CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation
CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation
复制标题
CRISPR/Cas9敲除百脉根豆血红蛋白基因揭示了它们在共生固氮中的协同作用
DOI：
10.1111/nph.16077
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发表时间：
2019-10-01
期刊：
影响因子：
9.4
通讯作者：
Duanmu, Deqiang
中科院分区：
文献类型：
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作者：
Wang, Longlong;Rubio, Maria Carmen;Duanmu, Deqiang
豆科植物根瘤含有由多个基因编码的高浓度豆类血红蛋白。造成这种多样性的原因尚不清楚。利用CRISPR/Cas9技术获得了3个莲花LBS的稳定突变体。从生理、生化和分子水平对其表型进行了鉴定。对三个突变体的根瘤进行电子显微镜观察和RNA测序(RNA-seq)分析。互补研究表明，Lbs协同作用维持最佳的N-2固定。三个突变体的根瘤均产生过多的超氧阴离子自由基和过氧化氢，这可能与NADPH氧化酶的激活和超氧化物歧化酶亚型表达的变化有关。突变的根瘤表现出主要的超微结构变化，包括空泡化、聚β-羟基丁酸酯的积聚和线粒体的破坏。约20000个基因的RNA-SEQ显示，碳、氮代谢基因、转录因子和蛋白酶的表达发生了显著变化。缺乏Lb的根瘤的血红素含量减少了约30-50倍，但血红素生物合成基因的转录水平相似，这表明了血红素合成的翻译后调节机制。我们得出结论，Lbs在根瘤中的作用是相加的，Lbs的缺乏导致了根瘤的早期衰老。我们的观察还提供了与Lb缺乏相关的基因表达网络的重新编程的洞察力，可能是由于细胞内游离O-2浓度不受控制所致。
Legume nodules contain high concentrations of leghemoglobins (Lbs) encoded by several genes. The reason for this multiplicity is unknown. CRISPR/Cas9 technology was used to generate stable mutants of the three Lbs of Lotus japonicus. The phenotypes were characterized at the physiological, biochemical and molecular levels. Nodules of the triple mutants were examined by electron microscopy and subjected to RNA-sequencing (RNA-seq) analysis. Complementation studies revealed that Lbs function synergistically to maintain optimal N-2 fixation. The nodules of the triple mutants overproduced superoxide radicals and hydrogen peroxide, which was probably linked to activation of NADPH oxidases and changes in superoxide dismutase isoforms expression. The mutant nodules showed major ultrastructural alterations, including vacuolization, accumulation of poly-beta-hydroxybutyrate and disruption of mitochondria. RNA-seq of c. 20 000 genes revealed significant changes in expression of carbon and nitrogen metabolism genes, transcription factors, and proteinases. Lb-deficient nodules had c. 30-50-fold less heme but similar transcript levels of heme biosynthetic genes, suggesting a post-translational regulatory mechanism of heme synthesis. We conclude that Lbs act additively in nodules and that the lack of Lbs results in early nodule senescence. Our observations also provide insight into the reprogramming of the gene expression network associated with Lb deficiency, probably as a result of uncontrolled intracellular free O-2 concentration.