With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.

• MeSH
• Arabidopsis genetika   MeSH
• cytosol imunologie   metabolismus   MeSH
• fosfotransferasy biosyntéza   genetika   imunologie   MeSH
• mapy interakcí proteinů genetika   imunologie   MeSH
• proteinkinasy biosyntéza   genetika   imunologie   MeSH
• proteiny huseníčku biosyntéza   genetika   imunologie   MeSH
• protilátky aplikace a dávkování   imunologie   MeSH
• regulace genové exprese u rostlin MeSH
• rekombinantní proteiny aplikace a dávkování   imunologie   MeSH
• signální transdukce MeSH
• umlčování genů imunologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cytokinins comprise a group of phytohormones with an organ-specific mode of action. Although the mechanisms controlling the complex networks of cytokinin metabolism are partially known, the role of individual cytokinin types in the maintenance of cytokinin homeostasis remains unclear. Utilizing the overproduction of single-chain Fv antibodies selected for their ability to bind trans-zeatin riboside and targeted to the endoplasmic reticulum, we post-synthetically modulated cytokinin ribosides, the proposed transport forms of cytokinins. We observed asymmetric activity of cytokinin biosynthetic genes and cytokinin distribution in wild-type tobacco seedlings with higher cytokinin abundance in the root than in the shoot. Antibody-mediated modulation of cytokinin ribosides further enhanced the relative cytokinin abundance in the roots and induced cytokinin-related phenotypes in an organ-specific manner. The activity of cytokinin oxidase/dehydrogenase in the roots was strongly up-regulated in response to antibody-mediated formation of the cytokinin pool in the endoplasmic reticulum. However, we only detected a slight decrease in the root cytokinin levels. In contrast, a significant decrease of cytokinins occurred in the shoot. We suggest the roots as the main site of cytokinin biosynthesis in tobacco seedlings. Conversely, cytokinin levels in the shoot seem to depend largely on long-range transport of cytokinin ribosides from the root and their subsequent metabolic activation.

• MeSH
• cytokininy fyziologie   MeSH
• fenotyp * MeSH
• homeostáza * MeSH
• isopentenyladenosin analogy a deriváty   metabolismus   MeSH
• protilátky produkované rostlinami fyziologie   MeSH
• regulátory růstu rostlin fyziologie   MeSH
• semenáček fyziologie   MeSH
• tabák fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• autofagie * fyziologie   MeSH
• biotest metody   normy   MeSH
• lidé MeSH
• počítačová simulace MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Extramural MeSH
• směrnice MeSH