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.