Molecular-genetic analysis of natural variation in photoperiodic flowering of Arabidopsis thaliana

In Arabidopsis thaliana, the focus of my research, three developmental switches controlling the life cycle can be recognised. The first is germination that separates embryonic from post-embryonic development. The second signals the transition from the juvenile to the adult vegetative phase while the third, flowering, marks the initiation of the reproductive phase (Isabel Baurle and Caroline Dean, Cell 2006). All three exhibit both external (environmental) and endogenous (hormones) regulation. Natural genetic variation, namely phenotypic diversity due to genetic differences between individuals of the same species, has been reported both for germination and flowering initiation (Bentsink et al., PNAS 2006; O Neill et al., TAG 2008). Since individuals of Arabidopsis, commonly referred to as accessions, are collected from a variety of locations, it is believed that this genetic diversity reflects differences in the seasonal oscillations of environmental cues among the collection sites leading to local adaptation. Although natural genetic variation as a tool has been used in the study of flowering initiation in Arabidopsis (Alonso-Blanco and Maarten Koornneef, Trends in Plant Science 2000) a systematic survey that focuses mainly on the photoperiodic aspect of this regulation has been lacking. In order to expand the current knowledge two approaches were designed. First a survey for natural genetic variation in the flowering responses of phylogenetically distant Arabidopsis accessions under six different photoperiods was made. In parallel the transgenic equivalents of the same accessions, carrying a promoter fusion of the flowering time and circadian clock gene GIGANTEA (GI) were screened in the same photoperiods as for flowering time in order to detect for the first time trans-specific natural variation in the circadian regulation of an evening gene. Here I present evidence that natural genetic variation is present in a wide range of photoperiods both for the circadian clock and for flowering initiation per se. The flowering time responses are compared with the ones of mutants and transgenic lines of previously identified flowering time genes and I show that the affected known genes cannot fully cover the different patterns of day length discrimination that the natural accessions exhibit. Five different mapping populations were constructed by selecting interesting accessions from both screens, which led to the identification of new as well as known QTL, which alter various circadian and flowering responses between short and long days of similar duration. Generating advanced genetic material allows fine mapping and eventually cloning of some of the loci, while identification of genome-wide patterns of genetic interactions reveals additional loci that classical QTL mapping approaches cannot detect. Using RT-PCR and in situ hybridisation, I link this novel natural genetic variation between similar long day lengths with molecular variability in the temporal and spatial expression of flowering time genes FT and SOC1 thereby also demonstrating the tight dependence of the SAM floral commitment on the FT florigen. Finally I show that in nature, genetic variability in the property of enhanced photoperiod discrimination under similar long days, is enough to prevent winter flowering in a plant without any requirements for vernalization. Cologne, 200

Viruses affecting lentil (Lens culinaris Medik.) in Greece; incidence and genetic variability of Bean leafroll virus and Pea enation mosaic virus

In Greece, lentil (Lens culinaris Medik.) crops are mainly established with non-certified seeds of local landraces, implying high risks for seed transmitted diseases. During April and May of the 2007–2012 growing seasons, surveys were conducted in eight regions of Greece (Attiki, Evros, Fthiotida, Korinthos, Kozani, Larissa, Lefkada and Viotia) to monitor virus incidence in lentil fields. A total of 1216 lentil samples, from plants exhibiting symptoms suggestive of virus infection, were analyzed from 2007 to 2009, using tissue-blot immunoassays (TBIA). Pea seed-borne mosaic virus (PSbMV) overall incidence was 4.9%, followed by Alfalfa mosaic virus (AMV) (2.4%) and Bean yellow mosaic virus (BYMV) (1.0%). When 274 of the samples were tested for the presence of luteoviruses, 38.8% were infected with Bean leafroll virus (BLRV). Since BLRV was not identified in the majority of the samples collected from 2007 to 2009, representative symptomatic plants (360 samples) were collected in further surveys performed from 2010 to 2012 and tested by ELISA. Two viruses prevailed in those samples: BLRV (36.1%) was associated with stunting, yellowing, and reddening symptoms and Pea enation mosaic virus-1 (PEMV-1) (35.0%) was associated with mosaic and mottling symptoms. PSbMV (2.2%), AMV (2.2%), BYMV (3.9%) and CMV (2.8%) were also detected. When the molecular variability was analyzed for representative isolates, collected from the main Greek lentil production areas, five BLRV isolates showed 95% identity for the coat protein (CP) gene and 99% for the 3’ end region. Three Greek PEMV isolates co-clustered with an isolate from Germany when their CP sequence was compared with isolates with no mutation in the aphid transmission gene. Overall, limited genetic variability was detected among Greek isolates of BLRV and PEMV

