ABSTRACT Iron (Fe) is an essential element for most organisms since it is involved in various biological reactions. Fe deficiency is a major constraint for plant growth and leads to low crop yields and poor nutritional quality. Fe uptake and translocation in plants require the coordinated expression of multiple genes, which is regulated by various transcription factors (TFs), especially members of the basic helix-loop-helix (bHLH) family. OsIRO3 was first reported as a repressor related to Fe homeostasis gene regulation in roots based on evidence in overexpressing rice. However, its functions in regulating rice growth and Fe homeostasis between roots and shoots during Fe deficiency is poorly understood. Here, we developed osiro3 knockout mutants by the CRISPR/Cas9 system. The leaves of osiro3 knockout mutants showed abnormal growth and development after Fe-deficiency treatment. Strong lesions occurred on the young leaves of osiro3 knockout mutants, which even led to plant death during severe Fe deficiency. Furthermore, accumulation of reactive oxygen species (ROS), Fe and manganese concentrations were all elevated in the shoots of osiro3 knockout mutants under the Fe deficiency stress. Quantitative RT-PCR showed that OsNAC4 for ROS induced plant hypersensitive cell death and OsFerritins for Fe storage and overload were all significantly activated in osiro3 knockout shoots after Fe deficiency treatment. In osiro3 knockout mutants, Fe-deficiency response genes were induced in roots, but suppressed in shoots, and furthermore Fe sufficiency/overload genes were induced in shoots under Fe-deficient conditions according to DNA microarray. Yeast two-hybrid analysis revealed a positive interaction between OsIRO3 and the homologous protein OsbHLH062, which was also transcriptionally increased in Fe-deficient roots. OsIRO3 is essential for maintaining plant survival in rice under Fe-deficient conditions and plays an important role in signal transmission from shoots to roots which is critical for plants to prevent Fe overload.

中文翻译：

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bHLH 蛋白 OsIRO3 对于缺铁条件下水稻 (Oryza sativa L.) 的植物存活和铁 (Fe) 稳态至关重要

摘要 铁 (Fe) 是大多数生物体的必需元素，因为它参与各种生物反应。铁缺乏是植物生长的主要制约因素，导致作物产量低和营养质量差。植物中铁的吸收和易位需要多个基因的协同表达，这些基因受各种转录因子 (TF) 的调控，尤其是基本螺旋-环-螺旋 (bHLH) 家族的成员。基于过度表达水稻的证据，OsIRO3 首次被报道为与根中 Fe 稳态基因调控相关的抑制因子。然而，人们对其在缺铁期间调节水稻生长和根和芽之间铁稳态的功能知之甚少。在这里，我们通过 CRISPR/Cas9 系统开发了 osiro3 敲除突变体。osiro3基因敲除突变体叶片在缺铁处理后生长发育异常。osiro3 基因敲除突变体的幼叶发生强烈病害，严重缺铁时甚至导致植株死亡。此外，在缺铁胁迫下，osiro3 敲除突变体的枝条中活性氧 (ROS) 的积累、Fe 和锰浓度均升高。定量RT-PCR表明，在缺铁处理后osiro3敲除芽中，用于ROS诱导的植物过敏性细胞死亡的OsNAC4和用于铁储存和过载的OsFerritins均被显着激活。在 osiro3 敲除突变体中，缺铁反应基因在根中被诱导，但在芽中被抑制，此外，根据DNA微阵列，在缺铁条件下在芽中诱导了Fe充足/过载基因。酵母双杂交分析显示 OsIRO3 与同源蛋白 OsbHLH062 之间存在正相互作用，该蛋白在缺铁根中也转录增加。OsIRO3对于在缺铁条件下维持水稻植物存活至关重要，并且在从芽到根的信号传递中起重要作用，这对于植物防止铁过载至关重要。