Cellular Complexity in MAPK Signaling in Plants: Questions and Emerging Tools to Answer Them

Abstract

Mitogen activated protein kinase (MAPK) cascades play an important role in many aspects of plant growth, development, and environmental response. Because of their central role in many important processes, MAPKs have been extensively studied using biochemical and genetic approaches. This work has allowed for the identification of the MAPK genes and proteins involved in a number of different signaling pathways. Less well developed, however, is our understanding of how MAPK cascades and their corresponding signaling pathways are organized at subcellular levels. In this review, we will provide an overview of plant MAPK signaling, including a discussion of what is known about cellular mechanisms for achieving signaling specificity. Then we will explore what is currently known about the subcellular localization of MAPK proteins in resting conditions and after pathway activation. Finally, we will discuss a number of new experimental methods that have not been widely deployed in plants that have the potential to provide a deeper understanding of the spatial and temporal dynamics of MAPK signaling.

Keywords: MAPK; activity sensors; microscopy; phosphorylation; plant; signaling cascade.

FIGURE 1

Microcopy based tools to study and manipulate MAPK signaling. (A) Substrate based activity sensors (SBAS) are constituted of a pair of fluorescent proteins able to perform FRET (for example YFP and CFP) separated by a phosphosite-containing peptide derived from a kinase substrate and a phospho-amino acid-binding domain. Upon phosphorylation of the substrate domain, the sensor undergoes a conformational change triggering a FRET effect. (B) Probes for conformation changes (PCC) are constituted of a pair of fluorescent proteins able to perform FRET separated by a kinase which, upon activation, undergoes conformational changes triggering a FRET effect. (C) Kinase translocation reporters (KTRs) is composed of a fluorophore whose localization is driven by a NES/NLS sequence carrying a MAPK phosphosite. Upon phosphorylation, KTR relocalizes in the nucleus. An additive fluorophore is co-expressed in the cell allowing a ratiometric quantification of the relocalization. (D) Photo-switchable kinases is composed of a constitutive active kinase (for example, a MAP2K carrying 2 phospho-mimicking mutations on the residues which are phosphorylated by upstream MAP3Ks) which is neutralized by two dimerising pdDROMPA domains. Upon illumination at 500 nm pdDROMPAs dissociate and the kinase active site becomes accessible able to phosphorylate downstream targets (ON). This process is reversible by using an illumination at 400 nm (OFF).