Symbiotic nitrogen fixation within nitrogen-fixing clade (NFC) plants is thought to have arisen from a single gain followed by massive losses in the genomes of ancestral non-nodulating plants. However, molecular evidence supporting this model is limited. Here, we confirm through bioinformatic analysis that NODULES WITH ACTIVATED DEFENSE1 (NAD1) is present only in NFC plants and is thus an NFC-specific gene. Moreover, NAD1 was specifically expressed in nodules. We identified three conserved nodulation-associated cis-regulatory elements (NACE1–3) in the promoter of LjNAD1 from Lotus japonicus that are required for its nodule specific expression. A survey of NFC plants revealed that NACE1 and NACE2 are specific to the Fabales and Papilionoideae, respectively, while NACE3 is present in all NFC plants. Moreover, we found that nodule inception (NIN) directly binds to all three NACEs to activate NAD1 expression. Mutation of L. japonicus LjNAD1 resulted in the formation of abnormal symbiosomes with enlarged symbiosome space and frequent breakdown of bacteroids in nodules, resembling phenotypes reported for Medicago truncatula Mtnad1 and Mtnin mutants. These data point to NIN–NAD1 as an important module regulating rhizobial accommodation in nodules. The regulation of NAD1 by NIN in the NFC ancestor represent an important evolutionary adaptation for nodulation.

中文翻译：

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保守的顺式元件使结节在结节过程中通过 NODULE INCEPTION 激活DEFENSE1调节结节


固氮进化枝 （NFC） 植物内的共生固氮被认为是由祖先非结瘤植物基因组的单次增益和大量损失引起的。然而，支持该模型的分子证据有限。在这里，我们通过生物信息学分析证实，具有活化DEFENSE1的结节 （NAD1） 仅存在于 NFC 植物中，因此是 NFC 特异性基因。此外，NAD1 在结节中特异性表达。我们在 Lotus japonicus 的 LjNAD1 启动子中鉴定了三个保守的结瘤相关顺式调节元件 （NACE1-3），这是其根瘤特异性表达所必需的。对 NFC 植物的调查显示，NACE1 和 NACE2 分别是 Fabales 和 Papilionoideae 的特异性，而 NACE3 存在于所有 NFC 植物中。此外，我们发现结节起始 （NIN） 直接与所有三种 NACE 结合以激活 NAD1 表达。L. japonicus LjNAD1 突变导致异常共生体的形成，共生体空间扩大，结节中类杆菌频繁分解，类似于苜蓿 Mtnad1 和 Mtnin 突变体报道的表型。这些数据表明 NIN-NAD1 是调节结节中根瘤调节的重要模块。NFC 祖先中 NIN 对 NAD1 的调节代表了对结瘤的重要进化适应。