Measuring Cytokinin Levels in the Root Tip by the Zeptomole

Perseverance, a competitive spirit, and luck helped Folke Skoog and colleagues isolate and characterize the first cytokinin (CK) from degraded herring sperm DNA samples some 60 years ago (described in Amasino, 2005). Since then, much has been learned about this vital class of phytohormones, which have both local and long-distance effects on multiple processes throughout the plant. CKs interact with several other classes of phytohormones, most famously auxins. The precise balance between CKs and auxins underlies their critical, antagonistic roles in regulating organ initiation, embryogenesis, meristem function, and other crucial processes. Phytohormone levels are thought to be tightly regulated both temporally and spatially. Indeed, Petersson et al. (2009) detected the presence of an auxin gradient in Arabidopsis thaliana roots. However, determining the precise localization of CK is more challenging because CKs are present at extremely low levels (pmol·g⁻¹ fresh weight, ∼100-fold lower than auxin levels) and consist of several related molecules and derived metabolites, different forms of which can interconvert via enzymatic reactions. Promoter-reporter studies have predicted the presence of CK gradients within the root apex, but this approach is limited, as it reflects an indirect measure of CK levels, and the reporters employed are potentially subject to additional layers of regulation.

Antoniadi et al. (2015) used a powerful technique to construct a map of the intracellular distribution of CKs and CK metabolites in the Arabidopsis root tip based on precise measurements of CK pools in different cell types. To construct this map, the authors measured CK levels in protoplasts prepared from four different populations of root tip cells. The cell populations were labeled by expressing green fluorescent protein (GFP) in root tips under the control of four different cell-type-specific promoters driving expression in different regions of the root tip, including the distal root (root cap, columella, columella initials, and quiescent center), endodermis, stele, and cortex/epidermis regions. Protoplasts were isolated from these four lines and sorted into GFP⁺ and GFP^– populations using fluorescence-activated cell sorting. Cytokinins were then extracted by multilayer solid-phase microextraction and quantified by ultra-high-sensitivity mass spectrometry.

An intracellular gradient of CKs and CK metabolites was detected in the apical part of the primary root, with maximum levels in the lateral root cap, columella, columella initials, and quiescent center cells (see figure). Strikingly, the authors estimated the concentration of CK metabolites in a single root cell to be in the zeptomole range (between 3 × 10⁻²¹ and 100 × 10⁻²¹ mol per cell), highlighting the exquisite sensitivity of their detection system. A similar CK gradient was indirectly revealed by comparing data from microarray, proteomic, and promoter-reporter studies. The presence of this CK gradient and the previously detected auxin gradient implies that the antagonistic interactions between these phytohormones are cell type specific. Unraveling the mechanisms underlying the effects of these interactions on individual cell types will require persistence, luck, and perhaps the development of even more powerful techniques.

Cytokinin gradient map showing a concentration maximum in the lateral root cap, columella, columella initials, and quiescent center cells. The red color scale indicates the CK content in GFP⁺ protoplasts relative to that in the GFP^– reference population for each GFP cell line. N/A, not analyzed. (Reprinted from Antoniadi et al. [2015], Figure 5A.)