Dual regulation of the Arabidopsis high-affinity root iron uptake system by local and long-distance signals

Abstract

Regulation of the root high-affinity iron uptake system by whole-plant signals was investigated at the molecular level in Arabidopsis, through monitoring FRO2 and IRT1 gene expression. These two genes encode the root ferric-chelate reductase and the high-affinity iron transporter, respectively, involved in the iron deficiency-induced uptake system. Recovery from iron-deficient conditions and modulation of apoplastic iron pools indicate that iron itself plays a major role in the regulation of root iron deficiency responses at the mRNA and protein levels. Split-root experiments show that the expression of IRT1 and FRO2 is controlled both by a local induction from the root iron pool and through a systemic pathway involving a shoot-borne signal, both signals being integrated to tightly control production of the root iron uptake proteins. We also show that IRT1 and FRO2 are expressed during the day and down-regulated at night and that this additional control is overruled by iron starvation, indicating that the nutritional status prevails on the diurnal regulation. Our work suggests, for the first time to our knowledge, that like in grasses, the root iron acquisition in strategy I plants may also be under diurnal regulation. On the basis of the new molecular insights provided in this study and given the strict coregulation of IRT1 and FRO2 observed, we present a model of local and long-distance regulation of the root iron uptake system in Arabidopsis.

Figure 1.

Time-course response of IRT1 and FRO2 expression to iron starvation in Suc-free medium. Plants were grown under iron-sufficiency (100 μm iron) for 5 weeks (lane C) and then washed for 5 min with fresh iron-deficient medium before being transferred to iron deficiency. Plants were harvested after 1, 3, 5, or 7 d of iron deficiency. Seven-day-starved plants were also resupplied with 100 μm iron for 24 h (lane R). A, Northern-blot analyses. IRT1 and FRO2 probes were sequentially hybridized to a blot containing 12 μg of total RNA extracted from roots of Arabidopsis. B, Immunoblot analysis. IRT1 protein was detected by hybridizing the blot with an IRT1-specific antibody.

Figure 2.

Effect of apoplastic iron on IRT1 and FRO2 expression. A, Plant growth conditions. Plants were grown under iron-sufficiency (100 μm iron) for 5 weeks (lane C), transferred to a medium containing various concentrations of iron as indicated for 2 d, and then washed with bipyridyl before being transferred to iron-free medium. Roots were harvested 1, 3, 5, or 7 d after transfer to –Fe solution. When indicated, 7-d iron-starved plants were also resupplied with 100 μm iron for 24 h (B, lane R). B through D, Northern-blot analyses. IRT1 and FRO2 probes were sequentially hybridized to a blot containing 10 μg of total RNA extracted from roots of Arabidopsis grown as described in A, with a 2-d preculture in 100 μm iron (B), 500 μm (C), or 50 to 500 μm as indicated on the figure (D) before the root wash with bipyridyl.

Figure 3.

Time course analysis of IRT1 and FRO2 expression upon recovery from iron deficiency. Plants were grown under iron-sufficiency (100 μm iron) for 4 weeks, washed with bipyridyl, transferred to iron-deficient medium for 7 d, and then supplied with an excess of iron (500 μm) before being harvested at the times indicated on the figure. IRT1 and FRO2 probes were sequentially hybridized to a blot containing 15 μg of total RNA extracted from roots of Arabidopsis grown as described above.

Figure 4.

Response of IRT1 and FRO2 expression to localized supply of iron. One side of the split-root system of plants grown for 5 weeks in iron sufficiency (100 μm) was washed with bipyridyl and transferred to iron-deficient medium. Total RNA (A and C) and protein (B and D) was extracted from roots harvested separately from the +Fe and the –Fe sides after 3 d (A and B) and 7 d (C and D) of deficiency. The northern blots (A and C) contained 15 μg of total RNA and were sequentially hybridized with the IRT1 and FRO2 probes. The western blots (B and D) contained 10 μg of total protein extract and were hybridized with an IRT1 specific antibody.

Figure 5.

Diurnal regulation of IRT1 and FRO2. A, Northern-blot analyses. Wild-type plants were grown hydroponically in a medium containing 100 μm iron under short-day conditions. B, Northern-blot analysis. Plants grown as in A except that 24 h before the beginning of sample collection, plants were transferred to low-iron conditions (5 μm iron). A northern blot containing 15 μg of total RNA extracted from roots harvested at the time points indicated was sequentially hybridized with IRT1, FRO2, and EF1α probes. Blots presented in this figure are representative of three independent experiments.

Figure 6.

Schematic representation of two hypothetical models of regulation by the coordinated action of local and systemic signals. In the promotive model, the shoot-borne signal is produced in response to iron deficiency and, in conjunction with the local iron induction, results in activation of root iron uptake genes. In this scheme, iron-sufficient status in the shoot would prevent release of this systemic signal, and genes could not be expressed. Conversely, the repressive model accounts for a situation in which the systemic signal is produced in response to shoot-sufficient iron status. In that case, this signal could turn on a pathway leading to negative regulation of root iron uptake genes; its absence under iron deficiency would thus allow derepression of gene expression, given that iron molecules are present locally to trigger the response.