Non-Coding Variants in Cancer: Mechanistic Insights and Clinical Potential for Personalized Medicine

The cancer genome is characterized by extensive variability, in the form of Single Nucleotide Polymorphisms (SNPs) or structural variations such as Copy Number Alterations (CNAs) across wider genomic areas. At the molecular level, most SNPs and/or CNAs reside in non-coding sequences, ultimately affecting the regulation of oncogenes and/or tumor-suppressors in a cancer-specific manner. Notably, inherited non-coding variants can predispose for cancer decades prior to disease onset. Furthermore, accumulation of additional non-coding driver mutations during progression of the disease, gives rise to genomic instability, acting as the driving force of neoplastic development and malignant evolution. Therefore, detection and characterization of such mutations can improve risk assessment for healthy carriers and expand the diagnostic and therapeutic toolbox for the patient. This review focuses on functional variants that reside in transcribed or not transcribed non-coding regions of the cancer genome and presents a collection of appropriate state-of-the-art methodologies to study them

Crosstalk mechanisms between the WNT signaling pathway and long non-coding RNAs

The WNT/β-catenin signaling pathway controls a plethora of biological processes throughout animal development and adult life. Because of its fundamental role during animal lifespan, the WNT pathway is subject to strict positive and negative multi-layered regulation, while its aberrant activity causes a wide range of pathologies, including cancer. At present, despite the inroads into the molecules involved in WNT-mediated transcriptional responses, the fine-tuning of WNT pathway activity and the totality of its target genes have not been fully elucidated. Over the past few years, long non-coding RNAs (lncRNAs), RNA transcripts longer that 200nt that do not code for proteins, have emerged as significant transcriptional regulators. Recent studies show that lncRNAs can modulate WNT pathway outcome by affecting gene expression through diversified mechanisms, from the transcriptional to post-translational level. In this review, we selectively discuss those lncRNA-mediated mechanisms we believe the most important to WNT pathway modulation

Distinct Patterns of Genetic Variation Alter Flowering Responses of Arabidopsis Accessions to Different Daylengths1[C][W][OA]

Many plants flower in response to seasonal changes in daylength. This response often varies between accessions of a single species. We studied the variation in photoperiod response found in the model species Arabidopsis (Arabidopsis thaliana). Seventy-two accessions were grown under six daylengths varying in 2-h intervals from 6 to 16 h. The typical response was sigmoidal, so that plants flowered early under days longer than 14 h, late under days shorter than 10 h, and at intermediate times under 12-h days. However, many accessions diverged from this pattern and were clustered into groups showing related phenotypes. Thirty-one mutants and transgenic lines were also scored under the same conditions. Statistical comparisons demonstrated that some accessions show stronger responses to different daylengths than are found among the mutants. Genetic analysis of two such accessions demonstrated that different quantitative trait loci conferred an enhanced response to shortening the daylength from 16 to 14 h. Our data illustrate the spectrum of daylength response phenotypes present in accessions of Arabidopsis and demonstrate that similar phenotypic variation in photoperiodic response can be conferred by different combinations of loci

Development of Toehold Switches as a Novel Ribodiagnostic Method for West Nile Virus

West Nile virus (WNV) is an emerging neurotropic RNA virus and a member of the genus Flavivirus. Naturally, the virus is maintained in an enzootic cycle involving mosquitoes as vectors and birds that are the principal amplifying virus hosts. In humans, the incubation period for WNV disease ranges from 3 to 14 days, with an estimated 80% of infected persons being asymptomatic, around 19% developing a mild febrile infection and less than 1% developing neuroinvasive disease. Laboratory diagnosis of WNV infection is generally accomplished by cross-reacting serological methods or highly sensitive yet expensive molecular approaches. Therefore, current diagnostic tools hinder widespread surveillance of WNV in birds and mosquitoes that serve as viral reservoirs for infecting secondary hosts, such as humans and equines. We have developed a synthetic biology-based method for sensitive and low-cost detection of WNV. This method relies on toehold riboswitches designed to detect WNV genomic RNA as transcriptional input and process it to GFP fluorescence as translational output. Our methodology offers a non-invasive tool with reduced operating cost and high diagnostic value that can be used for field surveillance of WNV in humans as well as in bird and mosquito populations

