Silver ions increase auxin efflux independently of effects on ethylene response

Abstract

Silver nitrate and aminoethoxyvinylglycine (AVG) are often used to inhibit perception and biosynthesis, respectively, of the phytohormone ethylene. In the course of exploring the genetic basis of the extensive interactions between ethylene and auxin, we compared the effects of silver nitrate (AgNO(3)) and AVG on auxin responsiveness. We found that although AgNO(3) dramatically decreased root indole-3-acetic acid (IAA) responsiveness in inhibition of root elongation, promotion of DR5-beta-glucuronidase activity, and reduction of Aux/IAA protein levels, AVG had more mild effects. Moreover, we found that that silver ions, but not AVG, enhanced IAA efflux similarly in root tips of both the wild type and mutants with blocked ethylene responses, indicating that this enhancement was independent of ethylene signaling. Our results suggest that the promotion of IAA efflux by silver ions is independent of the effects of silver ions on ethylene perception. Although the molecular details of this enhancement remain unknown, our finding that silver ions can promote IAA efflux in addition to blocking ethylene signaling suggest that caution is warranted in interpreting studies using AgNO(3) to block ethylene signaling in roots.

Figure 1.

AgNO₃ Dampens IAA Responses in Roots. (A) Silver nitrate and AVG are similarly effective in restoring eto1 mutant phenotypes. Wild-type (Col-0), eto1-1, and eto2-1 seedlings were grown on medium supplemented with various concentrations of AgNO₃ or AVG. Hypocotyls were measured 4 d after transfer of 1-d-old seedlings to the dark (top panels). Primary roots of 8-d-old seedlings were measured after growth under continuous white light (bottom panels). Error bars represent se (n = 12). (B) Root elongation inhibition response of the wild type (Col-0), aux1-7, ein2-1, and eir1-1 to IAA and 2,4-D in the presence and absence of AgNO₃ or AVG. Primary root lengths of 8-d-old seedlings grown under continuous yellow-filtered light on mock (ethanol)-supplemented medium or medium supplemented with 600 nM IAA or 100 nM 2,4-D with or without 5 μM AgNO3 or 10 μM AVG are shown. Error bars represent se (n ≥ 12). (C) Silver nitrate decreases IAA-induced DR5-GUS expression. Eight-day-old light-grown wild-type (Col-0) seedlings carrying the DR5-GUS transgene (Ulmasov et al., 1997) were mock treated or treated with 1 μM IAA for 2 h in medium lacking or containing 10 μM AgNO₃ or 10 μM AVG and then stained for GUS activity. Bar = 0.5 mm. (D) Silver nitrate decreases IAA-induced IAA28myc degradation. Ten-day-old wild-type (Col-0) seedlings carrying the IAA28myc construct (Strader et al., 2008a) were treated for 10 min with the indicated combinations of IAA (top panel), 2,4-D (bottom panel), AgNO₃, and AVG in liquid media. Anti-myc and anti-HSC70 antibodies were used to detect IAA28myc and HSC70 (loading control), respectively, on immunoblots of protein prepared from roots of treated seedlings.

Figure 2.

AgNO₃ Promotes [³H]-IAA Efflux from Root Tips. (A) Root tips of Col-0 (Wt) seedlings were incubated for 1 h in uptake buffer containing 25 nM [³H]-IAA, 25 nM [³H]-IAA and 10 μM AgNO₃, or 25 nM [³H]-IAA and 10 μM KNO₃, rinsed three times with uptake buffer, and then removed and analyzed by scintillation counting. (B) Root tips of Col-0 (Wt), aux1-7, ein2-1, and eir1-1 seedlings were incubated for 1 h in uptake buffer containing 25 nM [³H]-IAA, 25 nM [³H]-IAA and 10 μM AgNO₃, or 25 nM [³H]-IAA and 10 μM AVG, rinsed three times with uptake buffer, and then removed and analyzed by scintillation counting. (C) Root tips of Col-0 (Wt), pen3-4, etr1-1, and ers2-1 seedlings were incubated for 1 h in uptake buffer containing 25 nM [³H]-IAA or 25 nM [³H]-IAA and 10 μM AgNO₃, rinsed three times with uptake buffer, and then removed and analyzed by scintillation counting. (D) Root tips of Col-0 (Wt), mdr1-3, mdr4-1, pgp1-100, Ws-2 (Wt), and pgp1-1 mdr1-1 (in the Wassilewskija background) seedlings were incubated for 1 h in uptake buffer containing 25 nM [³H]-IAA or 25 nM [³H]-IAA and 10 μM AgNO₃, rinsed three times with uptake buffer, and then removed and analyzed by scintillation counting. (E) Root tips of Col-0 (Wt), aux1-7, ein2-1, and eir1-1 seedlings were incubated for 1 h in 80 μL uptake buffer containing 25 nM [³H]-IAA, rinsed three times, incubated for an additional 30 or 60 min in 400 μL buffer with or without 10 μM AgNO₃ or 10 μM AVG, and then removed and analyzed by scintillation counting. (F) Root tips of Col-0 (Wt) seedlings were incubated for 1 h in 80 μL uptake buffer containing 25 nM [³H]-IAA, rinsed three times, incubated for an additional 1 h in 400 μL buffer containing 0, 0.5, 1, 5, or 10 μM AgNO₃, and then removed and analyzed by scintillation counting. (G) Root tips of Col-0 (Wt) seedlings were incubated for 1 h in 80 μL uptake buffer containing 25 nM [³H]-IAA, rinsed three times, incubated for an additional 30 min in 400 μL buffer containing mock (ethanol), 100 μM NPA, or 100 μM TIBA, and then removed and analyzed by scintillation counting. For all experiments, data are from six replicates of assays with five root tips (5-mm sections) of 8-d-old light-grown seedlings of each genotype. Error bars represent se. (H) A model for the effects on AgNO₃ and AVG on auxin and ethylene signaling. IAA is transported into cells by AUX1 and related transporters and via diffusion through the membrane and is removed by effluxers such as EIR1/PIN2 and the ABCB proteins. In the cell, IAA stimulates the degradation of Aux/IAA proteins to relieve repression of auxin-responsive transcription (reviewed in Woodward and Bartel, 2005), leading to various responses, including induction of ACS transcription and ethylene production (reviewed in Yang and Hoffman, 1984; Tsuchisaka and Theologis, 2004). ACS activity can be blocked with AVG (Yang and Hoffman, 1984), and signaling by ethylene receptors, such as ETR1 and ERS2, can be blocked with Ag⁺ (Rodriguez et al., 1999; Zhao et al., 2002; Binder et al., 2007). EIN2 is required for ethylene signaling and acts downstream of ethylene perception (Alonso et al., 1999). The double-headed gray arrow represents extensive crosstalk between auxin and ethylene pathways. Solid black arrows depict signaling, dashed black arrows depict hormone synthesis, and dotted black arrows represent transport. Silver ions appear to stimulate IAA efflux independently of known IAA efflux components, such as EIR1/PIN2 and the ABCB proteins. Whether the Ag⁺-stimulated IAA efflux is transporter mediated or results from an effect on membrane permeability is unknown.