Data_Sheet_1_A Plant Gene Encoding One-Heme and Two-Heme Hemoglobins With Extreme Reactivities Toward Diatomic Gases and Nitrite.pdf

In plants, symbiotic hemoglobins act as carriers and buffers of O2 in nodules, whereas nonsymbiotic hemoglobins or phytoglobins (Glbs) are ubiquitous in tissues and may perform multiple, but still poorly defined, functions related to O2 and/or nitric oxide (NO). Here, we have identified a Glb gene of the model legume Medicago truncatula with unique properties. The gene, designated MtGlb1-2, generates four alternative splice forms encoding Glbs with one or two heme domains and 215–351 amino acid residues. This is more than double the size of any hemoglobin from plants or other organisms described so far. A combination of molecular, cellular, biochemical, and biophysical methods was used to characterize these novel proteins. RNA-sequencing showed that the four splice variants are expressed in plant tissues. MtGlb1-2 is transcriptionally activated by hypoxia and its expression is further enhanced by an NO source. The gene is preferentially expressed in the meristems and vascular bundles of roots and nodules. Two of the proteins, bearing one or two hemes, were characterized using mutants in the distal histidines of the hemes. The Glbs are extremely reactive toward the physiological ligands O2, NO, and nitrite. They show very high O2 affinities, NO dioxygenase activity (in the presence of O2), and nitrite reductase (NiR) activity (in the absence of O2) compared with the hemoglobins from vertebrates and other plants. We propose that these Glbs act as either NO scavengers or NO producers depending on the O2 tension in the plant tissue, being involved in the fast and fine tuning of NO concentration in the cytosol in response to sudden changes in O2 availability.

在植物体内，共生血红蛋白充当结瘤中氧气的携带者和缓冲剂，而非共生血红蛋白或植物血红蛋白（Glbs）则普遍存在于组织中，可能执行与氧气和/或一氧化氮（NO）相关的多种但尚未明确的功能。本研究中，我们鉴定了一种具有独特特性的Glbs基因，来自模式豆科植物Medicago truncatula。该基因被命名为MtGlb1-2，可产生四种不同的剪接形式，编码具有一个或两个血红素结构域和215至351个氨基酸残基的Glbs。这一尺寸是迄今为止所描述的来自植物或其他生物的任何血红蛋白的两倍以上。通过结合分子、细胞、生化及生物物理方法，我们对这些新型蛋白进行了特性表征。RNA测序显示，这四种剪接变异体在植物组织中均有表达。在缺氧条件下，MtGlb1-2基因的转录被激活，其表达在NO源存在时进一步增强。该基因在根和结瘤的分生组织和维管束中优先表达。通过使用血红素远端组氨酸的突变体，对带有一个或两个血红素的两种蛋白进行了特性研究。与脊椎动物和其他植物中的血红蛋白相比，这些Glbs对生理配体氧气、NO和亚硝酸盐具有极高的反应性。它们表现出极高的氧气亲和力、NO双加氧酶活性（在氧气存在时）以及亚硝酸盐还原酶（NiR）活性（在氧气不存在时）。我们提出，这些Glbs根据植物组织中的氧气张力，可作为NO清除剂或NO生成剂，参与对细胞质中NO浓度的快速和精细调节，以响应氧气供应的突然变化。