Cytokinin-dependent expression of the ARR5::GUS construct during transgenic arabidopsis growth

Growth and glucuronidase (GUS) activity were followed in the cotyledons and rosette leaves of Arabidopsis thaliana (L.) Heynh (ecotype Wassilewskija) plants transformed with the GUS gene under the control of the cytokinin-dependent promoter of the ARR5 gene. The presence of active cytokinins in plant tissues was assessed from GUS activity. Plants were grown for three weeks on the nitrate-or ammonium-containing nutrient medium. In plants grown on ammonium nutrition, cotyledon and leaf growth was substantially suppressed as compared with plants feeding with nitrates. In correspondence with this growth inhibition, GUS activity was markedly lower in plant leaves grown on the ammonium-containing medium. This indicated a reduction in these leaves of active cytokinin forms capable of activation of the promoter for the ARR5 gene. On both nitrogen sources, GUS activity increased during leaf growth and dropped sharply after growth ceasing. This indicated that leaf growth depended on the cytokinin content in them. High GUS activity was detected in petioles and leaf conductive system, indicating leaf providing with cytokinins along the conductive vessels. A sharp drop in the GUS activity after leaf growth stoppage coincided in time with GUS activation in the leaf positioned above this leaf. This indicated possible cytokinin redistribution in the plant; its content could be a limiting factor for leaf growth. A higher growth rate in plants on nitrate nitrogen nutrition and corresponding high GUS activity in them are discussed in terms of cytokinin signaling role in leaf growth regulation mediated by nitrate.     

Cytokinin activity of zeatin allylic phosphate, a natural compound

Zeatin allylic phosphate (ZAP) retarded chlorophyll loss in the barleyleaf senescence assay at a concentration 20 times higher than for6-benzyladenine (BA): the effective concentrations for ZAP and BA were 10 and 0.5 , respectively. Sodium molybdate,an inhibitor of phosphatases, decreased the ZAP effective concentration to 0.5 without affecting leaf senescence andtrans-zeatin activity in the control. This demonstrates theimportance of the phosphate group for ZAP activity or its penetration into leafcells. ZAP up-regulated the protein kinase activity of the barley leaf chromatinwith concentration dependence similar to that oftrans-zeatin. Conversely, ZAP was 1000 times less activethan trans-zeatin in the competition with anti-idiotypeantibodies (raised against antibody to zeatin) for binding with atrans-zeatin-binding site oftrans-zeatin-binding protein ZBP67 isolated from barleyleaves. In contrast to trans-zeatin, ZAP did not activateRNA synthesis in the presence of ZBP in the in vitro systemcontaining chromatin and RNA polymerase I isolated from barley leaves. Insummary, data presented show that ZAP possesses cytokinin activity asdemonstrated by the retardation of barley leaf senescence, but moleculartarget(s) for ZAP in barley leaf cells differs, at least partially, from thesefor trans-zeatin. It seems possible that the cytokininactivity of ZAP results from its hydrolysis while producing zeatin.     

Fast changes in growth rate and cytokinin content of the shoot following rapid cooling of roots of wheat seedling

Changes in the concentration of cytokinins were studied following root cooling. Simultaneously, the growth rate of the second leaf was monitored with a highly sensitive growth sensor attached to its tip. Cytokinins were separated by thin layer chromatography and immunoassayed using antibodies to zeatin riboside. The extension rate of the second leaf decreased within 15 minutes of cooling the nutrient medium from 24 °C to 4 °C. The concentration of cytokinins in shoots decreased with similar rapidity. In contrast cytokinins in roots increased slightly during the initial period of cooling before declining. The sharp decrease in cytokinin concentrations in shoots 15 minutes after cooling of roots may contribute to the abrupt inhibition of shoot growth.