Functional characterisation of cis-acting regulatory elements in the promoters of MSL family genes from the moss Physcomitrium patens

Abstract

All organisms use mechanosensation to sense and respond to their immediate environment by translating physical stimulus to biochemical information. Plants being sessile in nature, rely heavily on mechanosensation to enable them to adapt and survive in a variety of environmental conditions. Mechanosensation is mostly mediated by a set of membrane channel proteins called Mechanosensitive (MS) ion channels. One of the most well-characterised MS ion channels is the Mechano-sensitive channel small conductance (MscS), originally reported in E. coli. The homologs of MscS-like channels (MSLs) have been found in all plant lineages and are extensively studied in angiosperms like Arabidopsis thaliana, Oryza sativa etc. Recently, the study of such developmentally crucial genes in extant land plants has gained popularity due to their significance in the course of land plant evolution. Physcomitrium patens is one such land plant and an excellent model organism that has a simple life cycle and a highly efficient homologous recombination system. Comparative studies of the molecular processes in P. patens can help establish an evolutionary relationship between MSLs of vascular and non-vascular plants. Our in silico studies reveal that P. patens has 16 MSL genes, of which 4 MSLs were differentially regulated under dehydration stress. The differential gene expression of these MSLs was validated by RT-qPCR analysis, suggesting a possible regulatory switch particularly activated during the dehydration stress. In-silico analyses of the four PpMSL promoters revealed that they house key dehydration-responsive regulatory elements. Based on these preliminary analyses, two PpMSLs were selected for further studies. To analyse the physiological function of the dehydration-responsive regulatory motifs through reporter fusion assays, we generated independent vector constructs having full-length and truncated promoters of PpMSL3 and PpMSL15 upstream to the GUS reporter gene. Stable transgenic lines are being generated in moss using these vectors to explore the gene expression dynamics under dehydration stress. Quantification of relative GUS activity in these transgenic lines will reveal the minimal functional promoters for the PpMSL genes and the regulatory mechanism of gene expression by the various motifs under dehydration-rehydration stress conditions.