A Positive Regulatory Loop between a Wnt-Regulated Non-coding RNA and ASCL2 Controls Intestinal Stem Cell Fate

The canonical Wnt pathway plays a central role in stem cell maintenance, differentiation, and proliferation in the intestinal epithelium. Constitutive, aberrant activity of the TCF4/β-catenin transcriptional complex is the primary transforming factor in colorectal cancer. We identify a nuclear long non-coding RNA, termed WiNTRLINC1, as a direct target of TCF4/β-catenin in colorectal cancer cells. WiNTRLINC1 positively regulates the expression of its genomic neighbor ASCL2, a transcription factor that controls intestinal stem cell fate. WiNTRLINC1 interacts with TCF4/β-catenin to mediate the juxtaposition of its promoter with the regulatory regions of ASCL2. ASCL2, in turn, regulates WiNTRLINC1 transcriptionally, closing a feedforward regulatory loop that controls stem cell-related gene expression. This regulatory circuitry is highly amplified in colorectal cancer and correlates with increased metastatic potential and decreased patient survival. Our results uncover the interplay between non-coding RNA-mediated regulation and Wnt signaling and point to the diagnostic and therapeutic potential of WiNTRLINC1

Antioxidant and Anticancer Activities of Synthesized Methylated and Acetylated Derivatives of Natural Bromophenols

Natural bromophenols are important secondary metabolites in marine algae. Derivatives of these bromophenol are potential candidates for the drug development due to their biological activities, such as antioxidant, anticancer, anti-diabetic and anti-inflammatory activity. In our present study, we have designed and synthesized a series of new methylated and acetylated bromophenol derivatives from easily available materials using simple operation procedures and evaluated their antioxidant and anticancer activities on the cellular level. The results showed that 2.,3-dibromo-1-(((2-bromo-4,5-dimethoxybenzyl)oxy)methyl)-4,5-dimethoxybenzene (3b-9) and (oxybis(methylene))bis(4-bromo-6-methoxy-3,1-phenylene) diacetate (4b-3) compounds ameliorated H2O2-induced oxidative damage and ROS generation in HaCaT keratinocytes. Compounds 2.,3-dibromo-1-(((2-bromo-4,5-dimethoxybenzyl)oxy)methyl)-4,5-dimethoxybenzene (3b-9) and (oxybis(methylene) )bis(4-bromo-6-methoxy-3,1-phenylene) diacetate (4b-3) also increased the TrxR1 and HO-1 expression while not affecting Nrf2 expression in HaCaT. In addition, compounds (oxybis(methylene)bis(2-bromo-6-methoxy-4,1-phenylene) diacetate (4b-4) inhibited the viability and induced apoptosis of leukemia K562 cells while not affecting the cell cycle distribution. The present work indicated that some of these bromophenol derivatives possess significant antioxidant and anticancer potential, which merits further investigation

Antioxidant and Anticancer Activities of Synthesized Methylated and Acetylated Derivatives of Natural Bromophenols

Natural bromophenols are important secondary metabolites in marine algae. Derivatives of these bromophenol are potential candidates for the drug development due to their biological activities, such as antioxidant, anticancer, anti-diabetic and anti-inflammatory activity. In our present study, we have designed and synthesized a series of new methylated and acetylated bromophenol derivatives from easily available materials using simple operation procedures and evaluated their antioxidant and anticancer activities on the cellular level. The results showed that 2.,3-dibromo-1-(((2-bromo-4,5-dimethoxybenzyl)oxy)methyl)-4,5-dimethoxybenzene (3b-9) and (oxybis(methylene))bis(4-bromo-6-methoxy-3,1-phenylene) diacetate (4b-3) compounds ameliorated H2O2-induced oxidative damage and ROS generation in HaCaT keratinocytes. Compounds 2.,3-dibromo-1-(((2-bromo-4,5-dimethoxybenzyl)oxy)methyl)-4,5-dimethoxybenzene (3b-9) and (oxybis(methylene) )bis(4-bromo-6-methoxy-3,1-phenylene) diacetate (4b-3) also increased the TrxR1 and HO-1 expression while not affecting Nrf2 expression in HaCaT. In addition, compounds (oxybis(methylene)bis(2-bromo-6-methoxy-4,1-phenylene) diacetate (4b-4) inhibited the viability and induced apoptosis of leukemia K562 cells while not affecting the cell cycle distribution. The present work indicated that some of these bromophenol derivatives possess significant antioxidant and anticancer potential, which merits further investigation