Transcriptome Analysis of ppdnmt2 and Identification of Superoxide Dismutase as a Novel Interactor of DNMT2 in the Moss Physcomitrella patens

Abstract

DNMT2 is a DNA/tRNA cytosine methyltransferase that is highly conserved in structure and function in eukaryotes. In plants however, limited information is available on the function of this methyltransferase. We have previously reported that in the moss Physcomitrella patens , DNMT2 plays a crucial role in stress recovery and tRNA Asp transcription/stability under salt stress. To further investigate the role of PpDNMT2 at genome level, in this study we have performed RNA sequencing of ppdnmt2 . Transcriptome analysis reveals a number of genes and pathways to function differentially and suggests a close link between PpDNMT2 function and osmotic and ionic stress tolerance. We propose PpDNMT2 to play a pivotal role in regulating salt tolerance by affecting molecular networks involved in stress perception and signal transduction that underlie maintenance of ion homeostasis in cells. We also examined interactome of PpDNMT2 using affinity purification (AP) coupled to mass spectrometry (AP-MS). Quantitative proteomic analysis reveals several chloroplast proteins involved in light reactions and carbon assimilation and proteins involved in stress response and some not implicated in stress to co-immunoprecipitate with PpDNMT2. Comparison between transcriptome and interactome datasets has revealed novel association between PpDNMT2 activity and the antioxidant enzyme Superoxide dismutase (SOD), protein turnover mediated by the Ubiquitin-proteasome system and epigenetic gene regulation. PpDNMT2 possibly exists in complex with CuZn-SODs in vivo and the two proteins also directly interact in the yeast nucleus as observed by yeast two-hybrid assay. Taken together, the work presented in this study sheds light on diverse roles of PpDNMT2 in maintaining molecular and physiological homeostasis in P. patens . This is a first report describing transcriptome and interactome of DNMT2 in any land plant.

Role of TRDMT1/DNMT2 in stress adaptation and its influence on transcriptome and proteome dynamics under osmotic stress in Physcomitrium patens

“…Transcript levels of genes encoding peroxidases were also observed to be down-regulated in ppdnmt2 under stress. We have previously shown that Cu, Zn-SODs exist in an immunocomplex with PpTRDMT1/PpDNMT2 under oxidative stress (Singh et al, 2020) and in the present study we also found these proteins to co-purify with PpTRDMT1/PpDNMT2 under osmotic stress. PpTRDMT1/ PpDNMT2 and the Cu, Zn-SOD, PpCSD2 also directly interact with one another (Singh et al, 2020).…”

“…Wild‐type and PpTRDMT1/PpDNMT2 loss‐of‐function plants ppdnmt2#8 , hereafter referred to as ppdnmt2 , used previously for stress tolerance and transcriptome studies (Arya et al, 2016; Singh et al, 2020) were propagated and exposed to 900 mM mannitol for 24 h. This concentration of mannitol was used since wild‐type P. patens are known to tolerate and recover from osmotic stress under these conditions (Arya et al, 2016; Saavedra et al, 2006). Changes in apical and sub‐apical cells of filamentous protonemata were examined under the microscope.…”

“…To validate the direct interactions between PpTRDMT1/PpDNMT2 and selected immunoprecipitated proteins we took note of the follow- T A B L E 2 Comparison of gene expression datasets for ppdnmt2 generated under non-stress conditions (Singh et al, 2020) and osmotic stress (present study). (Continues) the light harvesting complex II chlorophyll a/b binding protein (Pp1s4_321V6.1) and the other around the core proteins of the glycolytic pathway (Figure 7B, Table S10), b.…”

TRDMT1 exhibited protective effects against LPS‐induced inflammation in rats through TLR4‐NF‐κB/MAPK‐TNF‐α pathway

“…Growing evidence illustrates that Trdmt1 plays a potential role in stress stimulation. 13 , 19 , 21 , 22 , 38 Here, we applied the LPS‐induced sepsis model to analyze Trdmt1 gene function. We first measured the expression of Trdmt1 in different tissues after administering SD rats with LPS.…”

“…Growing evidence illustrates that Trdmt1 plays a potential role in stress stimulation 13,19,21,22,38 . Here, we applied the LPS‐induced sepsis model to analyze Trdmt1 gene function.…”