Binary cloning vectors for efficient genetic transformation of rice plants

The availability of effective vector systems is a prerequisite for genetic manipulation of plants through recombinant DNA technology. We report here construction of a series of binary vectors that have cauliflower mosaic virus 35S promoter-driven genes encoding either resistance to hygromycin or phosphinothricin for selection of the transformants, and high strength constitutive promoters of either ubiquitin1 or actin1 genes for efficient expression of the transgenes. The efficacy of the constructs is tested in stably transformed Pusa Basmati 1 rice plants through β-glucuronidase reporter gene activity. Availability of vectors with variable promoters and selectable marker genes provides flexibility in stacking two genes. The vectors constructed in this study are suitable for both particle gun and Agrobacterium-based transformation protocols

Distinctive Core Histone Post-Translational Modification Patterns in Arabidopsis thaliana

Post-translational modifications of histones play crucial roles in the genetic and epigenetic regulation of gene expression from chromatin. Studies in mammals and yeast have found conserved modifications at some residues of histones as well as non-conserved modifications at some other sites. Although plants have been excellent systems to study epigenetic regulation, and histone modifications are known to play critical roles, the histone modification sites and patterns in plants are poorly defined. In the present study we have used mass spectrometry in combination with high performance liquid chromatography (HPLC) separation and phospho-peptide enrichment to identify histone modification sites in the reference plant, Arabidopsis thaliana. We found not only modifications at many sites that are conserved in mammalian and yeast cells, but also modifications at many sites that are unique to plants. These unique modifications include H4 K20 acetylation (in contrast to H4 K20 methylation in non-plant systems), H2B K6, K11, K27 and K32 acetylation, S15 phosphorylation and K143 ubiquitination, and H2A K144 acetylation and S129, S141 and S145 phosphorylation, and H2A.X S138 phosphorylation. In addition, we found that lysine 79 of H3 which is highly conserved and modified by methylation and plays important roles in telomeric silencing in non-plant systems, is not modified in Arabidopsis. These results suggest distinctive histone modification patterns in plants and provide an invaluable foundation for future studies on histone modifications in plants

Experimentation in biology of plant abiotic stress responses

During the course of growth under natural field conditions, crop plants are exposed to a number of different abiotic stresses (such as water stress, temperature stress, salt stress, flooding stress, chemical stress and oxidative stress). These stresses exert adverse effects on metabolism, growth and yield of the crops. The intensity of the abiotic stresses is on the rise, implying that various possible solutions for mitigating the damage caused by such stresses must be combined for future increase in crop production. At the level of plant genetics, there are indications that it may be possible to improve plants against such stress factors. However, the practical success in this regard depends on how well we understand the biochemistry. physiology and molecular biology of the plant abiotic stress responses. The cellular response of plants to abiotic stresses is of complex nature involving simultaneous interplay of several mechanisms. Although there is a great deal of progress in cataloguing the biochemical reactions that are associated with plant abiotic stress responses, precise understanding of the defense reactions leading to acquisition of stress tolerance remains a challenge. A number of different experimental systems including lower and higher plants as well as microbes have been analyzed for examining the plant abiotic stress responses. The molecular analysis of the stress response has been carried out at the level of stress proteins, stress genes, stress promoters, trans-acting factors that bind to stress promoters and signal transduction components involved in mediation of stress responses. The functional relevance of the stress - associated genes is being tested in different trans-systems including yeast as well as higher plant species. In this article, we discuss selective features of experimentation in biology of plant abiotic stress responses

Histone H3.3 and its proteolytically processed form drive a cellular senescence programme.

