AhNRAMP1 iron transporter is involved in iron acquisition in peanut

Abstract

Peanut/maize intercropping is a sustainable and effective agroecosystem to alleviate iron-deficiency chlorosis. Using suppression subtractive hybridization from the roots of intercropped and monocropped peanut which show different iron nutrition levels, a peanut gene, AhNRAMP1, which belongs to divalent metal transporters of the natural resistance-associated macrophage protein (NRAMP) gene family was isolated. Yeast complementation assays suggested that AhNRAMP1 encodes a functional iron transporter. Moreover, the mRNA level of AhNRAMP1 was obviously induced by iron deficiency in both roots and leaves. Transient expression, laser microdissection, and in situ hybridization analyses revealed that AhNRAMP1 was mainly localized on the plasma membrane of the epidermis of peanut roots. Induced expression of AhNRAMP1 in tobacco conferred enhanced tolerance to iron deprivation. These results suggest that the AhNRAMP1 is possibly involved in iron acquisition in peanut plants.

Fig. 1.

Phylogenetic tree of the NRAMP proteins from Arabidopsis, rice, tomato, and peanut. Accession numbers are as follows: AhNRAMP1, JQ581595; AtNRAMP1, AEE36455.1; AtNRAMP2, AEE32142.1; AtNRAMP3, AAF13278.1; AtNRAMP4, AAF13279.1; AtNRAMP5, NP_193614.1; AtNRAMP6, AEE29390.1; OsNRAMP1, AAB36424.1; OsNRAMP2, Q10Q65; OsNRAMP3, Q653V6; OsNRAMP4, Os02g0131800; OsNRAMP5, Os07g0257200; OsNRAMP6, Os01g0503400; OsNRAMP7, Os12g0581600; LeNRAMP1, NP_001234318; LeNRAMP3, NP_001233770.

Fig. 2.

Expression pattern of AhNRAMP1 in response to Fe-deficient and Fe-sufficient conditions. Peanut plants were treated without Fe or with normal amounts of Fe (100 μM) for 1, 4, 7, and 11 d. 1d, 4d, 7d, and 11d represent the day of treatments. On day 12 (12d), the Fe-deficient peanut plants were transferred to 500 μM Fe for the treatment with excess Fe (++Fe) for 1 d. The samples were harvested from roots (A), young leaves (B), and old leaves (C) of the treated peanut plants. The vertical bars indicate the expression level of the genes relative to that of the control AhUbiquitin gene. Values are the means of three replications. Error bars indicate the SD.

Fig. 3.

Functional complementation of the fet3fet4 yeast mutant with AhNRAMP1. Empty pDR195 vector was used as a negative control and OsNRAMP1 cDNA as a positive control. Serial dilutions of the transformed yeast cells with OD_{600 nm} 1 to 0.001 were plated onto SD medium at pH 5 (A), pH 6 (B), and pH 7 (C).

Fig. 4.

Subcellular localization of AhNRAMP1 in onion epidermal cells. (A) AhNRAMP1 fused to GFP was transiently expressed in onion epidermal cells. (B) The red fluorescence detected from the FM4-64 dye (2 μM), as a plasma membrane marker. (C) Confocal microscopy images illustrating AhNRAMP1–GFP co-localization with the FM4-64 dye at the plasma membrane. (D) GFP alone was transiently expressed in onion epidermal cells. (E) Overlay with the transmission image shown in (D). Scale bars represent 50 μm.

Fig. 5.

Tissue localization of AhNRAMP1 in peanut roots by LMD analysis and in situ hybridization. (A) LMD and expression analysis of AhNRAMP1. Peanut plants were cultured with or without Fe for 7 d. Stele, cortex, and epidermis/endodermis of peanut roots were separated by LMD. The vertical bars indicate the relative expression level of AhNRAMP1 as compared with the control AhUbiquitin gene. Three biological replications were performed for each treatment. Error bars represent the SD. (B–E) In situ hybridization analysis of AhNRAMP1 in the roots of Fe-deficient peanut. AhNRAMP1 antisense probes were hybridized in a cross-section (B) or longitudinal section (D) of peanut root. Sense probes of AhNRAMP1 were used as a control (C, E). Bars = 100 μm. Arrowheads indicate representative hybridization signals.

Fig. 6.

The response to Fe deficiency in AhNRAMP1-induced tobacco lines. The phenotype of AhNRAMP1-induced lines grown on MS medium (A) or hydroponics (B) without Fe. NT represents non-transformed tobacco. IN is AhNRAMP1-induced tobacco. (C) Fe and (D) Mn concentration in the new leaves of NT and IN tobacco in hydroponics treated under Fe deficiency for 9 d. The results are presented as the means ±SD of triplicate samples. Asterisks indicate a significant difference at P < 0.05 by t-test.