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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 m5C38 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. [ABSTRACT FROM AUTHOR]

OsMADS29 (M29) is a crucial regulator of seed development in rice. The expression of M29 is strictly regulated at transcriptional as well as post‐transcriptional levels. The MADS‐box proteins are known to bind to DNA as dimers. However, in the case of M29, the dimerization also plays a vital role in its localization into the nucleus. The factor(s) that affect oligomerization and nuclear transport of MADS proteins have not yet been characterized. By using BiFC in transgenic BY‐2 cell lines and Yeast‐2‐hybrid assay (Y2H), we show that calmodulin (CaM) interacts with M29 in a Ca2+‐dependent manner. This interaction specifically takes place in the cytoplasm, probably in association with the endoplasmic reticulum. By generating domain‐specific deletions, we show that both sites in M29 are involved in this interaction. Further, by using BiFC‐FRET‐FLIM, we demonstrate that CaM may also help in the dimerization of two M29 monomers. Since most MADS proteins have CaM binding domains, the interaction between these proteins could be a general regulatory mechanism for oligomerization and nuclear transport. [ABSTRACT FROM AUTHOR]

Abstract Background Important developmental processes in both plants and animals are partly regulated by genes whose expression is modulated at the post-transcriptional level by processes such as RNA interference (RNAi). Dicers, Argonautes and RNA-dependent RNA polymerases (RDR) form the core components that facilitate gene silencing and have been implicated in the initiation and maintenance of the trigger RNA molecules, central to process of RNAi. Investigations in eukaryotes have revealed that these proteins are encoded by variable number of genes with plants showing relatively higher number in each gene family. To date, no systematic expression profiling of these genes in any of the organisms has been reported. Results In this study, we provide a complete analysis of rice Dicer-like, Argonaute and RDR gene families including gene structure, genomic localization and phylogenetic relatedness among gene family members. We also present microarray-based expression profiling of these genes during 14 stages of reproductive and 5 stages of vegetative development and in response to cold, salt and dehydration stress. We have identified 8 Dicer-like (OsDCLs), 19 Argonaute (OsAGOs) and 5 RNA-dependent RNA polymerase (OsRDRs) genes in rice. Based on phylogeny, each of these genes families have been categorized into four subgroups. Although most of the genes express both in vegetative and reproductive organs, 2 OsDCLs, 14 OsAGOs and 3 OsRDRs were found to express specifically/preferentially during stages of reproductive development. Of these, 2 OsAGOs exhibited preferential up-regulation in seeds. One of the Argonautes (OsAGO2) also showed specific up-regulation in response to cold, salt and dehydration stress. Conclusion This investigation has identified 23 rice genes belonging to DCL, Argonaute and RDR gene families that could potentially be involved in reproductive development-specific gene regulatory mechanisms. These data provide an insight into probable domains of activity of these genes and a basis for further, more detailed investigations aimed at understanding the contribution of individual components of RNA silencing machinery during reproductive phase of plant development.

Summary: In flowering plants, LIKE HETEROCHROMATIN PROTEIN 1 (LHP1)/TERMINAL FLOWER 2 (TFL2) is known to interact with polycomb group (PcG) and non‐PcG proteins and control developmental programs. LHP1/TFL2 is an ancient protein and has been characterized in the early‐divergent plant Physcomitrella patens. However, interacting partners of PpLHP1 other than the chromomethylase PpCMT have not been identified to date. Also, while functional polycomb repressive complex 2 (PRC2) is known to exist in P. patens, there is no experimental evidence to support the existence of PRC1‐like complexes in these mosses. In this study, using protein−protein interaction methods, transient expression assays and targeted gene knockout strategy, we report the conserved properties of LHP1/TFL2 using the Physcomitrella system. We show that a PRC1‐like core complex comprising of PpLHP1 and the putative PRC1 Really Interesting New Gene (RING)‐finger proteins can form in vivo. Also, the interaction between PpRING and the PRC2 subunit PpCLF further sheds light on the possible existence of combinatorial interactions between the Polycomb Repressive Complex (PRC) in early land plants. Based on the interaction between PpLHP1 and putative hnRNP PpLIF2‐like in planta, we propose that the link between PpLHP1 regulation and RNA metabolic processes was established early in plants. The conserved subnuclear distribution pattern of PpLHP1 in moss protonema further provides insight into the manner in which LHP1/TFL2 are sequestered in the nucleoplasm in discrete foci. The PpLHP1 loss‐of‐function plants generated in this study share some of the pleiotropic defects with multiple aberrations reported in lhp1/tfl2. Taken together, this work documents an active role for PpLHP1 in epigenetic regulatory network in P. patens. Significance Statement: We provide evidence for the existence of a PRC1‐like complex in P. patens and conservation of LHP1 function in mosses and Arabidopsis. Interaction of PpLHP1 with PRC1 Really Interesting New Gene (RING)‐finger proteins that further interact with PRC2 PpCLF advocates the existence of a transcriptional repression mechanism in these early plants. Association of PpLHP1 with hnRNP PpLIF2‐like 1 suggests the conservation of a link between PpLHP1 regulation and RNA processes in land plants. pplhp1 plants share morphological aberrations with Arabidopsis lhp1/tfl2. [ABSTRACT FROM AUTHOR]

