Root Growth and Leaf Senescence Assays as Physiological Readouts of Cytokinin Response in Arabidopsis thaliana.

Cytokinin (CK) is a plant hormone that regulates multiple aspects of growth and development throughout the plant. Here we provide protocols to examine these effects in roots and leaves. In roots, basal levels of CK, in conjunction with other hormones like auxin, are key in normative structural organization by maintaining the boundary between the meristematic zone and the elongation zone and post-transition cell wall stiffening. To quantify CK effects on root growth, we present a simple plate-based assay in which Arabidopsis thaliana seeds are surface-sterilized, plated on phytagel media, and grown vertically with or without CK. In this protocol, 6-benzylaminopurine (BA) is used as a representative CK (though other CKs can be substituted), and primary root length is measured after a defined growth period. Exogenous CK treatment suppresses root elongation, therefore, differences in root length between treated and nontreated roots directly reflect hormone sensitivity and signaling. In leaves, CKs act to preserve photosynthetic capacity by delaying senescence. To assess this function, this chapter presents a simple detached-leaf bioassay wherein excised Arabidopsis leaves are incubated in CK-containing solutions. CK efficacy is evaluated by measuring chlorophyll fluorescence and pigment retention, which reflect photosystem II activity and chlorophyll stability, respectively. Further analyses of cell damage and oxidative stress allow for comprehensive assessment of the state of senescence. Thus, these quantitative assays provide robust methods to determine how Arabidopsis plants respond to CK in a dose-dependent manner, and can be used as a proxy of CK signaling. Furthermore, these assays can be used to determine how CK perception and signaling responds to environmental or genetic perturbations, and provide reproducible measurements that can help elucidate hormone physiology and signaling in either root and shoot tissues.