The Auxin Binding Protein 1 (ABP1) is one of the most studied proteins in plants. Since decades ago, it has been the prime receptor candidate for the plant hormone auxin with a plethora of described functions in auxin signaling and development. The developmental importance of ABP1 has recently been questioned by identification of Arabidopsis thaliana abp1 knock-out alleles that show no obvious phenotypes under normal growth conditions. In this study, we examined the contradiction between the normal growth and development of the abp1 knock-outs and the strong morphological defects observed in three different ethanol-inducible abp1 knock-down mutants ( abp1-AS, SS12K, SS12S). By analyzing segregating populations of abp1 knock-out vs. abp1 knock-down crosses we show that the strong morphological defects that were believed to be the result of conditional down-regulation of ABP1 can be reproduced also in the absence of the functional ABP1 protein. This data suggests that the phenotypes in abp1 knock-down lines are due to the off-target effects and asks for further reflections on the biological function of ABP1 or alternative explanations for the missing phenotypic defects in the abp1 loss-of-function alleles.

• Klíčová slova
• AUXIN BINDING PROTEIN 1 (ABP1), Arabidopsis, auxin, knock-down mutant, off-target,
• Publikační typ
• časopisecké články MeSH

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
• Názvy látek
• AHP3 protein, Arabidopsis MeSH Prohlížeč
• CKI1 protein, Arabidopsis MeSH Prohlížeč
• fosfotransferasy MeSH
• proteinkinasy MeSH
• proteiny huseníčku MeSH
• protilátky MeSH
• rekombinantní proteiny MeSH

BACKGROUND: Auxin binding protein 1 (ABP1) is a putative auxin receptor and its function is indispensable for plant growth and development. ABP1 has been shown to be involved in auxin-dependent regulation of cell division and expansion, in plasma-membrane-related processes such as changes in transmembrane potential, and in the regulation of clathrin-dependent endocytosis. However, the ABP1-regulated downstream pathway remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: Using auxin transport assays and quantitative analysis of cellular morphology we show that ABP1 regulates auxin efflux from tobacco BY-2 cells. The overexpression of ABP1can counterbalance increased auxin efflux and auxin starvation phenotypes caused by the overexpression of PIN auxin efflux carrier. Relevant mechanism involves the ABP1-controlled vesicle trafficking processes, including positive regulation of endocytosis of PIN auxin efflux carriers, as indicated by fluorescence recovery after photobleaching (FRAP) and pharmacological manipulations. CONCLUSIONS/SIGNIFICANCE: The findings indicate the involvement of ABP1 in control of rate of auxin transport across plasma membrane emphasizing the role of ABP1 in regulation of PIN activity at the plasma membrane, and highlighting the relevance of ABP1 for the formation of developmentally important, PIN-dependent auxin gradients.

• MeSH
• Arabidopsis cytologie   metabolismus   MeSH
• buněčné linie MeSH
• FRAP MeSH
• konfokální mikroskopie MeSH
• kyseliny indoloctové metabolismus   MeSH
• modulátory membránového transportu metabolismus   MeSH
• receptory buněčného povrchu biosyntéza   metabolismus   MeSH
• rostlinné proteiny biosyntéza   metabolismus   MeSH
• tabák cytologie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• auxin-binding protein 1 MeSH Prohlížeč
• endoplasmic reticulum auxin-binding protein 4, Zea mays MeSH Prohlížeč
• kyseliny indoloctové MeSH
• modulátory membránového transportu MeSH
• receptory buněčného povrchu MeSH
• rostlinné proteiny MeSH