ENOD93 interacts with cytochrome c oxidase altering respiratory ATP production and root growth in plants – blue native gel protein complex identification

The early nodulin 93 (ENOD93) gene family in plants can regulate biological nitrogen fixation in legumes and nitrogen use efficiency in cereals but its molecular function is unknown. We show profile hidden Markov models define ENOD93 as a distant homolog of the N-terminal domain of RESPIRATORY SUPERCOMPLEX FACTOR 2 (RCF2). RCF2 is reported to regulate cytochrome oxidase (CIV) influencing the generation of a mitochondria proton motive force in yeast. Knockout of enod93 in Arabidopsis leads to a short root phenotype. ENOD93 is associated with a protein complex the size of CIV in isolated mitochondria but neither CIV abundance nor its activity in ruptured organelles changed in enod93. However, a progressive loss of ADP-dependent respiration rate was observed in enod93 mitochondria which could be fully recovered in complemented lines. Mitochondrial membrane potential was higher in enod93 but ATP synthesis and ADP depletion rates progressively decreased. Respiration rate of whole enod93 seedlings was elevated and root ADP content was nearly double that in WT without a change in ATP content. These altered energetic states correlated with elevated respiratory substrate levels in roots of enod93 compared to WT and complemented lines. Overexpression of ENOD93 lowered ATP content in roots and increased the abundance of a range of amino acids in both roots and leaves. We propose that two previously unconnected gene families in plants, ENOD93 and HYPOXIA INDUCED GENE DOMAIN, are the functional equivalent of yeast RCF2 but have remained undiscovered in many eukaryotic lineages because they are encoded in two separate genes.

植物中的早期结瘤素93（early nodulin 93, ENOD93）基因家族可调控豆科植物的生物固氮以及谷类作物的氮利用效率，但其分子功能至今尚未明确。本研究借助剖面隐马尔可夫模型（profile hidden Markov models）鉴定发现，ENOD93是呼吸超复合体因子2（RESPIRATORY SUPERCOMPLEX FACTOR 2, RCF2）N端结构域的远缘同源蛋白。已有研究表明，RCF2可调控细胞色素氧化酶（cytochrome oxidase, CIV），进而影响酵母线粒体质子动力势的产生。在拟南芥（Arabidopsis）中敲除enod93基因会导致植株出现根系缩短的表型。ENOD93与分离线粒体中分子量与CIV相当的蛋白质复合物相关，但enod93突变体中CIV的丰度及其在破裂细胞器中的活性均未发生改变。然而，在enod93突变体的线粒体中观察到ADP依赖性呼吸速率呈进行性下降，该表型可在互补转基因株系中完全恢复。enod93突变体的线粒体膜电位更高，但ATP合成速率与ADP消耗速率呈进行性降低。整株enod93突变体幼苗的呼吸速率升高，且根系中的ADP含量约为野生型（wild type, WT）的两倍，但其ATP含量未发生变化。上述能量代谢状态的改变与enod93突变体根系相较于野生型及互补株系中呼吸底物水平升高存在显著相关性。过表达ENOD93会降低根系的ATP含量，并提升根系与叶片中多种氨基酸的丰度。本研究提出，植物中两个此前未被发现存在关联的基因家族——ENOD93与低氧诱导基因结构域（HYPOXIA INDUCED GENE DOMAIN）——是酵母RCF2的功能等价物，但由于它们由两个独立的基因编码，因此在诸多真核生物谱系中尚未被鉴定出来。