Profiling the transcriptome of Gracilaria changii (Rhodophyta) in response to light deprivation

Light regulates photosynthesis, growth and reproduction, yield and properties of phycocolloids, and starch contents in seaweeds. Despite its importance as an environmental cue that regulates many developmental, physiological, and biochemical processes, the network of genes involved during light deprivation are obscure. In this study, we profiled the transcriptome of Gracilaria changii at two different irradiance levels using a cDNA microarray containing more than 3,000 cDNA probes. Microarray analysis revealed that 93 and 105 genes were up- and down-regulated more than 3-fold under light deprivation, respectively. However, only 50% of the transcripts have significant matches to the nonredundant peptide sequences in the database. The transcripts that accumulated under light deprivation include vanadium chloroperoxidase, thioredoxin, ferredoxin component, and reduced nicotinamide adenine dinucleotide dehydrogenase. Among the genes that were down-regulated under light deprivation were genes encoding light harvesting protein, light harvesting complex I, phycobilisome 7.8 kDa linker polypeptide, low molecular weight early light-inducible protein, and vanadium bromoperoxidase. Our findings also provided important clues to the functions of many unknown sequences that could not be annotated using sequence comparison

Profiling of spatial metabolite distributions in wheat leaves under normal and nitrate limiting conditions

The control and interaction between nitrogen and carbon assimilatory pathways is essential in both photosynthetic and non-photosynthetic tissue in order to support metabolic processes without compromising growth. Physiological differences between the basal and mature region of wheat (Triticum aestivum) primary leaves confirmed that there was a change from heterotrophic to autotrophic metabolism. Fourier Transform Infrared (FT-IR) spectroscopy confirmed the suitability and phenotypic reproducibility of the leaf growth conditions. Principal Component–Discriminant Function Analysis (PC–DFA) revealed distinct clustering between base, and tip sections of the developing wheat leaf, and from plants grown in the presence or absence of nitrate. Gas Chromatography-Time of Flight/Mass Spectrometry (GC-TOF/MS) combined with multivariate and univariate analyses, and Bayesian network (BN) analysis, distinguished different tissues and confirmed the physiological switch from high rates of respiration to photosynthesis along the leaf. The operation of nitrogen metabolism impacted on the levels and distribution of amino acids, organic acids and carbohydrates within the wheat leaf. In plants grown in the presence of nitrate there was reduced levels of a number of sugar metabolites in the leaf base and an increase in maltose levels, possibly reflecting an increase in starch turnover. The value of using this combined metabolomics analysis for further functional investigations in the future are discussed

Proteomic Analysis of Grape Berry Cell Cultures Reveals that Developmentally Regulated Ripening Related Processes Can Be Studied Using Cultured Cells

The original publication is available at http:/www.plosone.orgBackground: This work describes a proteomics profiling method, optimized and applied to berry cell suspensions to evaluate organ-specific cultures as a platform to study grape berry ripening. Variations in berry ripening within a cluster(s) on a vine and in a vineyard are a major impediment towards complete understanding of the functional processes that control ripening, specifically when a characterized and homogenous sample is required. Berry cell suspensions could overcome some of these problems, but their suitability as a model system for berry development and ripening needs to be established first. Methodology/Principal Findings: In this study we report on the proteomic evaluation of the cytosolic proteins obtained from synchronized cell suspension cultures that were established from callus lines originating from green, véraison and ripe Vitis vinifera berry explants. The proteins were separated using liquid phase IEF in a Microrotofor cell and SDS PAGE. This method proved superior to gel-based 2DE. Principal component analysis confirmed that biological and technical repeats grouped tightly and importantly, showed that the proteomes of berry cultures originating from the different growth/ripening stages were distinct. A total of twenty six common bands were selected after band matching between different growth stages and twenty two of these bands were positively identified. Thirty two % of the identified proteins are currently annotated as hypothetical. The differential expression profile of the identified proteins, when compared with published literature on grape berry ripening, suggested common trends in terms of relative abundance in the different developmental stages between real berries and cell suspensions. Conclusions: The advantages of having suspension cultures that accurately mimic specific developmental stages are profound and could significantly contribute to the study of the intricate regulatory and signaling networks responsible for berry development and ripening. © 2011 Sharathchandra et al.Publishers' Versio

Reference genes for gene expression studies in wheat flag leaves grown under different farming conditions

