Arabidopsis T-DNA mutants affected in TRDMT1/DNMT2 show differential protein synthesis and compromised stress tolerance.

TRDMT1/DNMT2 belongs to the conserved family of nucleic acid methyltransferases. Unlike the animal systems, studies on TRDMT1/DNMT2 in land plants have been limited. We show that TRDMT1/DNMT2 is strongly conserved in the green lineage. Studies in mosses have previously shown that TRDMT1/DNMT2 plays a crucial role in modulating molecular networks involved in stress perception and signalling and in transcription/stability of specific tRNAs under stress. To gain deeper insight into its biological roles in a flowering plant, we examined more closely the previously reported Arabidopsis SALK_136635C line deficient in TRDMT1/DNMT2 function [Goll MG et al. (2006) Science 311, 395-398]. RNAs derived from Arabidopsis Dnmt2-deficient plants lacked m5 C38 in tRNAAsp . In this study, by transient expression assays we show that Arabidopsis TRDMT1/DNMT2 is distributed in the nucleus, cytoplasm and RNA-processing bodies, suggesting a role for TRDMT1/DNMT2 in RNA metabolic processes possibly by shuttling between cellular compartments. Bright-field and high-resolution SEM and qPCR analysis reveal roles of TRDMT1/DNMT2 in proper growth and developmental progression. Quantitative proteome analysis by LC-MS/MS coupled with qPCR shows AtTRDMT1/AtDNMT2 function to be crucial for protein synthesis and cellular homeostasis via housekeeping roles and proteins with poly-Asp stretches and RNA pol II activity on selected genes are affected in attrdmt1/atdnmt2. This shift in metabolic pathways primes the mutant plants to become increasingly sensitive to oxidative and osmotic stress. Taken together, our study sheds light on the mechanistic role of TRDMT1/DNMT2 in a flowering plant.

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

Early land plants such as the moss Physcomitrium patens lack several morphological traits that offer protection to tracheophytes from environmental stresses. These plants instead have evolved several physiological and biochemical mechanisms that facilitate them to adapt to terrestrial stresses such as drought. We have previously shown that loss-of-function mutants of tRNA (cytosine(38)-C(5))-methyltransferase TRDMT1/DNMT2 in P. patens are highly sensitive to oxidative and osmotic stress. To gain insight into the role of PpTRDMT1/PpDNMT2 in modulating genetic networks under osmotic stress, genome-wide transcriptome and proteome studies were undertaken in wild-type and ppdnmt2 plants. Transcriptome analysis revealed 375 genes to be differentially expressed in the ppdnmt2 under stress compared to the WT. Most of these genes are affiliated with carbohydrate metabolic pathways, photosynthesis, cell wall biogenesis, pathways related to isotropic and polarised cell growth and transcription factors among others. Histochemical staining showed elevated levels of reactive oxygen species in ppdnmt2 while transmission electron microscopy revealed no distinct defects in the ultrastructure of chloroplasts. Immunoprecipitation using PpDNMT2-specific antibody coupled with mass spectrometry revealed core proteins of the glycolytic pathway, antioxidant enzymes, proteins of amino acid biosynthetic pathways and photosynthesis-related proteins among others to co-purify with PpTRDMT1/PpDNMT2 under osmotic stress. Yeast two-hybrid assays, protein deletion and α-galactosidase assays showed the cytosol glycolytic protein glyceraldehyde 3-phosphate dehydrogenase to bind to the catalytic motifs in PpTRDMT1/PpDNMT2. Results presented in this study allow us to better understand genetic networks linking enzymes of energy metabolism, epigenetic processes and RNA pol II-mediated transcription during osmotic stress tolerance in P. patens.

Epigenetic variation: A major player in facilitating plant fitness under changing environmental conditions.

Recent research in plant epigenetics has increased our understanding of how epigenetic variability can contribute to adaptive phenotypic plasticity in natural populations. Studies show that environmental changes induce epigenetic switches either independently or in complementation with the genetic variation. Although most of the induced epigenetic variability gets reset between generations and is short-lived, some variation becomes transgenerational and results in heritable phenotypic traits. The short-term epigenetic responses provide the first tier of transient plasticity required for local adaptations while transgenerational epigenetic changes contribute to stress memory and help the plants respond better to recurring or long-term stresses. These transgenerational epigenetic variations translate into an additional tier of diversity which results in stable epialleles. In recent years, studies have been conducted on epigenetic variation in natural populations related to various biological processes, ecological factors, communities, and habitats. With the advent of advanced NGS-based technologies, epigenetic studies targeting plants in diverse environments have increased manifold to enhance our understanding of epigenetic responses to environmental stimuli in facilitating plant fitness. Taking all points together in a frame, the present review is a compilation of present-day knowledge and understanding of the role of epigenetics and its fitness benefits in diverse ecological systems in natural populations.

Comprehending the dynamism of B chromosomes in their journey towards becoming unselfish.

Investigated for more than a century now, B chromosomes (Bs) research has come a long way from Bs being considered parasitic or neutral to becoming unselfish and bringing benefits to their hosts. B chromosomes exist as accessory chromosomes along with the standard A chromosomes (As) across eukaryotic taxa. Represented singly or in multiple copies, B chromosomes are largely heterochromatic but also contain euchromatic and organellar segments. Although B chromosomes are derived entities, they follow their species-specific evolutionary pattern. B chromosomes fail to pair with the standard chromosomes during meiosis and vary in their number, size, composition and structure across taxa and ensure their successful transmission through non-mendelian mechanisms like mitotic, pre-meiotic, meiotic or post-meiotic drives, unique non-disjunction, self-pairing or even imparting benefits to the host when they lack drive. B chromosomes have been associated with cellular processes like sex determination, pathogenicity, resistance to pathogens, phenotypic effects, and differential gene expression. With the advancements in B-omics research, novel insights have been gleaned on their functions, some of which have been associated with the regulation of gene expression of A chromosomes through increased expression of miRNAs or differential expression of transposable elements located on them. The next-generation sequencing and emerging technologies will further likely unravel the cellular, molecular and functional behaviour of these enigmatic entities. Amidst the extensive fluidity shown by B chromosomes in their structural and functional attributes, we perceive that the existence and survival of B chromosomes in the populations most likely seem to be a trade-off between the drive efficiency and adaptive significance versus their adverse effects on reproduction.

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

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 tRNAAsp 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.