Cytokinin flow from roots to shoots can serve as a long-distance signal important for root-to-shoot communication. In the past, changes in cytokinin flow from roots to shoots have been mainly attributed to changes in the rate of synthesis or breakdown in the roots. The present research tested the possibility that active uptake of cytokinin by root cells may also influence its export to shoots. To this end, we collapsed the proton gradient across root membranes using the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to inhibit secondary active uptake of exogenous and endogenous cytokinins. We report the impact of CCCP on cytokinin concentrations and delivery in xylem sap and on accumulation in shoots of 7-day-old wheat plants in the presence and absence of exogenous cytokinin applied as zeatin. Zeatin treatment increased the total accumulation of cytokinin in roots and shoots but the effect was smaller for the shoots. Immunohistochemical localization of cytokinins using zeatin-specific antibodies showed an increase in immunostaining of the cells adjacent to xylem in the roots of zeatin-treated plants. Inhibition of secondary active cytokinin uptake by CCCP application decreased cytokinin accumulation in root cells but increased both flow from the roots and accumulation in the shoots. The possible importance of secondary active uptake of cytokinins by root cells for the control of their export to the shoot is discussed.

Although nutrient deprivation alters the concentrations of several plant hormones, the role of each in decreasing shoot-to-root ratio is not clear. A 10-fold dilution of the nutrient concentration supplied to hydroponically-grown 7-day-old durum wheat (Triticum turgidum L. ssp. durum Desf.) plants decreased shoot growth, shoot-to-root ratio and shoot and root cytokinin concentrations, increased shoot ABA concentration and shoot cytokinin oxidase activity, but had no effect on xylem sap ABA and cytokinin concentrations. Nutrient deprivation also increased xylem concentrations of conjugated ABA. The role of ABA in these responses was addressed by adding 11.4 µm ABA to the nutrient solution of well fertilised plants, or 1.2 mm fluridone (an inhibitor of ABA biosynthesis) to the nutrient solution of nutrient-deprived plants. The former induced similar changes in shoot-to-root ratio (by inhibiting shoot growth), shoot ABA concentration, shoot and root cytokinin concentrations and shoot cytokinin oxidase activity as nutrient deprivation. Conversely, fluridone addition to nutrient-deprived plants restored shoot-to-root ratio (by inhibiting root growth), shoot ABA concentration, shoot and root cytokinin concentrations to levels similar to well fertilised plants. Although root growth maintenance during nutrient deprivation depends on a threshold ABA concentration, shoot growth inhibition is independent of shoot ABA status. Although fluridone decreased shoot cytokinin oxidase activity of nutrient-deprived plants, it was still 1.7-fold greater than well fertilised plants, implying that nutrient deprivation could also activate shoot cytokinin oxidase independently of ABA. These data question the root signal basis of cytokinin action, but demonstrate that changes in ABA status can regulate shoot cytokinin concentrations via altering their metabolism.

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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.

Although changes in root architecture in response to the environment can optimize mineral and water nutrient uptake, mechanisms regulating these changes are not well-understood. We investigated whether P deprivation effects on root development are mediated by abscisic acid (ABA) and its interactions with other hormones. The ABA-deficient barley mutant Az34 and its wild-type (WT) were grown in P-deprived and P-replete conditions, and hormones were measured in whole roots and root tips. Although P deprivation decreased growth in shoot mass similarly in both genotypes, only the WT increased primary root length and number of lateral roots. The effect was accompanied by ABA accumulation in root tips, a response not seen in Az34. Increased ABA in P-deprived WT was accompanied by decreased concentrations of cytokinin, an inhibitor of root extension. Furthermore, P-deficiency in the WT increased auxin concentration in whole root systems in association with increased root branching. In the ABA-deficient mutant, P-starvation failed to stimulate root elongation or promote branching, and there was no decline in cytokinin and no increase in auxin. The results demonstrate ABA’s ability to mediate in root growth responses to P starvation in barley, an effect linked to its effects on cytokinin and auxin concentrations.