Functional characterization of Arabidopsis orthologs of PAT1 gene in Physcomitrella patens (P. patens)

Abstract

Plant-specific GRAS proteins are the putative transcription factors that play critical and diverse roles in plant growth and development. GRAS family comprises of seven clades in moss with GRAS domain containing proteins- SCR, SHR, DELLA, LAS/Ls, HAM, SCL and PAT1. Previous studies have showed that, AtPAT1 plays a role in phytochrome signal transduction in A. thaliana, while VaPAT1 is involved in abiotic stress responses in V. amurensis. However, the role of PAT1 have been unexplored in the lower group of plants viz bryophytes. We are interested in understanding the functional role of PAT1 proteins in P. patens. A comprehensive analysis of PpPAL1A and PpPAL1B genes, including gene structure, motif search, sequence alignment, phylogeny, tissue specific expression profile and targeted mutagenesis were performed. The sequence alignment, motif search and phylogenetic tree analyses together confirmed that PpPAL1A and PpPAL1B are the members of the PAT1-branch of the GRAS domain protein family and they showed 82.95% identity at the level of amino acids. Based on the available in silico microarray data, we studied the expression levels of both PpPAL1A and PpPAL1B genes in moss. Interestingly, we observed that PpPAL1A showed constitutive expression in all tissue while PpPAL1B expression was dominated in the sporophytic phase. Our tissue specific gene expression analysis done by semi-quantitative RT-PCR supported the available in silico data, where both the genes showed relatively higher expression level in gametophore compared to protonema. Statistical significance was seen in relative expression levels, in protonema and gametophore tissues for both PpPAL1A and PpPAL1B. The full length coding sequences for PpPAL1A and PpPAL1B were successfully cloned and transformed in moss by Agrobacterium-mediated transformation. Putative transgenic lines are growing in BCDAT media and will be further characterized. Knockout construction of PpΔpal1a and PpΔpal1b vectors and generation of mutant lines by PEG-mediated transformation is currently under progress. These approaches would help us to understand the functional roles of PAT1-like proteins in the transcriptional regulation and modulatory functions of GRAS domain proteins in moss.