Microscale magnetic microparticle-based immunopurification of cytokinins from Arabidopsis root apex

0476413 - ÚEB 2018 RIV US eng J - Journal Article
Plačková, Lenka - Oklešťková, Jana - Pospíšková, K. - Poláková, K. - Buček, J. - Stýskala, J. - Zatloukal, M. - Šafařík, Ivo - Zbořil, R. - Strnad, Miroslav - Doležal, Karel - Novák, Ondřej
Microscale magnetic microparticle-based immunopurification of cytokinins from Arabidopsis root apex.
Plant Journal. Roč. 89, č. 5 (2017), s. 1065-1075. ISSN 0960-7412. E-ISSN 1365-313X
R&D Projects: GA MŠMT(CZ) LO1204; GA MŠMT LK21306; GA ČR GA14-34792S
Institutional support: RVO:61389030 ; RVO:60077344
Keywords : solid-phase extraction * tandem mass-spectrometry * endogenous cytokinins * plant-samples * cis-zeatin * quantitative-analysis * aromatic cytokinins * abscisic-acid * oryza-sativa * purification * cytokinins * magnetic microparticles * immunoaffinity separation * metabolite profiling * Arabidopsis thaliana * technical advance
OECD category: Plant sciences, botany
Impact factor: 5.775, year: 2017

Cytokinins (CKs) are pivotal plant hormones that have crucial roles in plant growth and development. However, their isolation and quantification are usually challenging because of their extremely low levels in plant tissues (pmol g(-1) fresh weight). We have developed a simple microscale magnetic immunoaffinity-based method for selective one-step isolation of CKs from very small amounts of plant tissue (less than 0.1 mg fresh weight). The capacity of the immunosorbent and the effect of the complex plant matrix on the yield of the rapid one-step purification were tested using a wide range of CK concentrations. The total recovery range of the new microscale isolation procedure was found to be 30-80% depending on individual CKs. Immunoaffinity extraction using group-specific monoclonal CK antibodies immobilized onto magnetic microparticles was combined with a highly sensitive ultrafast mass spectrometry-based method with a detection limit close to one attomole. This combined approach allowed metabolic profiling of a wide range of naturally occurring CKs (bases, ribosides and N-9-glucosides) in 1.0-mm sections of the Arabidopsis thaliana root meristematic zone. The magnetic immunoaffinity separation method was shown to be a simple and extremely fast procedure requiring minimal amounts of plant tissue.
Permanent Link: http://hdl.handle.net/11104/0272921