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.