Temporal relationships between nitrogenase and intercellular glycoprotein in developing white lupin nodules
Temporal relationships between nitrogenase and intercellular glycoprotein in developing white lupin nodules
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DOI：
10.1006/anbo.1996.0376
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发表时间：
1997-05-01
期刊：
影响因子：
4.2
通讯作者：
Sprent, JI
中科院分区：
文献类型：
--
作者：
James, EK;Minchin, FR;Sprent, JI
白羽扇豆(Lupinus albus L.)固氮共生研究进展CV。分别于感染后0、8、12、17和20d进行乙炔还原试验(ARA)、蛋白提取液免疫印迹和显微镜/免疫金标记法对多发性和慢型根瘤菌ISLU16进行跟踪观察。在0d、8d和12d，虽然肉眼可见的根瘤原基已经在根上形成，但在0、8和12d没有ARA。用固氮酶特异性抗体对免疫金标记法的否定信号证实了这些时间段固氮酶的缺乏。AR17d 6株中有3株有ARA，其中的根瘤与固氮酶抗体呈阳性反应。相比之下，17d时ARA(-)(FIX(-))结节较小(平均半径为0.49 mm，FIX(+)结节为1.01 mm)，且与固氮酶抗体呈阴性信号。用单抗MAC236和MAC265(识别与根瘤菌侵染和根瘤氧扩散调控有关的糖蛋白上的两个表位)提取的根瘤蛋白的Western blotting与20d植株的根瘤(FIX+)和17d的FIX(-)植株的根瘤(FIX+)有很强的信号。MAC236/MAC265的信号在17d的植株根瘤中明显较弱，而在0、8和12d收获的根瘤/节根中没有明显的信号。然而，进一步的免疫金标记法显示，MAC236和MAC265不仅在20d和17d的FIX(+)/FIX(-)结节的皮质细胞间隙中表达，而且在8d的新感染组织周围的发育中的皮质以及12d结节的皮质和感染组织中也有强烈的表达。这些数据表明，MAC‘36和MAC265识别的糖蛋白在固氮酶表达和功能开始之前就存在，但表位的表达似乎在N-2融合开始后增强。对所有收获的根瘤进行了侵染线的调查，因为MAC236/MAC265-1生态识别的糖蛋白也是其他豆科植物根瘤中侵染线基质的组成部分。除20d的结节外，所有收获的结节中均未见侵染线，且仅在连续切片后才能看到。后者显示，偶尔有短而宽的侵染线进入并释放根瘤菌到感染组织内的未感染细胞的小口袋中，但不是在分生组织内。这些侵染线的基质用MAC236和MAC265弱标记或根本不标记，由此得出结论，在羽扇豆瘤提取液中检测到的IMAC236/MAC265大部分来自皮质细胞间隙中的糖丙氨酸。(C)1997年植物学公司年鉴。
The development of the N-2-fixing symbiosis between white lupin (Lupinus albus L.) cv. Multolupa and Bradyrhizobium strain ISLU16 was followed using the acetylene reduction assay (ARA), immunoblots of protein extracts, and microscopy/immunogold labelling at 0, 8, 12, 17 and 20 d after infection. There was no ARA at 0, 8 and 12 d, although macroscopically visible nodule primordia had formed on roots by 8 d. The lack of nitrogenase at these times was confirmed by a negative signal to immunogold labelling with nitrogenase-specific antibodies. Ar 17 d three out of six plants had ARA, and nodules from these gave a positive signal with the nitrogenase antibody. By contrast, ARA(-) (fix(-)) nodules at 17 d were smaller (mean radius of 0.49 mm compared to 1.01 mm with fix(+) nodules) and gave a negative signal with the nitrogenase antibody. Western blots of nodule protein extracts using the monoclonal antibodies MAC236 and MAC265 (which recognize two epitopes on a glycoprotein which is considered to beinvolved in both rhizobial infection and the regulation ofnodule oxygen diffusion) gave a strong signal with nodules (fix+) from 20 d plants and with 17 d fix(-) plants. The signal with 1MAC236/MAC265 was substantially weaker with nodules from 17 d fix- plants, and there was no signal apparent from nodules/nodulated roots from the 0, 8 and 12d harvests. However, further investigation using immunogold labelling revealed that not only were MAC236 and MAC265 expressed within cortical intercellular spaces in 20 d and 17 d fix(+)/fix(-) nodules, but they were also strongly expressed in the developing cortex surrounding the newly-infected tissue in 8 d nodules, as well as in intercellular spaces within the cortex and infected tissue of 12 d nodules. These data demonstrate that the glycoprotein recognized by MAC'36 and MAC265 is present before the onset of nitrogenase expression and function, but expression of the epitopes appears to be enhanced from the onset of N-2 futation. Nodules at all harvests were investigated for the presence of infection threads, as the MAC236/MAC265-1 ecognized glycoprotein is also a component of the infection thread matrix in nodules from other legumes. Infection threads were not seen in nodules from any of the harvests except for the 20 d nodules, and then only after serial sectioning. The latter revealed occasional short wide infection threads entering and releasing rhizobia into small pockets of uninfected cells, within the infected tissue, but not within the meristems. The matrix of these infection threads labelled weakly, or not at all, with MAC236 and MAC265, and it was concluded that the majority of the IMAC236/MAC265 detected in lupin nodule extracts originated from glycoprolein within cortical intercellular spaces. (C) 1997 Annals of Botany Company.