The process of cellular senescence generates a repressive chromatin environment, however, the role of histone variants and histone proteolytic cleavage in senescence remains unclear. Here, using models of oncogene-induced and replicative senescence, we report novel histone H3 tail cleavage events mediated by the protease Cathepsin L. We find that cleaved forms of H3 are nucleosomal and the histone variant H3.3 is the preferred cleaved form of H3. Ectopic expression of H3.3 and its cleavage product (H3.3cs1), which lacks the first 21 amino acids of the H3 tail, is sufficient to induce senescence. Further, H3.3cs1 chromatin incorporation is mediated by the HUCA histone chaperone complex. Genome-wide transcriptional profiling revealed that H3.3cs1 facilitates transcriptional silencing of cell cycle regulators including RB/E2F target genes, likely via the permanent removal of H3K4me3. Collectively, our study identifies histone H3.3 and its proteolytically processed forms as key regulators of cellular senescence.This is the author's accepted manuscript. The final version is available from NPG at http://www.nature.com/ncomms/2014/141114/ncomms6210/full/ncomms6210.html

Analysis of Marker-Defined HNSCC Subpopulations Reveals a Dynamic Regulation of Tumor Initiating Properties

Head and neck squamous carcinoma (HNSCC) tumors carry dismal long-term prognosis and the role of tumor initiating cells (TICs) in this cancer is unclear. We investigated in HNSCC xenografts whether specific tumor subpopulations contributed to tumor growth. We used a CFSE-based label retentions assay, CD49f (α6-integrin) surface levels and aldehyde dehydrogenase (ALDH) activity to profile HNSCC subpopulations. The tumorigenic potential of marker-positive and -negative subpopulations was tested in nude (Balb/c nu/nu) and NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice and chicken embryo chorioallantoic membrane (CAM) assays. Here we identified in HEp3, SQ20b and FaDu HNSCC xenografts a subpopulation of G0/G1-arrested slow-cycling CD49fhigh/ALDH1A1high/H3K4/K27me3low subpopulation (CD49f+) of tumor cells. A strikingly similar CD49fhigh/H3K27me3low subpopulation is also present in primary human HNSCC tumors and metastases. While only sorted CD49fhigh/ALDHhigh, label retaining cells (LRC) proliferated immediately in vivo, with time the CD49flow/ALDHlow, non-LRC (NLRC) tumor cell subpopulations were also able to regain tumorigenic capacity; this was linked to restoration of CD49fhigh/ALDHhigh, label retaining cells. In addition, CD49f is required for HEp3 cell tumorigenicity and to maintain low levels of H3K4/K27me3. CD49f+ cells also displayed reduced expression of the histone-lysine N-methyltransferase EZH2 and ERK1/2phosphorylation. This suggests that although transiently quiescent, their unique chromatin structure is poised for rapid transcriptional activation. CD49f− cells can “reprogram” and also achieve this state eventually. We propose that in HNSCC tumors, epigenetic mechanisms likely driven by CD49f signaling dynamically regulate HNSCC xenograft phenotypic heterogeneity. This allows multiple tumor cell subpopulations to drive tumor growth suggesting that their dynamic nature renders them a “moving target” and their eradication might require more persistent strategies

Posttranscriptional Induction of Two Cu/Zn Superoxide Dismutase Genes in Arabidopsis Is Mediated by Downregulation of miR398 and Important for Oxidative Stress Tolerance

MicroRNAs (miRNAs) are a class of regulatory RNAs of ∼21 nucleotides that posttranscriptionally regulate gene expression by directing mRNA cleavage or translational inhibition. Increasing evidence points to a potential role of miRNAs in diverse physiological processes. miR398 targets two closely related Cu/Zn superoxide dismutases (cytosolic CSD1 and chloroplastic CSD2) that can detoxify superoxide radicals. CSD1 and CSD2 transcripts are induced in response to oxidative stress, but the regulatory mechanism of the induction is unknown. Here, we show that miR398 expression is downregulated transcriptionally by oxidative stresses, and this downregulation is important for posttranscriptional CSD1 and CSD2 mRNA accumulation and oxidative stress tolerance. We also provide evidence for an important role of miR398 in specifying the spatial and temporal expression patterns of CSD1 and CSD2 mRNAs. Our results suggest that CSD1 and CSD2 expression is fine-tuned by miR398-directed mRNA cleavage. Additionally, we show that transgenic Arabidopsis thaliana plants overexpressing a miR398-resistant form of CSD2 accumulate more CSD2 mRNA than plants overexpressing a regular CSD2 and are consequently much more tolerant to high light, heavy metals, and other oxidative stresses. Thus, relieving miR398-guided suppression of CSD2 in transgenic plants is an effective new approach to improving plant productivity under oxidative stress conditions

