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.

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.

Cytokinin producing bacteria stimulate amino acid deposition by wheat roots.

Phytohormone production is one mechanism by which rhizobacteria can stimulate plant growth, but it is not clear whether the bacteria gain from this mechanism. The hypothesis that microbial-derived cytokinin phytohormones stimulate root exudation of amino acids was tested. The rhizosphere of wheat plants was drenched with the synthetic cytokinin trans-zeatin or inoculated with Bacillus subtilis IB-22 (which produces zeatin type cytokinins) or B. subtilis IB-21 (which failed to accumulate cytokinins). Growing plants in a split root system allowed spatial separation of zeatin application or rhizobacterial inoculation to one compartment and analyses of amino acid release from roots (rhizodeposition) into the other compartment (without either microbial inoculation or treatment with exogenous hormone). Supplying B. subtilis IB-22 or zeatin to either the whole root system or half of the roots increased concentrations of amino acids in the soil solution although the magnitude of the increase was greater when whole roots were treated. There was some similarity in amino acid concentrations induced by either bacterial or zeatin treatment. Thus B. subtilis IB-22 increased amino acid rhizodeposition, likely due to its ability to produce cytokinins. Furthermore, B. subtilis strain IB-21, which failed to accumulate cytokinins in culture media, did not significantly affect amino acid concentrations in the wheat rhizosphere. The ability of rhizobacteria to produce cytokinins and thereby stimulate rhizodeposition may be important in enhancing rhizobacterial colonization of the rhizoplane.

Effect of partial root excision on transpiration, root hydraulic conductance and leaf growth in wheat seedlings.

Removal of four out of five roots did not lower transpiration and stomatal conductivity of wheat (Triticum durum Desf.) seedlings. Water content of mature expanded leaf lamina remained constant at control levels. The results suggest that the only remaining root was capable to supply the shoot with water. This was evidenced by an increase in hydraulic conductivity of the root system following partial root excision measured at low subatmospheric pressures induced by vacuum. In the absence of a hydrostatic gradient, water flow from reduced root system was initially not higher than from an intact system, but increased subsequently. ABA content was increased in roots 1 h after partial root excision, which might contribute to the increase in hydraulic conductivity.

Effect of partial root excision on shoot water relations.

Removing 4 out of 5 serminal roots from 7-day-old wheat seedlings arrested leaf elongation for 1.5 h. This effect can be explained by an initial decrease in foliar water content resulting from the smaller root surface area available for water uptake. Subsequently, leaf hydration increased with time and came to equal that of intact plants within 2 h. The rehydration was seemingly effected by an increasing conductivity of the one remaining root axis, since transpiration of the partially de-rooted plants did not fall below that of controls. With time, leaf elongation resumed, but at a slower rate than in intact plants. This slower growth may be attributed to a decrease in leaf extensibility since this was found to be reduced when measured by a counterweight technique involving linear displacement transducers. Loss of extensibility was associated with decreased IAA concentration in the leaf elongation zone.