Cell-to-cell communication is tightly regulated in response to environmental stimuli in plants. We previously used a photoconvertible fluorescent protein Dendra2 as a model reporter to study this process. This experiment revealed that macromolecular trafficking between protonemal cells in Physcomitrella patens is suppressed in response to abscisic acid (ABA). However, it remains unknown which ABA signaling components contribute to this suppression and how. Here, we show that ABA signaling components SUCROSE NON-FERMENTING 1-RELATED PROTEIN KINASE 2 (PpSnRK2) and ABA INSENSITIVE 3 (PpABI3) play roles as an essential and promotive factor, respectively, in regulating ABA-induced suppression of Dendra2 diffusion between cells (ASD). Our quantitative imaging analysis revealed that disruption of PpSnRK2 resulted in defective ASD onset itself, whereas disruption of PpABI3 caused an 81-min delay in the initiation of ASD. Live-cell imaging of callose deposition using aniline blue staining showed that, despite this onset delay, callose deposition on cross walls remained constant in the PpABI3 disruptant, suggesting that PpABI3 facilitates ASD in a callose-independent manner. Given that ABA is an important phytohormone to cope with abiotic stresses, we further explored cellular physiological responses. We found that the acquisition of salt stress tolerance is promoted by PpABI3 in a quantitative manner similar to ASD. Our results suggest that PpABI3-mediated ABA signaling may effectively coordinate cell-to-cell communication during the acquisition of salt stress tolerance. This study will accelerate the quantitative study for ABA signaling mechanism and function in response to various abiotic stresses.
An important approach to investigate intercellular connectivity via plasmodesmata is to visualize and track the movement of fluorescent proteins between cells. The intercellular connectivity is largely controlled by the size exclusion limit of the pores. Over the past few decades, the technique to observe and analyze intercellular movement of a fluorescent protein has been developed mainly in angiosperms such as Arabidopsis thaliana. We recently applied the corresponding system to track the intercellular movement of the fluorescent protein Dendra2 in the moss Physcomitrium (Physcomitrella) patens. The protonemal tissues are -2 -particularly suited for observation of the intercellular movement due to the simple organization.Here, we describe a protocol suitable for the analysis of Dendra2 movement between cells in P. patens.
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