Plants are the principal source of iron in most diets, yet iron availability often limits plant growth. In response to iron deficiency, Arabidopsis roots induce the expression of the divalent cation transporter IRT1. Here, we present genetic evidence that IRT1 is essential for the uptake of iron from the soil. An Arabidopsis knockout mutant in IRT1 is chlorotic and has a severe growth defect in soil, leading to death. This defect is rescued by the exogenous application of iron. The mutant plants do not take up iron and fail to accumulate other divalent cations in low-iron conditions. IRT1-green fluorescent protein fusion, transiently expressed in culture cells, localized to the plasma membrane. We also show, through promoter:: ␤ -glucuronidase analysis and in situ hybridization, that IRT1 is expressed in the external cell layers of the root, specifically in response to iron starvation. These results clearly demonstrate that IRT1 is the major transporter responsible for high-affinity metal uptake under iron deficiency.

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The iron-storage protein ferritin has been purified to homogeneity from maize seeds, allowing to determine the sequence of the first 29 NH2-terminal amino acids of its subunit and to raise specific rabbit polyclonal antibodies. Addition of 500 microM Fe-EDTA/75 microM Fe-citrate to hydroponic culture solutions of maize plantlets, previously starved for iron, led to a significant increase of the iron concentration of roots and leaves, albeit root iron was mainly found associated with the apoplast. Immunodetection of ferritin by western blots indicated that this iron treatment induced ferritin protein accumulation in roots and leaves over a period of 3 days. In order to investigate this induction at the ferritin mRNA level, various ferritin cDNA clones were isolated from a cDNA library prepared from poly(A)+ mRNA isolated from roots 48 h after iron treatment. These cDNAs were classified into two groups called FM1 and FM2. Upstream of the sequence encoding the mature ferritin subunit, both of these cDNAs contained an in-frame coding sequence with the characteristics of a transit peptide for plastid targeting. Two members of the FM1 subfamily, both partial at their 5' extremity, were characterized. They are identical, except in their 3' untranslated region: FM1A extends 162 nucleotides beyond the 3' terminus of FM1B. These two mRNAs could arise from the use of two different polyadenylation signals. FM2 is 96% identical to FM1 and contains 45 nucleotides of 5' untranslated region. Northern analyses of root and leaf RNAs, at different times after iron treatment, revealed ferritin mRNA accumulation in response to iron. Ferritin mRNA accumulation was transient and particularly abundant in leaves, reaching a maximum at 24 h. The level of ferritin mRNA in roots was affected to a lesser extent than in leaves.

SummaryIntracellular iron concentration requires tight control and is regulated both at the uptake and storage levels. Our knowledge of the role that the iron-storage protein ferritins play in plants is still very limited. Overexpression of this protein, either in the cytoplasm or the plastids of transgenic tobacco, was obtained by placing soybean ferritin cDNA cassettes under the control of the CAMV 35S promoter. The protein accumulated in 4-and 6-dayold seedlings and in leaves of 3-week-old plants but not in dry seeds or in 2-day-old seedlings, which is consistent with previous reports describing a post-transcriptional control of ferritin amounts during the germination process. Overaccumulated ferritin in leaves was correctly assembled as 24-mers. Transformants were more resistant to methylviologen toxicity, indicating that the transgenic ferritins were functional in vivo. Ferritin overaccumulation in transgenic tobacco leaves leads to an illegitimate iron sequestration. As a consequence, these transgenic plants behave as iron deficient and activate iron transport systems as revealed by an increase in root ferric reductase activity and in leaf iron content.