Study of cytokinin transport from shoots to roots of wheat plants is informed by a novel method of differential localization of free cytokinin bases or their ribosylated forms by means of their specific fixation

Abstract

The aim of the present report was to demonstrate how a novel approach for immunohistochemical localization of cytokinins in the leaf and particularly in the phloem may complement to the study of their long-distance transport. Different procedures of fixation were used to conjugate either cytokinin bases or their ribosides to proteins of cytoplasm to enable visualization and differential localization of these cytokinins in the leaf cells of wheat plants. In parallel to immunolocalization of cytokinins in the leaf cells, we immunoassayed distribution of free bases of cytokinins, their nucleotides and ribosides between roots and shoots of wheat plants as well as their presence in phloem sap after incubation of leaves in a solution supplemented with either trans-zeatin or isopentenyladenine. The obtained data show ribosylation of the zeatin applied to the leaves and its elevated level in the phloem sap supported by in vivo localization showing the presence of ribosylated forms of zeatin in leaf vessels. This suggests that conversion of zeatin to its riboside is important for the shoot-to-root transport of zeatin-type cytokinins in wheat. Exogenous isopentenyladenine was not modified, but diffused from the leaves as free base. These metabolic differences may not be universal and may depend on the plant species and age. Although the measurements of cytokinins in the phloem sap and root tissue is the most defining for determining cytokinin transport, study of immunolocalization of either free cytokinin bases or their ribosylated forms may be a valuable source of information for predicting their transport in the phloem and to the roots.

Ethylene-Cytokinin Interaction Determines Early Defense Response of Wheat against Stagonospora nodorum Berk.

“…Different forms of cytokinins present in an aliquot of aqueous residue were concentrated on a C18 column (Waters Corporation, Milford, MA, USA), eluted with 5 mL of 80% ethanol and then evaporated to dryness. Cytokinin bases and their derivatives from the dry residue were separated by thin layer chromatography on silufol plates (Merck KGaA, Fluka, Darmstadt, Germany) in the system of solvents butanol: ammonium hydrate: water (6:1:2) according to the work of [40]. This procedure enabled separation and assay of cytokinin nucleotide (Rf 0-0.1), cytokinin glucosides (Rf 0.1-0.2), riboside of zeatin (ZR, Rf 0.4-0.5), isopentenyladenosine (iPA, Rf 0.5-0.6), zeatin (Z, Rf 0.6-0.7), and isopentenyladenine (iP, Rf 0.7-0.8).…”

“…The material from different zones was eluted with 0.1 M PB, pH 7.4 for 16 h. Then, silica gel was removed by 10-min centrifugation at 10,000× g in an Eppendorf 5415 K centrifuge. In the supernatant, phytohormone was assayed by means of ELISA as earlier described using specific antibodies [40]. Anti-trans-ZR and anti-iPA sera were used for the assay of cytokinins of Z and iP types, correspondingly [40].…”

“…In the supernatant, phytohormone was assayed by means of ELISA as earlier described using specific antibodies [40]. Anti-trans-ZR and anti-iPA sera were used for the assay of cytokinins of Z and iP types, correspondingly [40]. Their specificity has been described previously [40,41].…”

Rhizobacteria Inoculation Effects on Phytohormone Status of Potato Microclones Cultivated In Vitro under Osmotic Stress

“…Zeatin and isopentenyl adenine derivatives (free bases and ribosides) were assayed in the eluates of the zones corresponding to the position of standards with antibodies raised against ribosides of zeatin and isopentenyl adenine as described [ 30 ]. The reliability of the method was confirmed by comparison of its results with the data obtained with the help of HPLC combined with mass-spectrometry [ 31 , 32 ].…”

Limitation of Cytokinin Export to the Shoots by Nucleoside Transporter ENT3 and Its Linkage with Root Elongation in Arabidopsis

“…Antibodies had high immunoreactivity towards corresponding zeatin and their derivatives (ribosides, 9-Nglucosides, and nucleotides) and low cross-reactivity to cis-Z and iP-type cytokinins (less than 1%). This method has proven to be reliable by testing its results against physicochemical assay [28,29,33].…”