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Summary Plants have evolved two strategies to acquire ferrous (Strategy I) or ferric (Strategy II) iron from soil. The iron‐related bHLH transcription factor 2 (IRO2) has been identified as a key regulator of iron acquisition (Strategy II) in rice. However, its mode of action, subcellular localisation and binding partners are not clearly defined. Using RNA‐seq analyses, we identified a novel bHLH‐type transcription factor, OsbHLH156. The function of OsbHLH156 in Fe homeostasis was analysed by characterisation of the phenotypes, elemental content, transcriptome, interaction and subcellular localisation of OsbHLH156 and IRO2. OsbHLH156 is primarily expressed in the roots and transcript abundance is greatly increased by Fe deficiency. Loss of function of OsbHLH156 resulted in Fe‐deficiency‐induced chlorosis and reduced Fe concentration in the shoots under upland or Fe(III) supplied conditions. Transcriptome analyses revealed that the expression of most Fe‐deficiency‐responsive genes involved in Strategy II were not induced in the osbhlh156‐1 mutant. Furthermore, OsbHLH156 was required for nuclear localisation of IRO2. We conclude that OsbHLH156 is required for a Strategy II uptake mechanism in rice, partnering with a previously identified ‘master’ regulator IRO2. Mechanistically it is required for the nuclear localisation of IRO2.

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