Suzuki Yoshihito, Mizuno Toru, Urakami Eriko, Yamaguchi Isomaro, Asami Tadao
Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Plant Biotechnol J. 2008 May;6(4):355-67. doi: 10.1111/j.1467-7652.2008.00325.x. Epub 2008 Feb 14.
Immunomodulation is a means to modulate an organism's function by antibody production to capture either endogenous or exogenous antigens. This method was applied to plants to repress the function of gibberellins (GAs), a class of phytohormones responsible for plant elongation, by anti-bioactive GA antibodies. Two different antibodies were produced in Arabidopsis as single-chain variable fragment (scFv) fused to green fluorescent protein (GFP) with four different subcellular localizations: endoplasmic reticulum (ER), cytosol, apoplastic space or the outer surface of the plasma membrane. When targeting scFv-GFP to ER, plants showed the highest accumulation of scFv-GFP, with binding activity, strong GFP fluorescence in ER-derived compartments and mild but clear GA-deficient phenotypes, including a smaller leaf size, delayed bolting, shorter inflorescence length and decreased germination. Plants expressing scFv-GFP in ER responded to exogenous GA(4) and contained 15-40 times greater endogenous GA(4) than wild-type plants. They also showed increased gene expression for GA3ox1, GA20ox1 and GA20ox2, but decreased expression for GA2ox1, which are feedback and feedforward regulated by GA signalling, respectively. These results suggest that the level of free functional GA(4) decreased when trapped in the ER with scFv to the extent that mild GA-deficient phenotypes were created. A dramatic increase in the total sum of GA(4) (free plus scFv-GFP bound) was detected as a result of the up-regulation of GA biosynthesis (feedback regulated), and a decrease in GA(4) catabolism as a result of protection by scFv-GFP binding. This study demonstrates that the use of immunomodulation to inhibit the action of bioactive GAs is an effective method of creating GA-deficient plants.
免疫调节是一种通过产生抗体来捕获内源性或外源性抗原从而调节生物体功能的手段。该方法被应用于植物，通过抗生物活性赤霉素（GA）抗体来抑制赤霉素（一类负责植物伸长的植物激素）的功能。在拟南芥中产生了两种不同的抗体，它们是与绿色荧光蛋白（GFP）融合的单链可变片段（scFv），具有四种不同的亚细胞定位：内质网（ER）、细胞质、质外体空间或质膜外表面。当将scFv-GFP靶向内质网时，植物表现出scFv-GFP的最高积累量，具有结合活性，在内质网衍生的区室中有强烈的GFP荧光以及轻微但明显的GA缺陷表型，包括较小的叶片尺寸、抽薹延迟、较短的花序长度和发芽率降低。在内质网中表达scFv-GFP的植物对外源GA(4)有反应，并且其内源GA(4)含量比野生型植物高15 - 40倍。它们还显示出GA3ox1、GA20ox1和GA20ox2的基因表达增加，但GA2ox1的表达降低，这些分别受GA信号的反馈和前馈调节。这些结果表明，当与scFv一起被困在内质网中时，游离功能性GA(4)的水平降低到足以产生轻微GA缺陷表型的程度。由于GA生物合成的上调（反馈调节），检测到GA(4)（游离的加上与scFv-GFP结合的）总量急剧增加，并且由于scFv-GFP结合的保护，GA(4)分解代谢减少。这项研究表明，利用免疫调节来抑制生物活性GA的作用是创建GA缺陷植物的有效方法。