Production and phenotypic analysis of rice transgenics with altered levels of pyruvate decarboxylase and alcohol dehydrogenase proteins

Pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) enzymes are responsible for the operation of ethanolic fermentation pathway that appears to correlate to an extent with anoxia tolerance in plants. This study was undertaken with the objective of (a) analysing the rice pdc gene family and (b) altering the efficacy of the ethanolic fermentation process, through production of transgenic rice plants over- and under-expressing pyruvate decarboxylase (employing Ospdc1 gene from rice) as well as over-expressing alcohol dehydrogenase (employing Ghadh2 gene from cotton) proteins. Correlations noted in this study between the pattern of expression of the Pdc α-subunit and Ospdc2 transcript as well as between the Pdc β-subunit and Ospdc1 transcript suggest the possibility that α-subunit is encoded by Ospdc2 and that β-subunit is encoded by Ospdc1. The fact that levels of Pdc β-subunit were particularly high in pUH-sPdc1 (plasmid construct designed for over-expression of Ospdc1) seedlings while levels of β-subunit levels were negligible or lower in pUH-asPdc1 (plasmid construct designed for under-expression of Ospdc1) seedlings also support these observations. Transgenics raised for over-expression of Pdc and Adh and under-expression of Pdc were confirmed for the transgene presence and effects by PCR, Southern blotting, Northern blotting, Western blotting and isozyme assays. Pdc and Adh over-expressing rice transgenics at early seedling stage under unstressed control growth conditions showed slight, consistent advantage in root vigour as compared to that of wild-type seedlings

Role of Arabidopsis AGO6 in siRNA accumulation, DNA methylation and transcriptional gene silencing

Argonautes (AGOs) are conserved proteins that contain an RNA-binding PAZ domain and an RNase H-like PIWI domain. In Arabidopsis, except for AGO1, AGO4 and AGO7, the roles of seven other AGOs in gene silencing are not known. We found that a mutation in AGO6 partially suppresses transcriptional gene silencing in the DNA demethylase mutant ros1-1. In ago6-1ros1-1 plants, RD29A promoter short interfering RNAs (siRNAs) are less abundant, and cytosine methylation at both transgenic and endogenous RD29A promoters is reduced, compared to that in ros1-1. Interestingly, the ago4-1 mutation has a stronger suppression of the transcriptional silencing phenotype of ros1-1 mutant. Analysis of cytosine methylation at the endogenous MEA-ISR, AtREP2 and SIMPLEHAT2 loci revealed that the CpNpG and asymmetric methylation levels are lower in either of the ago6-1 and ago4-1 single mutants than those in the wild type, and the levels are the lowest in the ago6-1ago4-1 double mutant. These results suggest that AGO6 is important for the accumulation of specific heterochromatin-related siRNAs, and for DNA methylation and transcriptional gene silencing, this function is partly redundant with AGO4

Incorporating Sustainability: A Comprehensive Review of Factors Influencing Consumer Acceptance of Mobile Wallets

Mobile wallets have gained widespread popularity as a convenient, secure, and user-friendly payment method embraced by consumers. However, the pace of mobile wallet adoption has exhibited variations across different markets, primarily due to a range of factors. In this study, we present an all-encompassing examination of existing literature, aiming to pinpoint the fundamental elements influencing the sustainable acceptance of mobile wallets by consumers. Through an exhaustive analysis of 80 research papers published between 2010 and 2022, we discern the prevailing factors that hold sway over the adoption of mobile wallets. Our scrutinization highlights that factor such as perceived sustainability, usefulness, ease of use, security, social influence, trustworthiness, and compatibility stand out as the most formidable propellants of mobile wallet adoption. Furthermore, our investigation uncovers hurdles that hinder the wider acceptance of mobile wallets, encompassing insufficient awareness, perceived intricacies, and lingering uncertainties regarding the technology’s sustainability. Our in-depth evaluation underscores the necessity of comprehending consumer perspectives and dispositions towards mobile wallets to galvanize their adoption sustainably. The culmination of our inquiry involves a discourse on the implications drawn from our discoveries, catering to researchers and practitioners vested in fostering the sustainable adoption of mobile wallets