growth and coordinated expression of genes induced by Pi starvation were analyzed. Phi suppressed many of the Pi starvation responses that are commonly observed in plants. Enhanced root growth and root to shoot ratio, a hallmark of Pi stress response, was strongly inhibited by Phi. The negative effects of Phi were not obvious in plants supplemented with Pi. The expression of Pi starvation-induced genes such as LePT1, LePT2, AtPT1, and AtPT2 (high-affinity Pi transporters); LePS2 (a novel acid phosphatase); LePS3 and TPSI1 (novel genes); and PAP1 (purple acid phosphatase) was suppressed by Phi in plants and cell cultures. Expression of luciferase reporter gene driven by the Pi starvation-induced AtPT2 promoter was also suppressed by Phi. These analyses showed that suppression of Pi starvation-induced genes is an early response to addition of Phi. These data also provide evidence that Phi interferes with gene expression at the level of transcription. Synchronized suppression of multiple Pi starvation-induced genes by Phi points to its action on the early molecular events, probably signal transduction, in Pi starvation response.

Figure 1

Phi inhibits growth of Pi-starved tomato. Three-week-old tomato plants were transferred to one-half-strength Hoagland solution. After 7 d of growth, plants were treated with different concentrations of Phi (0–3 mm) in the presence or absence of Pi. A, Difference in total fresh weight (g) measured at the beginning and the end of 5 d of Phi treatment was expressed as gain in weight. Error bars indicate ± sd. B, Plant height (cm) was measured from the crown region to the apex of plants at the end of 5 d of Phi treatment. Error bars indicate ± sd. C, Root to shoot ratio of hydroponically grown tomato plants treated with different concentrations of Phi. Error bars indicate ±sd.

Figure 2

Phi inhibits root growth of Pi-starved Arabidopsis seedlings. Arabidopsis seedlings were grown for 5 d on membranes placed on one-half-strength Murashige and Skoog agar medium. Plates were inclined vertically to allow the roots to grow down. A, The membranes along with seedlings were transferred to plates containing Phi in the presence (P+) or absence (P−) of Pi and allowed to grow for 5 more d. Plates were inverted such that the roots were pointing up to monitor root growth and bending. B, The membranes along with seedlings were transferred to plates containing different concentrations of Phi in the presence (P+) or absence (P−) of Pi. After 5 d of transfer, root length of plants was measured. The table represents the mean ± sd of root lengths of 20 seedlings.

Figure 3

Phi suppresses Pi starvation-induced gene expression in tomato. A, Three-week-old tomato seedlings were transferred to one-half-strength Hoagland nutrient solution. After 7 d, they were treated with Phi (0, 1, 2, and 3 mm) in the presence (+) or absence (−) of Pi for 5 d. Total RNA extracted from the roots was used for northern-blot analysis of Pi starvation-induced genes. cDNAs of genes indicated on the left of the figure were used as probes. Tomato cyclophilin (C-philin) served as a control. B, Tomato cell cultures grown in full-strength Murashige and Skoog medium were transferred to medium containing Phi (0, 1, 2, and 3 mm) with (+) or without (−) Pi. Cells were harvested after 48 h for RNA isolation and northern-blot analysis. C, Tomato cells grown in full-strength Murashige and Skoog medium were transferred to medium containing relatively low concentrations of Phi (0, 0.3, and 0.5 mm) in the presence (+) or absence (−) of Pi. Cells were harvested after 48 h for RNA isolation and northern-blot analysis.

Figure 4

Suppression of Pi starvation-induced gene expression in Arabidopsis by Phi. A, AtPT2-LUC transgenic Arabidopsis were grown in one-half-strength Murashige and Skoog liquid medium for 6 d. The media was replaced with fresh solution containing indicated concentrations of Phi either in the presence (P+) or absence (P−) of Pi. After 5 d of Phi treatment plants were analyzed for Luc activity. Luc activity was expressed as relative luminescence units per microgram of protein. Values represent the mean of three replicates ± sd. B, Six-day-old Arabidopsis seedlings grown in one-half-strength Murashige and Skoog liquid medium were transferred to medium containing increasing concentrations of Phi (0–3 mm) in the presence (P+) or absence (P−) of Pi. After 5 d of transfer, plants were harvested for total RNA isolation and northern-blot analysis. cDNAs of genes representing AtPT1, AtPT2, PAP1, and LUC were used as probes. Arabidopsis tubulin gene probe served as a control.

Figure 5

Inhibition of Pi starvation-induced genes is an early response to Phi treatment. Arabidopsis seedlings were grown in one-half-strength Murashige and Skoog liquid media for 6 d and transferred to P− media without (0) Phi (white bars) or with 3 mm Phi (black bars). Replicated samples were harvested at indicated time (1–4 d) after Phi treatment for measuring LUC activity (A), and isolating RNA for northern-blot analysis (B). In a separate experiment, 6-d-old Arabidopsis seedlings were transferred to Pi-deficient medium for 2 d (P−) to activate Pi starvation-induced gene expression. Sets of Pi-starved plants received (R) an additional day of Pi stress in the absence (0) or presence of 3 mm of Phi. After 24 h of treatment, plants were harvested for measuring LUC activity (C) and isolating RNA for northern-blot analysis (D). The northern blots were probed with ³²P-labeled cDNAs of AtPT1, AtPT2, PAP1, LUC, and tubulin.