Characterization of FRO1, a Pea Ferric-Chelate Reductase Involved in Root Iron Acquisition

Abstract

To acquire iron, many plant species reduce soil Fe(III) to Fe(II) by Fe(III)-chelate reductases embedded in the plasma membrane of root epidermal cells. The reduced product is then taken up by Fe(II) transporter proteins. These activities are induced under Fe deficiency. We describe here the FRO1 gene from pea (Pisum sativum), which encodes an Fe(III)-chelate reductase. Consistent with this proposed role, FRO1 shows similarity to other oxidoreductase proteins, and expression of FRO1 in yeast conferred increased Fe(III)-chelate reductase activity. Furthermore, FRO1 mRNA levels in plants correlated with Fe(III)-chelate reductase activity. Sites of FRO1 expression in roots, leaves, and nodules were determined. FRO1 mRNA was detected throughout the root, but was most abundant in the outer epidermal cells. Expression was detected in mesophyll cells in leaves. In root nodules, mRNA was detected in the infection zone and nitrogen-fixing region. These results indicate that FRO1 acts in root Fe uptake and they suggest a role in Fe distribution throughout the plant. Characterization of FRO1 has also provided new insights into the regulation of Fe uptake. FRO1 expression and reductase activity was detected only in Fe-deficient roots of Sparkle, whereas both were constitutive in brz and dgl, two mutants with incorrectly regulated Fe accumulation. In contrast, FRO1 expression was responsive to Fe status in shoots of all three plant lines. These results indicate differential regulation of FRO1 in roots and shoots, and improper FRO1 regulation in response to a shoot-derived signal of iron status in the roots of the brz and dgl mutants.Iron is required for many functions in plants, including heme and chlorophyll biosynthesis, photosynthesis, and as a component of Fe-S cluster containing enzymes (Marschner, 1995). Iron is also vital for the establishment and function of symbiotic root nodules of legumes involved in nitrogen fixation (Udvardi and Day, 1997). Although abundant in the environment, iron is often a limiting nutrient for plant growth due to the low solubility of the oxidized form of Fe, Fe(III), at near neutral soil pH. Thus, plants have evolved with efficient mechanisms of Fe acquisition that are directed at solubilizing Fe. Strategy II plants, which includes all of the grasses, release Fe(III)-binding compounds called phytosiderophores into the surrounding soil that bind iron and are then taken up into the roots (Marschner, 1995). Strategy I plants (dicots and non-Graminaceous monocots) obtain Fe from the rhizosphere by first reducing Fe(III) to Fe(II) through the action of membrane-bound Fe(III)-chelate reductases. Iron reduction is then followed by uptake of Fe(II) into root cells by metal ion transporters. Reductase and transporter activities are inducible in roots under Fe deficiency. Furthermore, the roots of strategy I plants release more protons when Fe deficient, thereby lowering the rhizosphere pH and increasing Fe solubility.Many of the components of the Strategy I Fe acquisition system have recently been ...

Iron deficiency induces changes in riboflavin secretion and the mitochondrial electron transport chain in hairy roots of Hyoscyamus albus

“…In parallel with this change, biochemical changes have been observed, including proton extrusion by the activation of a proton-pump H + -ATPase, thereby reducing soil pH and increasing the solubility of Fe 3+ Waters et al, 2002;Hell and Stephan, 2003;Schmidt, 2003;Santi et al, 2005). Concomitantly, organic acids such as citric and malic acids that act as Fe 3+ -chelators (Ryan et al, 2001;Dakora and Phillips, 2002) are released, chelated Fe 3+ is reduced to Fe 2+ by the action of a membrane-bound Fe 3+ chelate reductase, and then Fe 2+ is transported into cells by a Fe-regulated transporter (Waters et al, 2002;Connolly et al, 2003). To support all these processes directly connected with the enhancement of Fe uptake, a supply of ATP as well as of 5 reducing agents such as NAD(P)H is essential (Zocchi, 2007).…”

Modulation of iron responsive gene expression and enzymatic activities in response to changes of the iron nutritional status in Cucumis sativus L.

“…On the contrary, it is in the absence of iron that genes encoding for FRO2 and FRO1 are up regulated (Waters et al, 2002;Connolly et al, 2003;Li et al, 2004) and it is also in this condition that IRT1 mRNA and protein accumulates in A. thaliana (Eide et al, 1996;Connolly et al, 2002;Vert et al, 2002).…”

“…CsFRO1 is hortologous to AtFRO2 (Robinson et al, 1999), LeFRO1 (Li et al, 2004) and PsFRO1 (Waters et al 2002), while CsIRT1 is hortologous to AtIRT1 (Eide et al, 1996) and LeIRT1 (Eckhardt et al, 2001). Concerning to the H + -ATPase, we considered the differential expression level of CsHA1 gene (Santi et al, 2005 (AF071788) (Sanchez & Cejudo, 2003).…”

“…It has been observed in Strategy I plants, that in response to iron starvation, there was an induction of the genes encoding for the Fe(III)-chelate reductase (AtFRO2, PsFRO1 and LeFRO1) which were already characterized (Robinson et al, 1999;Waters et al, 2002;Li et al, 2004). Enhancement of H + efflux, due to an increase in a P-type H + -ATPase activity in response to iron deprivation, was demonstrated in many Strategy I plants (Schmidt, 1999;Zocchi, 2006).…”

Functional analysis of transgenic rice (Oryza sativaL.) transformed with anArabidopsis thalianaferric reductase (AtFR02)

“…Total RNA was isolated from individual shoots and roots using the RNA-easy kit from Qiagen (Qiagen Inc., Valencia, Califomia, USA) according to the manufactur- , 1994Eide et al 1996;Higuchi et al 1999;Takahashi et al 1999;Curie et al 2001;Yun et al 2001;Waters et al 2002). In pmticular, one of the bestdescribed genes is the FR02 gene from Arahidopsis thaliana (AtFR02) (Robinson et al 1999;Connolly et al 2003).…”