Mutational reconstructed ferric chelate reductase confers enhanced tolerance in rice to iron deficiency in calcareous soil

Abstract

Iron (Fe) deficiency is a worldwide agricultural problem on calcareous soils with low-Fe availability due to high soil pH. Rice plants use a well documented phytosiderophore-based system (Strategy II) to take up Fe from the soil and also possess a direct Fe2+ transport system. Rice plants are extremely susceptible to low-Fe supply, however, because of low phytosiderophore secretion and low Fe3+ reduction activity. A yeast Fe3+ chelate-reductase gene refre1/372, selected for better performance at high pH, was fused to the promoter of the Fe-regulated transporter, OsIRT1, and introduced into rice plants. The transgene was expressed in response to a low-Fe nutritional status in roots of transformants. Transgenic rice plants expressing the refre1/372 gene showed higher Fe3+ chelate-reductase activity and a higher Fe-uptake rate than vector controls under Fe-deficient conditions. Consequently, transgenic rice plants exhibited an enhanced tolerance to low-Fe availability and 7.9x the grain yield of nontransformed plants in calcareous soils. This report shows that enhancing the Fe3+ chelate-reductase activity of rice plants that normally have low endogenous levels confers resistance to Fe deficiency.

Fig. 1.

The refre1/372 expression analyzed by RT-PCR. (A) Genomic PCR of DNA prepared from each transformant (lines 7, 8, and 11) and the vector control (VC). (B) Transcript of refre1/372 levels in roots grown under Fe-deficient and Fe-sufficient conditions was detected by using 2 μg of total RNA for each transformant (lines 7, 8, and 11), the vector control (VC), and plasmid containing refre1–372 (P). α-tubulin and OsIRT1 were internal standards.

Fig. 2.

Assays of Fe³⁺ chelate-reductase activity in vector control and the transformants. (A) Transformants (lines 7, 8, and 11) and vector control (V) were grown on standard culture solution for 3 weeks and then transferred to Fe-deficient culture solution for 5 days before the assay. pHs of assay buffers were 5.5 or 8.0. (B) Fe³⁺ chelate-reductase activity per root fresh weight in roots surface of transformants (lines 7 and 11) and vector control (V). Rice plants were grown for 3 weeks in normal nutrient solution and then transferred to Fe-deficient culture; roots were harvested 0, 3, and 7 days after the transfer (+2 and +5 indicate the number of days after Fe resupply; n = 9). (C) Total Fe³⁺ chelate-reductase activity in whole roots surface of transformants (lines 7 and 11) and vector control (V) (n = 9). (D) Degree of chlorosis of the fully expanded youngest leaf by using a SPAD-502 chlorophyll meter. The values followed by different letters are statistically different according to a Student-Newman-Keuls test (P < 0.05).

Fig. 3.

Uptake and transport of Fe as monitored by using PETIS. (A) Relative Fe-transition rate of transformant (TF; line 7) and vector control (V) in stem. (B) Bio-Imaging Analyzer System (BAS Bioanalytical Systems) image of the transformant. (C) Photograph of the transformant. (D) BAS Bioanalytical Systems image of the vector control. (E) Photograph of the vector control. Colors from blue through green, yellow, orange, and red indicate increasing Fe uptake in B and D.

Fig. 4.

Growth features and grain yield of transgenic rice plants containing the refre1/372 gene (line 7) and vector controls grown in calcareous soil (pH 8.5) and in bonsol (normal cultivated soil). (A) Transformant (TF, Left) and vector control (V, Center) after 4 weeks of growth in a calcareous soil; vector control (V, Right) in bonsol. (B) Transformant (TF, Left) and vector control (V, Right) after 17 weeks of growth in calcareous soil. (C) Plant height of transformant (TF; mean ± SD, n = 5) and vector control (V; mean ± SD, n = 3) for 70 days after transplanting into calcareous soil and bonsol. (D) SPAD-502 value (chlorophyll content) in leaves of the transformant (TF; mean ± SD, n = 5) and vector control (V; mean ± SD, n = 3) for 70 days after transplanting into calcareous soil and bonsol. (E) Dry weight of shoots (i.e., without grain) of transformant (TF; n = 5) and vector control (V; n = 3) after cultivation for 17 weeks in calcareous soil. (F) Grain yield of transformant (TF; n = 5) and vector control (V; n = 3) after cultivation for 17 weeks in calcareous soil. The values followed by different letters are statistically different according to a Student-Newman-Keuls test (P < 0.05).

Fig. 5.

Metal concentrations in transformants and vector control grown in calcareous soil. (A–D) Metal concentrations in shoots of transformants (lines 7 and 11, filled bars) and vector control (V, open bars). (E–H) Metal concentrations in seeds of transformants (TF; line 7, filled bars) and vector control (V, open bars).The concentrations of Fe, Zn, Mn, and Cu are expressed as micrograms per gram of dry weight (n = 3). The values followed by different letters are statistically different according to a Student-Newman-Keuls test (P < 0.05).