Bonilla I, Mergold-Villaseñor C, Campos M E, Sánchez N, Pérez H, López L, Castrejón L, Sánchez F, Cassab G I
Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Spain.
Plant Physiol. 1997 Dec;115(4):1329-40. doi: 10.1104/pp.115.4.1329.
B-deficient bean (Phaseolus vulgaris L.) nodules examined by light microscopy showed dramatic anatomical changes, mainly in the parenchyma region. Western analysis of total nodule extracts examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that one 116-kD polypeptide was recognized by antibodies raised against hydroxyproline-rich glycoproteins (HRGPs) from the soybean (Glycine max) seed coat. A protein with a comparable molecular mass of 116 kD was purified from the cell walls of soybean root nodules. The amino acid composition of this protein is similar to the early nodulin (ENOD2) gene. Immunoprecipitation of the soybean ENOD2 in vitro translation product showed that the soybean seed coat anti-HRGP antibodies recognized this early nodulin. Furthermore, we used these antibodies to localize the ENOD2 homolog in bean nodules. Immunocytochemistry revealed that in B-deficient nodules ENOD2 was absent in the walls of the nodule parenchyma. The absence of ENOD2 in B-deficient nodules was corroborated by performing hydroxyproline assays. Northern analysis showed that ENOD2 mRNA is present in B-deficient nodules; therefore, the accumulation of ENOD2 is not affected by B deficiency, but its assembly into the cell wall is. B-deficient nodules fix much less N2 than control nodules, probably because the nodule parenchyma is no longer an effective O2 barrier.
通过光学显微镜检查发现，缺硼菜豆（菜豆属普通菜豆）根瘤出现了显著的解剖学变化，主要发生在薄壁组织区域。对通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检查的根瘤总提取物进行蛋白质免疫印迹分析表明，一种116-kD的多肽能被针对大豆（大豆属）种皮富含羟脯氨酸糖蛋白（HRGPs）产生的抗体识别。从大豆根瘤细胞壁中纯化出了一种分子量相当的116 kD蛋白质。该蛋白质的氨基酸组成与早期结瘤素（ENOD2）基因相似。对大豆ENOD2体外翻译产物进行免疫沉淀表明，大豆种皮抗HRGP抗体能识别这种早期结瘤素。此外，我们用这些抗体在菜豆根瘤中定位ENOD2同源物。免疫细胞化学显示，在缺硼根瘤中，结节薄壁组织的细胞壁中不存在ENOD2。通过进行羟脯氨酸测定证实了缺硼根瘤中不存在ENOD2。Northern分析表明，缺硼根瘤中存在ENOD2 mRNA；因此，ENOD2的积累不受硼缺乏的影响，但其组装到细胞壁中受到影响。缺硼根瘤固定的N2比对照根瘤少得多，可能是因为根瘤薄壁组织不再是有效的氧气屏障。
