Cytokinins are pivotal plant hormones regulating the cell cycle and many components of development. They also transduce environmental signals such as nutrient deficiency and drought. The significance of cytokinin genes in agriculture has been highlighted by several reports, which associate them with improved crop performance. The aim of this study is to gain a better understanding of cytokinin metabolite distribution, in order to shed light on cytokinin transport, biochemistry and its function.udCytokinin metabolite levels were shown to vary amongst pea tissues and vascular saps, and across developmental stages. Detection was pushed to new limits by performing cytokinin measurements in distinct cell populations. For the first time, heterogeneous distribution was demonstrated within the Arabidopsis root apex and between the intra- and extra-cellular compartments.udA cytokinin gradient was revealed within the Arabidopsis root tip with maximal concentration in the columella, root cap, initials and QC cells. Cell-specific analysis of the TCSn:GFP cytokinin signaling reporter line indicated tZ as the only bioactive cytokinin related to intra-cellular cytokinin signaling. This result, coupled with identification of active cytokinins in the apoplast, indicates a significant role for cytokinin receptors at the plasma membrane. Cytokinin glucosides were largely intra-cellular, with their prevalence in cytokinin responsive cells indicating a role in cytokinin signaling. The riboside cZR was identified as a major transported cytokinin form while tZ-cytokinins were predominantly shoot compounds. The lateral root zone was also identified as a candidate site for cytokinin loading into the xylem.udComprehensive analysis of cytokinin’s distribution was also examined, particularly in response to strigolactone effects, another plant hormone which regulates branching. Strigolactone inhibited cytokinin degradative enzymes in root apices, thus acting as a local positive regulator of cytokinins. Furthermore, an RMS2-dependent mechanism regulating cytokinin homeostasis in the shoots was identified. In conclusion, this research provides new insights into the importance of cytokinin spatial distribution for understanding its roles in cell and developmental biology.
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