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Iron is an essential micronutrient for living organisms, but its solubility is extremely low under alkaline conditions. Plants often suffer from iron deficiency chlorosis in calcareous soils, which consist of approximately 30% of the world's cultivated area, severely limiting plant productivity. Iron deficiency anemia is also a widespread problem in humans, especially in Asian and African people who take up iron mainly from staple foods containing low iron concentrations. Transgenic manipulation of genes involved in plant iron uptake, translocation, and storage has made improvements in enhancing iron deficiency tolerance or iron accumulation in edible parts, but these two properties have been characterized separately. We previously produced transgenic rice lines, with concomitant improvement of iron deficiency tolerance and grain iron accumulation by knocking-down OsHRZ ubiquitin ligases, which negatively regulate iron deficiency response and iron accumulation in rice. In the present report, we aimed to further improve the iron deficiency tolerance and grain iron accumulation of OsHRZ knockdown rice by the simultaneous introduction of the engineered ferric-chelate reductase gene Refre1/372 under the control of the OsIRT1 promoter for further enhancement of iron uptake. We obtained several transgenic rice lines with repressed OsHRZ expression and induced Refre1/372 expression. These lines showed a variable degree of iron deficiency tolerance in calcareous soils, with increased iron accumulation in brown seeds under both iron-deficient and iron-sufficient soil cultures. Selected OsHRZ knockdown plus Refre1/372 lines showed similar or better growth compared with that of singly introduced OsHRZ knockdown or Refre1/372 lines in calcareous soils under both non-submerged and submerged conditions. After submerged calcareous soil cultivation, these OsHRZ knockdown plus Refre1/372 lines accumulated 2.5–4.3 times and 17–23 times more iron concentrations than that of non-transformants in brown rice and straw, respectively, which was comparable or superior to a single OsHRZ knockdown line. Our results indicate that the combined introduction of OsHRZ knockdown and OsIRT1 promoter-Refre1/372 is highly effective in further improving the iron deficiency tolerance without compromising the iron accumulation of the OsHRZ knockdown effects. [ABSTRACT FROM AUTHOR]