ABSTRACT
The two principal growth regulators cytokinins and ethylene are known to interact in the regulation of plant growth. However, information about underlying molecular mechanism and positional specificity of the cytokinin/ethylene crosstalk in root growth control is scarce. We have identified spatial specificity of cytokinin-regulated root elongation and root apical meristem (RAM) size, both of which we demonstrate to be dependent on ethylene biosynthesis. Upregulation of the cytokinin biosynthetic gene ISOPENTENYLTRANSFERASE (IPT) in proximal and peripheral tissues leads to both root and RAM shortening. In contrast, IPT activation in distal and inner tissues reduces RAM size while leaving the root length comparable to mock-treated controls. We show that cytokinins regulate two steps specific to ethylene biosynthesis, the production of ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) by ACC SYNTHASEs (ACSs), and its conversion to ethylene by ACC OXIDASEs (ACOs). We describe cytokinin- and ethylene-specific regulation controlling the activity of ACSs and ACOs that are spatially discrete along both proximo/distal and radial root axes. Using direct ethylene measurements, we identify ACO2, ACO3 and ACO4 as being responsible for ethylene biosynthesis and the ethylene-regulated root and RAM shortening in cytokinin-treated Arabidopsis. Direct interaction between ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2), a member of the multistep phosphorelay cascade and the C-terminal portion of ETHYLENE INSENSITIVE 2 (EIN2-C), a key regulator of canonical ethylene signaling is involved in the cytokinin-induced, ethylene-mediated control of ACO4. We propose tight cooperation between cytokinin and ethylene signaling in the spatial-specific regulation of ethylene biosynthesis as a key aspect of hormonal control over root growth.
ABSTRACT
The current repertoire of methods available for studying RNA-protein interactions in plants is somewhat limited. Employing an RNA-centric approach, particularly with less abundant RNAs, presents various challenges. Many of the existing methods were initially designed for different model systems, with their application in plants receiving limited attention thus far. The Comprehensive Identification of RNA-Binding Proteins by Mass Spectrometry (ChIRP-MS) technique, initially developed for mammalian cells, has been adapted in this study for application in Arabidopsis thaliana. The procedures have been meticulously modified and optimized for telomerase RNA, a notable example of a low-abundance RNA recently identified. Following these optimization steps, ChIRP-MS can serve as an effective screening method for identifying candidate proteins interacting with any target RNA of interest.
