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

Влияние дефицита фосфора на рост корней, продукцию активных форм кислорода и содержание гормонов в растениях ячменя

The article presents the results of research into the effect of removing phosphate from a nutrient medium on the content of auxins and cytokinins in roots, root elongation and content of reactive oxygen species in root tips of the barley ‘Steptoe’. In our experiments, the growth response was detected as root elongation after a 4-day exposure to a phosphate-free medium. Activation of linear root growth was preceded by changes in hormonal balance and in the level of reactive oxygen species. Auxin content in the roots increased after 6 h of phosphate starvation and a two-fold increase in the concentration of auxins in roots was detected by the end of the first day of the exposure to the phosphate deficit conditions. Staining with diaminobenzidine revealed an increased level of reactive oxygen species in the root tips of phosphate-starved plants after 6 h of exposure. However, after one day (24 h), a reverse pattern was observed: the level of staining was higher in the plants supplied with phosphates. Immunolocalisation of cytokinins in the root tips, where the zones of cell division and extension determining root elongation are located, showed a decreased content of zeatin in the cells under the effect of phosphorus deficit. The obtained data suggest that the detected rise in the amount of reactive oxygen species was due to the increased concentration of auxins accumulated as a result of the phosphate deficit effect on the barley plants. The increase in ROS and auxins contents could in turn influence the level of cytokinins and, in the end, affect root elongation. Further experiments are needed to test this hypothesisВ работе представлены результаты изучения влияния удаления фосфатов из питательного раствора на содержание ауксинов и цитокининов в корнях, удлинение корней и содержание активных форм кислорода в кончиках корней растений ячменя. Ростовая реакция, проявившаяся в наших экспериментах в удлинении корней растений ячменя сорта Steptoe, была отмечена после 4 суток воздействия бесфосфатной среды. Активации линейной скорости роста корней предшествовали изменения гормонального баланса и уровня реактивных форм кислорода. C помощью метода иммуноферментного анализа через 6 часов воздействия дефицита фосфора было отмечено достоверное возрастание, а к концу первых суток – двукратное увеличение концентрации ауксинов в корне. На фоне фосфатного голодания окрашивание корней диаминобензидином позволило выявить повышенный уровень реактивных форм кислорода в кончиках корней через 6 часов; по истечении первых суток (24 ч) наблюдали обратную картину – уровень окрашивания корней снабженных фосфатами растений был выше. Результаты иммунолокализации цитокининов в кончиках корней, где и находятся определяющие рост корней в длину зоны деления и растяжения, показали снижение содержания зеатина в клетках, находящихся под влиянием дефицита фосфора. Полученные данные позволяют предположить, что выявленное накопление в корнях активных форм кислорода может быть обусловлено повышенной концентрацией ауксинов, накопившихся в результате воздействия дефицита фосфора на растения ячменя. Повышение уровня ауксинов и реактивных форм кислорода в свою очередь могло повлиять на уровень цитокининов и, в конечном счете, на удлинение корней. Требуются дальнейшие исследования для проверки этого предположени

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

Abstract: 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