Effects of Salinity and Abscisic Acid on Lipid Transfer Protein Accumulation, Suberin Deposition and Hydraulic Conductance in Pea Roots.

Lipid transfer proteins (LTPs) participate in many important physiological processes in plants, including adaptation to stressors, e.g., salinity. Here we address the mechanism of this protective action of LTPs by studying the interaction between LTPs and abscisic acid (ABA, a "stress" hormone) and their mutual participation in suberin deposition in root endodermis of salt-stressed pea plants. Using immunohistochemistry we show for the first time NaCl induced accumulation of LTPs and ABA in the cell walls of phloem paralleled by suberin deposition in the endoderm region of pea roots. Unlike LTPs which were found localized around phloem cells, ABA was also present within phloem cells. In addition, ABA treatment resulted in both LTP and ABA accumulation in phloem cells and promoted root suberization. These results suggested the importance of NaCl-induced accumulation of ABA in increasing the abundance of LTPs and of suberin. Using molecular modeling and fluorescence spectroscopy we confirmed the ability of different plant LTPs, including pea Ps-LTP1, to bind ABA. We therefore hypothesize an involvement of plant LTPs in ABA transport (unloading from phloem) as part of the salinity adaptation mechanism.

Role of Pea LTPs and Abscisic Acid in Salt-Stressed Roots.

Lipid transfer proteins (LTPs) are a class of small, cationic proteins that bind and transfer lipids and play an important role in plant defense. However, their precise biological role in plants under adverse conditions including salinity and possible regulation by stress hormone abscisic acid (ABA) remains unknown. In this work, we studied the localization of LTPs and ABA in the roots of pea plants using specific antibodies. Presence of LTPs was detected on the periphery of the cells mainly located in the phloem. Mild salt stress (50 mM NaCI) led to slowing plant growth and higher immunostaining for LTPs in the phloem. The deposition of suberin in Casparian bands located in the endoderma revealed with Sudan III was shown to be more intensive under salt stress and coincided with the increased LTP staining. All obtained data suggest possible functions of LTPs in pea roots. We assume that these proteins can participate in stress-induced pea root suberization or in transport of phloem lipid molecules. Salt stress increased ABA immunostaining in pea root cells but its localization was different from that of the LTPs. Thus, we failed to confirm the hypothesis regarding the direct influence of ABA on the level of LTPs in the salt-stressed root cells.

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.

Accumulation of cytokinins in roots and their export to the shoots of durum wheat plants treated with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP).

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.