Chromomethylases ( CMTs) are plant-specific cytosine DNA methyltransferases that are involved in maintenance of CpNpG methylation. In seed plants, histone methylation and interaction of CMT with LIKE HETEROCHROMATIN PROTEIN 1 ( LHP1) is essential for recruitment of CMT to target sites. LHP1 has been characterized as a putative component of the POLYCOMB REPRESSIVE COMPLEX1 ( PRC1) in plants, and functions downstream of PRC2 to maintain genes in repressed state for orchestrated development. In the present study, we show that targeted disruption of Pp CMT results in an approximately 50% reduction in global cytosine methylation levels. This affects growth of apical cells, predominantly growth of side branch initials emerging from chloronema cells. In some places, these cells develop thick walls with plasmolyzed cellular contents. Transcript accumulation patterns of genes involved in apical cell extension and metabolism of hemicelluloses, such as xyloglucans, in the primary cell walls decreased many fold in ppcmt mutant lines, as determined by real-time PCR. Using yeast two-hybrid method and bimolecular fluorescence complementation assay, we show that Pp CMT and Pp LHP1 interact through their chromo domains, while Pp LHP1 homodimerizes through its chromo shadow domain. The results presented in this study provide insight into the role of the single chromomethylase, Pp CMT, in proliferation of protonema filaments, and shed light on the evolutionary conservation of proteins interacting with these methylases in the early land plant, Physcomitrella patens. [ABSTRACT FROM AUTHOR]

OsMADS29 (M29) is a crucial regulator of seed development in rice. The expression of M29 is strictly regulated at transcriptional as well as post-transcriptional levels. The MADS-box proteins are known to bind to DNA as dimers. However, in the case of M29, the dimerization also plays a vital role in its localization into the nucleus. The factor(s) that affect oligomerization and nuclear transport of MADS proteins have not yet been characterized. By using BiFC in transgenic BY-2 cell lines and Yeast-2-hybrid assay (Y2H), we show that calmodulin (CaM) interacts with M29 in a Ca 2+ -dependent manner. This interaction specifically takes place in the cytoplasm, probably in association with the endoplasmic reticulum. By generating domain-specific deletions, we show that both sites in M29 are involved in this interaction. Further, by using BiFC-FRET-FLIM, we demonstrate that CaM may also help in the dimerization of two M29 monomers. Since most MADS proteins have CaM binding domains, the interaction between these proteins could be a general regulatory mechanism for oligomerization and nuclear transport., (© 2023 Federation of European Biochemical Societies.)

Background and Objective: Controlled-rate freezing is a complicated, expensive, and time-consuming procedure. Therefore, there is a growing interest in uncontrolled-rate freezing (UCF) with -80°C mechanical freezers for cryopreservation of hematopoietic stem cells. This is a retrospective analysis of efficiency of UCF and outcome of autologous peripheral hematopoietic stem cell (PBSC) transplants at our center from December 2011 to June 2016., Materials and Methods: Cryoprotectant solutions used included 5% dimethyl sulfoxide and 5% albumin with 2% hydroxyethyl starch and stored at -80°C mechanical freezer till transplant. Evaluation of cryopreservation was studied by analyzing the variation in cellularity, viability, and CD34+ stem cell dose recovery as well as clinical follow-up with engraftment., Results: A total of 51 patients (23 females and 28 males) underwent autologous PBSC transplantations with a median age of 31 years (range: 3-60 years) for both hematological and nonhematological indications. Mean recovery post by UCF at -80°C mechanical was 92.9% ± 15.5% for nucleated cells, 86.6% ± 15.5% for viability, and 80% ± 21.5% in CD34+ dose. The median day to neutrophil engraftment was 10 (range 5-14 days) and platelets engraftment was 15 (range 8-45 days). The cryopreserved products were stored at -80°C for median 7 days (range 2-41 day) before transplant., Discussion/conclusion: Our analysis shows that PBSC can be successfully cryopreserved with mechanical uncontrolled rate freezing. This is a cheap and simple method to freeze the stem cells for a short period in resource-constrained setting., Competing Interests: There are no conflicts of interest.

Essential thrombocythemia (ET) is a clonal disorder of unknown etiology involving multipotent hematopoietic progenitor cell and belongs to the spectrum of chronic myeloproliferative disorders (CMPD). It is rarest of the CMPD with no case reported from India. The case being presented was detected incidentally during routine investigations in an adult female who presented with mild abdominal discomfort.