<p>Abstract</p> <p>Background</p> <p>Internal control genes with highly uniform expression throughout the experimental conditions are required for accurate gene expression analysis as no universal reference genes exists. In this study, the expression stability of 24 candidate genes from <it>Triticum aestivum </it>cv. Cubus flag leaves grown under organic and conventional farming systems was evaluated in two locations in order to select suitable genes that can be used for normalization of real-time quantitative reverse-transcription PCR (RT-qPCR) reactions. The genes were selected among the most common used reference genes as well as genes encoding proteins involved in several metabolic pathways.</p> <p>Findings</p> <p>Individual genes displayed different expression rates across all samples assayed. Applying geNorm, a set of three potential reference genes were suitable for normalization of RT-qPCR reactions in winter wheat flag leaves cv. Cubus: <it>TaFNRII </it>(ferredoxin-NADP(H) oxidoreductase; AJ457980.1), <it>ACT2 </it>(actin 2; TC234027), and <it>rrn26 </it>(a putative homologue to RNA 26S gene; AL827977.1). In addition of these three genes that were also top-ranked by NormFinder, two extra genes: <it>CYP18-2 </it>(Cyclophilin A, AY456122.1) and <it>TaWIN1 </it>(14-3-3 like protein, AB042193) were most consistently stably expressed.</p> <p>Furthermore, we showed that <it>TaFNRII, ACT2</it>, and <it>CYP18-2 </it>are suitable for gene expression normalization in other two winter wheat varieties (Tommi and Centenaire) grown under three treatments (organic, conventional and no nitrogen) and a different environment than the one tested with cv. Cubus.</p> <p>Conclusions</p> <p>This study provides a new set of reference genes which should improve the accuracy of gene expression analyses when using wheat flag leaves as those related to the improvement of nitrogen use efficiency for cereal production.</p

Effects of TWIN-OF-EYELESS on Clock Gene Expression and Central-Pacemaker Neuron Development in Drosophila.

Circadian oscillators are autonomous molecular rhythms that reside in cells to align whole organism physiology and behaviour to the 24h day. In flies, as in mammals, the oscillator operates in cells that co-express CLOCK (CLK) and CYCLE (CYC). Recent work in Drosophila has shown that CLK is unique in its ability to generate heterologous oscillators, indicating that Clk-gene-expression defines the circadian-cell-fate. Here, using standard in vitro and in vivo techniques, we show that TWIN-OF-EYELESS (TOY; dPax6) regulates Clk-expression in small ventrolateral neurons (s-LN(v)s) that coordinate sleep-wake cycles. Crucially, toy binds multiple sites at the Clk locus; is expressed independent of CLK-CYC in LN(v)s; regulates CLK protein levels under optimal photoperiodic conditions; and, sets clock-speed during endogenous free-run. Furthermore, TOY is necessary for the onset of Clk-expression in LN(v)s during embryogenesis. We propose that TOY contributes to a transcription complex that functions upstream of the oscillator to promote Clk-expression in s-LN(v)s

Effects of TWIN-OF-EYELESS on Clock

Circadian oscillators are autonomous molecular rhythms that reside in cells to align whole-organism physiology and behavior to the 24-h day. In flies, as in mammals, the oscillator operates in cells that coexpress CLOCK (CLK) and CYCLE (CYC). Recent work in Drosophila has shown that CLK is unique in its ability to generate heterologous oscillators, indicating that Clk gene expression defines the circadian cell fate. Here, using standard in vitro and in vivo techniques, we show that TWIN-OF-EYELESS (TOY; dPax6) regulates Clk expression in small ventrolateral neurons (s-LNvs) that coordinate sleep-wake cycles. Crucially, toy binds multiple sites at the Clk locus, is expressed independent of CLK-CYC in LNvs, regulates CLK protein levels under optimal photoperiodic conditions, and sets clock-speed during endogenous free-run. Furthermore, TOY is necessary for the onset of Clk expression in LNvs during embryogenesis. We propose that TOY contributes to a transcription complex that functions upstream of the oscillator to promote Clk expression in s-LNvs

Identification of a Major Epitope Recognized by PLA2R Autoantibodies in Primary Membranous Nephropathy.

Phospholipase A(2) receptor 1 (PLA2R) is a target autoantigen in 70% of patients with idiopathic membranous nephropathy. We describe the location of a major epitope in the N-terminal cysteine-rich ricin domain of PLA2R that is recognized by 90% of human anti-PLA2R autoantibodies. The epitope was sensitive to reduction and SDS denaturation in the isolated ricin domain and the larger fragment containing the ricin, fibronectin type II, first and second C-type lectin domains (CTLD). However, in nondenaturing conditions the epitope was protected against reduction in larger fragments, including the full-length extracellular region of PLA2R. To determine the composition of the epitope, we isolated immunoreactive tryptic fragments by Western blotting and analyzed them by mass spectrometry. The identified peptides were tested as inhibitors of autoantibody binding to PLA2R by surface plasmon resonance. Two peptides from the ricin domain showed strong inhibition, with a longer sequence covering both peptides (31-mer) producing 85% inhibition of autoantibody binding to PLA2R. Anti-PLA2R antibody directly bound this 31-mer peptide under nondenaturing conditions and binding was sensitive to reduction. Analysis of PLA2R and the PLA2R-anti-PLA2R complex using electron microscopy and homology-based representations allowed us to generate a structural model of this major epitope and its antibody binding site, which is independent of pH-induced conformational change in PLA2R. Identification of this major PLA2R epitope will enable further therapeutic advances for patients with idiopathic membranous nephropathy, including antibody inhibition therapy and immunoadsorption of circulating autoantibodies