<em>Arabidopsis thaliana</em> MIRO1 and MIRO2 GTPases Are Unequally Redundant in Pollen Tube Growth and Fusion of Polar Nuclei during Female Gametogenesis

MIRO GTPases have evolved to regulate mitochondrial trafficking and morphology in eukaryotic organisms. A previous study showed that T-DNA insertion in the Arabidopsis MIRO1 gene is lethal during embryogenesis and affects pollen tube growth and mitochondrial morphology in pollen, whereas T-DNA insertion in MIRO2 does not affect plant development visibly. Phylogenetic analysis of MIRO from plants revealed that MIRO 1 and 2 orthologs in dicots cluster in two separate groups due to a gene/genome duplication event, suggesting that functional redundancy may exists between the two MIRO genes. To investigate this possibility, we generated miro1(+/−)/miro2-2(−/−) plants. Compared to miro1(+/−)plants, the miro1(+/−)/miro2-2(−/−) plants showed increased segregation distortion. miro1(+/−)/miro2-2(−/−) siliques contained less aborted seeds, but more than 3 times the number of undeveloped ovules. In addition, reciprocal crosses showed that co-transmission through the male gametes was nearly absent, whereas co-transmission through the female gametes was severely reduced in miro1(+/−)/miro2-2(−/−) plants. Further investigations revealed that loss of MIRO2 (miro2(−/−)) function in the miro1(+/−) background enhanced pollen tube growth defects. In developing miro1(+/−)/miro2(−/−) embryo sacs, fusion of polar nuclei was further delayed or impaired compared to miro1 plants. This phenotype has not been reported previously for miro1 plants and coincides with studies showing that defects in some mitochondria-targeted genes results in the same phenotype. Our observations show that loss of function in MIRO2 in a miro1(+/−) background enhances the miro1(+/−) phenotype significantly, even though miro2(−/−) plants alone does not display any phenotypes. Based on these findings, we conclude that MIRO1 and MIRO2 are unequally redundant and that a proportion of the miro1(+/−)/miro2(−/−) plants haploid gametes displays the complete null phenotype of MIRO GTPase function at key developmental stages.MIRO GTP酶（MIRO GTPases）在真核生物中演化出调控线粒体运输与形态的功能。已有研究表明，拟南芥（Arabidopsis）MIRO1基因的T-DNA插入会导致胚胎发育致死，并影响花粉管生长及花粉内的线粒体形态；而MIRO2基因的T-DNA插入则不会对植物发育造成明显可见的影响。对植物来源MIRO的系统发育分析显示，双子叶植物中的MIRO1与MIRO2同源基因因一次基因/基因组复制事件，分别聚为两个独立的类群，这提示两个MIRO基因间可能存在功能冗余。为探究这一可能性，本研究构建了miro1(+/−)/miro2-2(−/−)双突变植株。与仅携带miro1(+/−)的植株相比，该双突变植株的分离扭曲程度显著升高。miro1(+/−)/miro2-2(−/−)的角果中败育种子更少，但未发育胚珠的数量是前者的3倍以上。此外，正反交实验显示，在miro1(+/−)/miro2-2(−/−)植株中，雄配子体的共传递几乎完全缺失，而雌配子体的共传递则显著受损。进一步研究发现，在miro1(+/−)遗传背景下，MIRO2功能缺失（miro2(−/−)）会加剧花粉管生长缺陷。与仅miro1(+/−)植株相比，发育中的miro1(+/−)/miro2(−/−)胚囊中，极核融合进一步延迟或受阻。该表型此前未在miro1单突变植株中被报道，且与部分线粒体靶向基因缺陷所引发的表型一致。本研究结果表明，在miro1(+/−)背景下缺失MIRO2功能，可显著增强miro1(+/−)的突变表型——即便单独的miro2(−/−)植株并无任何可见表型。基于上述发现，本研究得出结论：MIRO1与MIRO2存在不均等的功能冗余，且部分miro1(+/−)/miro2(−/−)植株的单倍体配子体在关键发育阶段会呈现MIRO GTP酶功能完全缺失的表型。